Patent Publication Number: US-2023151418-A1

Title: Joint detection method for lymphangioleio-myomatosis and use thereof

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims priority to PCT Application No. PCT/CN2019/129173, having a filing date of Dec. 27, 2019, which is based on Chinese Application No. 201911281998.6, having a filing date of Dec. 13, 2019, the entire contents both of which are hereby incorporated by reference. 
    
    
     SEQUENCE LISTING 
     This application includes a separate sequence listing in compliance with the requirement of 37 C.F.R.§.§ 1.824(a)(2)-1.824 (a)(6) and 1.824 (b), submitted under the file name “0102US01_Sequence_Listing”, created on Jun. 13, 2022, having a file size of 71.5 KB, the contents of which are hereby incorporated by reference. 
     FIELD OF TECHNOLOGY 
     The following relates to the technical field of genetic detection, particularly, it relates to a joint detection method for lymphangioleio-myomatosis and a use thereof. 
     BACKGROUND 
     Lymphangioleio-myomatosis (LAM) is a rare multisystem neoplastic disease that almost exclusively occurs in women. The main symptom of such disease is a disseminated thin-walled cystic lesions in the lung, which has progressed slowly. In the early stages, LAM patients experience mild symptoms, usually with gradually increasing dyspnea symptom, recurrent pneumothorax and chylothorax. About 60% to 70% of the patients develop a pneumothorax at some stages, while about 30% of the patients have chylothorax. The conventional extrapulmonary manifestation includes angiomyolipoma of the kidney, blockage of retroperitoneal lymphangion and blockage of pelvic lymph node, etc. 
     LAMs can be divided into two types, i.e., Sporadic LAM (S-LAM) and LAM associated with Tuberous Sclerosis Complex (TSC-LAM). The pathogenesis of LAM is unknown yet, but currently, it can be considered that patients suffering from LAM have gene mutations of TSC1 and TSC2, which result in continually activating cellular signaling pathway mediated by mammalian target of rapamycin (mTOR). Sirolimus can effectively inhibit the mTOR pathway and thus become an effective medicament for the treatment of LAM. It is also the first drug approved by FDA to treat LAM. And, it is reported that some LAM patients administrated with Sirolimus show improved lung function and decreased serum level of vascular endothelial growth factor D (VEGF-D) levels, and thus the symptoms of these patients are relieved and their life quality is improved; some LAM patients administrated with Sirolimus show continual exacerbation for the symptoms. Therefore, the primary cause of the difference in Sirolimus response is unclear. It is also shown in the researches that gene mutations of TSC1 and TSC2 cannot be detected in some patients, indicating that there may be another mechanism to participate in the occurrence and development of LAM. 
     Thus, it is significant for the diagnosis and treatment of LAM to develop a perfect LAM gene detection method and map a genetic mutation profile of a large samples for LAM patients so as to fully understand the pathogenesis of LAM. 
     SUMMARY 
     An aspect relates to a joint detection method for lymphangioleio-myomatosis and a use thereof. In the method, a probe capture method is firstly used to obtain coding regions sequences of TSC1/TSC2 genes and hotspot regions sequences of core genes related to the occurrence and development of tumors; and then paired-end sequencing is conducted on Illumina sequencing platform to detect sequence variant in the targeted region of samples; meanwhile, supplementary detections and result verification are performed in the methods, such as CMA, MLPA and Sanger, etc. to improve positive variant detection rate of LAM patients. Further, auxiliary diagnosis and pre-conception genetic counseling can be performed according to the detection results. 
     A joint detection method for lymphangioleio-myomatosis, comprises the steps as follows: 
     performing Target Sequencing based Hybridization capture: A Panel is designed for whole coding regions of TSC1 and TSC2 genes highly related to LAM and mutation genes closely related to solid tumors to construct a Genomic DNA (gDNA) library; sequencing is performed on a machine after a hybrid capture; 
     sorting: data obtained from the above sequencing is processed and analyzed through bioinformatics; when TSC1 and TSC2 genes are detected to be negative or there is only a one-hit mutated locus, a supplementary detection is performed by chromosomal microarray analysis (CMA) and Multiplex ligation-dependent probe amplification (MLPA); when a locus is detected to be an undefined locus originated from either a somatic mutation or a germline mutation, the locus can be verified by Sanger sequencing; 
     performing CMA to obtain loss of heterozygosity (LOH) and copy number variations; and 
     performing MLPA to obtain large fragment insertions and deletions; and 
     performing Sanger sequencing to test leukocyte samples corresponding to samples to be tested after taking the leukocyte samples, and to further determine whether the samples are S-LAM or TSC-LAM. 
     It has been determined that TSC1 and TSC2 genes are larger, including 23 and 42 exons, respectively, 0 with total coding region length of 9 kb, and have complex sequence regions. In conventional methods, such as Sanger sequencing, TSC1 and TSC2 genes are required to be segmented and amplified for several times and then sequenced, which consumes more workload, money, and samples. For the poor-quality Formalin-Fixed Paraffin-Embedded (FFPE) samples, the conventional amplification fails to achieve full coverage. 
     In the meanwhile, the research showed that the frequency of the somatic mutation for TSC1 and TSC2 genes was less than 10% for about 50% of LAM patients and the detection sensitivity of the conventional Sanger method was above 10%, leading to detection omission. Further, the occurrence and development of LAM is based on a Double-hit model, and the mutations of LAM occur in multiple forms, such as LOH and large fragment insertion and deletion. Therefore, a single detection method cannot meet the requirements of efficient detection. As LAM is heritable and sporadic, the hereditary patients with unobvious symptoms are often clinically misdiagnosed as sporadic LAM. 
     Based on the above research basis, the above joint detection method has been provided. In addition to the gene mutations of TSC1 and TSC2, another mechanism, which may be involved in the occurrence and development of LAM, is also taken into consideration in the method. Therefore, the detection of other tumor-related genes is included in the comprehensive detection of above-mentioned LAM, and meanwhile, methods such as CMA, MLPA, and Sanger, etc. are used for auxiliary diagnosis and result verification, thereby improving the positive mutation detection rate of LAM patients. Further, auxiliary diagnosis and pre-conception genetic counseling can be performed according to the detected result. 
     In one example, in the step of Target Sequencing based Hybridization capture, the Panel is designed to cover the following genes: ALDH1 gene, EGFR gene, FLT3 gene, MYC gene, PTEN gene, SDHD gene, AQP9 gene, ERBB2 gene, HRAS gene, MYCN gene, RET gene, TP53 gene, AR gene, ESR1 gene, KIT gene, NF1 gene, RICTOR gene, TSC1 gene, ATRX gene, FGFR1 gene, KRAS gene, NRAS gene, RUNX1 gene, TSC2 gene, BCL2 gene, FGFR2 gene, MDM2 gene, PDGFRA gene, SDHA gene, VHL gene, BRAF gene, FGFR3 gene, MAP2K1 gene, PGR gene, SDHB gene, CCND1 gene, FGFR4 gene, MET gene, POLE gene, SDHC gene; ABL1 gene, CDKN2A gene, FBXW7 gene, IDH2 gene, NOTCH1 gene, SMAD4 gene, AKT1 gene, CSF1R gene, GNA11 gene, JAK2 gene, NPM1 gene, SMARCB1 gene, ALK gene, CTNNB1 gene, GNAQ gene, JAK3 gene, PIK3CA gene, SMO gene, APC gene, DDR2 gene, GNAS gene, KDR gene, PTPN11 gene, SRC gene, ATM gene, ERBB4 gene, HNF1A gene, MLH1 gene, RB1 gene, STK11 gene, CDH1 gene, EZH2 gene, IDH1 gene, MPL gene, ROS1 gene, and TET2 gene. 
     With references to databases, such as COSMIC and TCGA etc., in combination with the latest guideline/consensus of National Comprehensive Cancer Network (NCCN), proto-oncogenes and tumor suppressor genes closely related to the occurrence and development of solid tumors are screened out and combined to form the above Panel, a gene panel. 
     In one example, in the step of Target Sequencing based Hybridization capture, probe sequences for the TSC1 and TSC2 genes include SEQ ID NO: 1 to SEQ ID NO: 276. 
     For the above-mentioned Panel design, the whole coding regions of TSC1 and TSC2 genes, which are basically highly related to LAM, are designed in a shingled form to ensure target regions of the two genes to be covered at least 2 times, with each probe length of 100 bp, thereby further increasing the detection rate. 
     In one example, in the step of Target Sequencing based Hybridization capture, the sequencing depth is more than 1000×. A mutated locus is identified at a mutation frequency of more than 1%, to avoid detection omission at a low mutation frequency. 
     It is another aspect to provide a use of the joint detection method for LAM in a study of pathogenesis of LAM and/or in a diagnosis and treatment of LAM. 
     In one example, it relates to a use of a specific detection reagent in the joint detection method in a preparation of a diagnostic reagent or a diagnostic equipment for jointly detecting LAM. 
     The present disclosure further discloses a joint detection kit for LAM, including a Panel covering the following genes: ALDH1 gene, EGFR gene, FLT3 gene, MYC gene, PTEN gene, SDHD gene, AQP9 gene, ERBB2 gene, HRAS gene, MYCN gene, RET gene, TP53 gene, AR gene, ESR1 gene, KIT gene, NF1 gene, RICTOR gene, TSC1 gene, ATRX gene, FGFR1 gene, KRAS gene, NRAS gene, RUNX1 gene, TSC2 gene, BCL2 gene, FGFR2 gene, MDM2 gene, PDGFRA gene, SDHA gene, VHL gene, BRAF gene, FGFR3 gene, MAP2K1 gene, PGR gene, SDHB gene, CCND1 gene, FGFR4 gene, MET gene, POLE gene, SDHC gene; ABL1 gene, CDKN2A gene, FBXW7 gene, IDH2 gene, NOTCH1 gene, SMAD4 gene, AKT1 gene, CSF1R gene, GNA11 gene, JAK2 gene, NPM1 gene, SMARCB1 gene, ALK gene, CTNNB1 gene, GNAQ gene, JAK3 gene, PIK3CA gene, SMO gene, APC gene, DDR2 gene, GNAS gene, KDR gene, PTPN11 gene, SRC gene, ATM gene, ERBB4 gene, HNF1A gene, MLH1 gene, RB1 gene, STK11 gene, CDH1 gene, EZH2 gene, IDH1 gene, MPL gene, ROS1 gene, and TET2 gene. 
     In one example, probe sequences of the Panel include SEQ ID NO: 1 to SEQ ID NO: 276. 
     In one example, the joint detection kit further includes an agent for CMA. It should be understood that the agent for CMA can be selected according to a practical experimental requirement, such as OncoScan® CNV FFRE Assay kit. 
     In one example, the joint detection kit further includes multiplex ligation-dependent probes for MLPA. It should be understood that the probes can be selected according to practical experimental requirements, 0 such as TSC1 and TSC2 probes from MRC-Holland. 
     It is another aspect to provide a joint detection system for LAM, comprising the modules as follows: 
     a detection module, comprising a module of Target Sequencing based Hybridization capture, a module of CMA, a module of MLPA, and a module of Sanger sequencing, wherein the module of Target Sequencing based Hybridization capture comprises a Panel designed for whole coding regions of TSC1 and TSC2 genes highly related to LAM and mutated genes closely related to solid tumors; and 
     an analysis module, firstly obtaining a detection result of Target Sequencing based Hybridization capture; requesting the module of chromosomal microarray analysis (CMA) and the module of Multiplex ligation-dependent probe amplification (MLPA) to perform a supplementary detection when TSC1 and TSC2 genes are detected to be negative or there is only a one-hit mutated locus; requesting the module of Sanger sequencing to verify the undefined locus when a locus is detected to be an undefined locus originated from either a somatic mutation or a germline mutation; and then analyzing and judging detection results from the detection modules, to draw a joint detection result of LAM. 
     In one example, the Panel covers genes as follows: ALDH1 gene, EGFR gene, FLT3 gene, MYC gene, PTEN gene, SDHD gene, AQP9 gene, ERBB2 gene, HRAS gene, MYCN gene, RET gene, TP53 gene, AR gene, ESR1 gene, KIT gene, NF1 gene, RICTOR gene, TSC1 gene, ATRX gene, FGFR1 gene, KRAS gene, NRAS gene, RUNX1 gene, TSC2 gene, BCL2 gene, FGFR2 gene, MDM2 gene, PDGFRA gene, SDHA gene, VHL gene, BRAF gene, FGFR3 gene, MAP2K1 gene, PGR gene, SDHB gene, CCND1 gene, FGFR4 gene, MET gene, POLE gene, SDHC gene; ABL1 gene, CDKN2A gene, FBXW7 gene, IDH2 gene, NOTCH1 gene, SMAD4 gene, AKT1 gene, CSF1R gene, GNA11 gene, JAK2 gene, NPM1 gene, SMARCB1 gene, ALK gene, CTNNB1 gene, GNAQ gene, JAK3 gene, PIK3CA gene, SMO gene, APC gene, DDR2 gene, GNAS gene, KDR gene, PTPN11 gene, SRC gene, ATM gene, ERBB4 gene, HNF1A gene, MLH1 gene, RB1 gene, STK11 gene, CDH1 gene, EZH2 gene, IDH1 gene, MPL gene, ROS1 gene, and TET2 gene. 
     In one example, in the module of Target Sequencing based Hybridization capture, probe sequences for the TSC1 and TSC2 genes include SEQ ID NO: 1 to SEQ ID NO: 276. 
     It should be understood, that the above-mentioned joint detection system can be a combination of equipments, instruments, or agents that can perform Target Sequencing based Hybridization capture, CMA, MPLA, and Sanger sequencing, as long as the functions of the system can be achieved. For example, the conventional equipments and instruments can be used with specific Panel of the present disclosure, to achieve the purpose of obtaining the whole coding regions sequences of TSC1 and TSC2 genes and the hotspot region sequences of the core genes related to the occurrence and development of the tumor, and performing supplementary detections and result verifications in the methods, such as CMA, MLPA, Sanger sequencing, etc. at the same time. 
     Compared with the conventional art, the present disclosure has the following benefits: 
     The present disclosure provides a joint detection method for lymphangioleio-myomatosis. In the method, a probe capture method is firstly used to obtain coding regions sequences of TSC1/TSC2 genes and hotspot regions sequences of the core gene related to the occurrence and development of the tumor; and then paired-end sequencing is conducted on Illumina sequencing platform to detect sequence variant in the targeted region of samples; meanwhile supplementary detections and result verifications are performed in the methods, such as CMA, MLPA and Sanger, to improve the positive variant detection rate of LAM patients. Further, auxiliary diagnosis and pre-conception genetic counseling can be performed according to the detection results. 
     In addition, for poor-quality FFPE samples, TSC1 and TSC2 genes and the hotspot regions of the core gene related to solid tumor can be perfectly obtained and sequenced in a single experiment, by using a 2×100 bp probe design scheme, combined with a liquid-phase hybridization capture method. 
     The present disclosure provides a joint detection kit for LAM, which can be used for detecting coding regions sequences of TSC1 and TSC2 genes and hotspot regions sequences of the core gene related to the occurrence and development of the tumor. The joint detection kit also includes an agent for CMA reagent and/or multiplex ligation-dependent probes for MLPA, etc., which can be selected according to the requirements. And meanwhile, supplementary detections and result verifications can be performed with the methods, such as CMA, MLPA, and Sanger sequencing to improve the positive mutation detection rate of LAM patients. Further, auxiliary diagnosis and pre-conception genetic counseling can be performed according to the detection results. 
     The present disclosure provides a joint detection system for LAM, comprising a detection module and an analysis module, wherein the detection module comprises a module of Target Sequencing based Hybridization capture, a module of chromosomal microarray analysis (CMA), a module of Multiplex ligation-dependent probe amplification (MLPA) and a module of Sanger sequencing, etc. The system can be used for detecting coding regions sequences of TSC1 and TSC2 genes and hotspot regions sequences of the core genes related to the occurrence and development of the tumor. In the system, methods, such as CMA, MLPA and Sanger, etc. can be selected according to the requirements, and meanwhile, supplementary detections and result verification can be performed with the methods, such as CMA, MLPA and Sanger, etc. to improve the positive mutation detection rate of LAM patients. Further, auxiliary diagnosis and pre-conception genetic counseling can be performed according to the detection results. 
    
    
     
       BRIEF DESCRIPTION 
       Some of examples will be described in detail, with reference to the following figures, wherein like designations denote like members, wherein: 
         FIG.  1    shows a flow diagram of a multi-method joint detection scheme for LAM; 
         FIG.  2    shows a design of targeted probes for the TSC1 and TSC2 genes; 
         FIG.  3    shows a pie chart of the proportion of variants detected by different detection methods; 
         FIG.  4    shows a detection advantage of the joint detection method over the single method; and 
         FIG.  5    shows LOH phenomena of patients with TSC2 gene detected by CMA. 
     
    
    
     DETAILED DESCRIPTION 
     For better understanding of the present disclosure, the present disclosure will be fully described below with reference to the relevant accompanying figures. Preferred embodiments are shown in the figures. However, the present disclosure can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided for the purpose of making the disclosed contents of the present disclosure more thorough and complete. 
     Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those normally understood by one skilled in the art in the technical field of the present disclosure. The terms used in the description of the present disclosure herein are only for the purpose of describing embodiments, and are not intended to limit the present disclosure. The term “and/or” used herein comprises anyone or all combinations of one or more corresponding items listed herein. 
     Example 1 
     LAM samples were detected as follows, according to a multi-method joint detection scheme for LAM, shown in  FIG.  1   . 
     I. Performing Target Sequencing Based Hybridization Capture, a Next-Generation Sequencing (NGS), with Probes: 
     1. Panel Design 
     The whole coding regions of TSC1 and TSC2 genes, which are basically highly related to LAM, were designed in a shingled form to ensure that the target regions of these two genes were covered at least 2 times, with each probe length of 100 bp, wherein the probes for TSC1 and TSC2 genes were designed as shown in  FIG.  2   . 
     At the same time, with reference to databases, such as COSMIC, TCGA, etc., in combination with the latest guidelines/consensus of NCCN, proto-oncogenes and tumor suppressor genes that were closely related to the occurrence and development of solid tumors were screened out, while probes were designed according to conventional method, that is, the probes were connected to the target region in an end-to-end manner at 1-fold coverage. The gene panel was listed as follows: 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Panel&#39;s Gene List 
               
            
           
           
               
               
            
               
                 Coverage 
                 Gene list 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Whole Coding 
                 ALDH1 
                 EGFR 
                 FLT3 
                 MYC 
                 PTEN 
                 SDHD 
               
               
                 Regions 
                 AQP9 
                 ERBB2 
                 HRAS 
                 MYCN 
                 RET 
                 TP53 
               
               
                 (40 genes) 
                 AR 
                 ESR1 
                 KIT 
                 NF1 
                 RICTOR 
                 TSC1 
               
               
                   
                 ATRX 
                 FGFR1 
                 KRAS 
                 NRAS 
                 RUNX1 
                 TSC2 
               
               
                   
                 BCL2 
                 FGFR2 
                 MDM2 
                 PDGFRA 
                 SDHA 
                 VHL 
               
               
                   
                 BRAF 
                 FGFR3 
                 MAP2K1 
                 PGR 
                 SDHB 
               
               
                   
                 CCND1 
                 FGFR4 
                 MET 
                 POLE 
                 SDHC 
               
               
                 Hotspot 
                 ABL1 
                 CDKN2A 
                 FBXW7 
                 IDH2 
                 NOTCH1 
                 SMAD4 
               
               
                 mutations 
                 AKT1 
                 CSF1R 
                 GNA11 
                 JAK2 
                 NPM1 
                 SMARCB1 
               
               
                 (36 genes) 
                 ALK 
                 CTNNB1 
                 GNAQ 
                 JAK3 
                 PIK3CA 
                 SMO 
               
               
                   
                 APC 
                 DDR2 
                 GNAS 
                 KDR 
                 PTPN11 
                 SRC 
               
               
                   
                 ATM 
                 ERBB4 
                 HNF1A 
                 MLH1 
                 RB1 
                 STK11 
               
               
                   
                 CDH1 
                 EZH2 
                 IDH1 
                 MPL 
                 ROS1 
                 TET2 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Design Table of Probes for TSC1 and TSC2 Genes 
               
            
           
           
               
               
               
               
               
               
            
               
                 SEQ 
                   
                   
                   
                   
                   
               
               
                 ID 
                   
                   
                   
                   
                   
               
               
                 NO: 
                 sequences 
                 Chr 
                 Start 
                 Stop 
                 Group 
               
               
                   
               
               
                   1 
                 GTCCCCATTCCTGTTTCGTTTGCACAGAGGGGTTTTCTGGTGCGT 
                 16 
                 20985 
                 2098 
                 TSC2 
               
               
                   
                 CCTGGTCCACCATGGCCAAACCAACAAGCAAAGATTCAGGCTTG 
                   
                    60 
                  660 
                   
               
               
                   
                 AAGGAGAAGTT 
                   
                   
                   
                   
               
               
                   
               
               
                   2 
                 TCCACCATGGCCAAACCAACAAGCAAAGATTCAGGCTTGAAGG 
                 16 
                 20986 
                 2098 
                 TSC2 
               
               
                   
                 AGAAGTTTAAGATTCTGTTGGGACTGGGAACACCGAGGCCAAAT 
                   
                    10 
                  710 
                   
               
               
                   
                 CCCAGGTCTGCAG 
                   
                   
                   
                   
               
               
                   
               
               
                   3 
                 TAAGATTCTGTTGGGACTGGGAACACCGAGGCCAAATCCCAGGT 
                 16 
                 20986 
                 2098 
                 TSC2 
               
               
                   
                 CTGCAGAGGGTAAACAGACGGAGTTTATCATCACCGCGGAAAT 
                   
                    60 
                  760 
                   
               
               
                   
                 ACTGAGAGTGAGT 
                   
                   
                   
                   
               
               
                   
               
               
                   4 
                 AGGGTAAACAGACGGAGTTTATCATCACCGCGGAAATACTGAG 
                 16 
                 20987 
                 2098 
                 TSC2 
               
               
                   
                 AGTGAGTGAGCTACCTGTGTCTTTGCTAGGCTAGAGGGAAATGC 
                   
                    10 
                  810 
                   
               
               
                   
                 AGAGAAGGCTGGG 
                   
                   
                   
                   
               
               
                   
               
               
                   5 
                 TTAAGGAGACCGTGGCCTGAGCACTGGCCCCTTTTTCTTCTTTCA 
                 16 
                 21003 
                 2100 
                 TSC2 
               
               
                   
                 TCTCTCTCCAGGAACTGAGCATGGAATGTGGCCTCAACAATCGC 
                   
                    44 
                  444 
                   
               
               
                   
                 ATCCGGATGAT 
                   
                   
                   
                   
               
               
                   
               
               
                   6 
                 CTCCAGGAACTGAGCATGGAATGTGGCCTCAACAATCGCATCCG 
                 16 
                 21003 
                 2100 
                 TSC2 
               
               
                   
                 GATGATAGGGCAGATTTGTGAAGTCGCAAAAACCAAGAAATTT 
                   
                    94 
                  494 
                   
               
               
                   
                 GAAGAGGTAGGTT 
                   
                   
                   
                   
               
               
                   
               
               
                   7 
                 AGGGCAGATTTGTGAAGTCGCAAAAACCAAGAAATTTGAAGAG 
                 16 
                 21004 
                 2100 
                 TSC2 
               
               
                   
                 GTAGGTTTATCCAGTTGAGCTACTAGAGAGAGGCACGTAGACTA 
                   
                    44 
                  544 
                   
               
               
                   
                 TTCAGAGCCTGAG 
                   
                   
                   
                   
               
               
                   
               
               
                   8 
                 TGCTCCAGTTGCCGGGGCCAGGGTTCTTGGAGAGCACATCCTCA 
                 16 
                 21032 
                 2103 
                 TSC2 
               
               
                   
                 CCGCTGTCCCCTCTGCTGGTGACAGCACGCAGTGGAAGCACTCT 
                   
                    73 
                  373 
                   
               
               
                   
                 GGAAGGCGGTCG 
                   
                   
                   
                   
               
               
                   
               
               
                   9 
                 TCCCCTCTGCTGGTGACAGCACGCAGTGGAAGCACTCTGGAAGG 
                 16 
                 21033 
                 2103 
                 TSC2 
               
               
                   
                 CGGTCGCGGATCTGTTGCAGCCGGAGCGGCCGCTGGAGGCCCGG 
                   
                    23 
                  423 
                   
               
               
                   
                 CACGCGGTGCTG 
                   
                   
                   
                   
               
               
                   
               
               
                  10 
                 CGGATCTGTTGCAGCCGGAGCGGCCGCTGGAGGCCCGGCACGC 
                 16 
                 21033 
                 2103 
                 TSC2 
               
               
                   
                 GGTGCTGGCTCTGCTGAAGGCCATCGTGCAGGGGCAGGTAAGGC 
                   
                    73 
                  473 
                   
               
               
                   
                 CCAGGGCGACGCT 
                   
                   
                   
                   
               
               
                   
               
               
                  11 
                 GCTCTGCTGAAGGCCATCGTGCAGGGGCAGGTAAGGCCCAGGG 
                 16 
                 21034 
                 2103 
                 TSC2 
               
               
                   
                 CGACGCTGGGATGGGTGACGTCAGGCTGCCCACTGACTGTCCTG 
                   
                    23 
                  523 
                   
               
               
                   
                 TCCCTGCTGGGCC 
                   
                   
                   
                   
               
               
                   
               
               
                  12 
                 CCGTGTGGGCGACGCTGGCAGGCTCTGCTGATCCTGTGGCTTTT 
                 16 
                 21042 
                 2104 
                 TSC2 
               
               
                   
                 GTCTTTAGGGCGAGCGTTTGGGGGTCCTCAGAGCCCTCTTCTTTA 
                   
                    44 
                  344 
                   
               
               
                   
                 AGGTCATCAAG 
                   
                   
                   
                   
               
               
                   
               
               
                  13 
                 AGGGCGAGCGTTTGGGGGTCCTCAGAGCCCTCTTCTTTAAGGTC 
                 16 
                 21042 
                 2104 
                 TSC2 
               
               
                   
                 ATCAAGGATTACCCTTCCAACGAAGACCTTCACGAAAGGCTGGA 
                   
                    94 
                  394 
                   
               
               
                   
                 GGTTTTCAAGGC 
                   
                   
                   
                   
               
               
                   
               
               
                  14 
                 GATTACCCTTCCAACGAAGACCTTCACGAAAGGCTGGAGGTTTT 
                 16 
                 21043 
                 2104 
                 TSC2 
               
               
                   
                 CAAGGCCCTCACAGACAATGGGAGACACATCACCTACTTGGAG 
                   
                    44 
                  444 
                   
               
               
                   
                 GAAGAGCTGGGTG 
                   
                   
                   
                   
               
               
                   
               
               
                  15 
                 CCTCACAGACAATGGGAGACACATCACCTACTTGGAGGAAGAG 
                 16 
                 21043 
                 2104 
                 TSC2 
               
               
                   
                 CTGGGTGGGTGCCACCTTGGGTTGGAGGTTTCTCTGGCCTTGAC 
                   
                    94 
                  494 
                   
               
               
                   
                 GATCAAGTGTAAC 
                   
                   
                   
                   
               
               
                   
               
               
                  16 
                 GGAGGGGGAGGTGAGTGGGAGATGTAGATTCGGCGTCCTCGCA 
                 16 
                 21053 
                 2105 
                 TSC2 
               
               
                   
                 AACTGCCGCCGCTTCTCCCCCAGCTGACTTTGTCCTGCAGTGGAT 
                   
                    36 
                  436 
                   
               
               
                   
                 GGATGTTGGCTT 
                   
                   
                   
                   
               
               
                   
               
               
                  17 
                 GCCGCTTCTCCCCCAGCTGACTTTGTCCTGCAGTGGATGGATGTT 
                 16 
                 21053 
                 2105 
                 TSC2 
               
               
                   
                 GGCTTGTCCTCGGAATTCCTTCTGGTGCTGGTGAACTTGGTCAAA 
                   
                    86 
                  486 
                   
               
               
                   
                 TTCAATAGCT 
                   
                   
                   
                   
               
               
                   
               
               
                  18 
                 GTCCTCGGAATTCCTTCTGGTGCTGGTGAACTTGGTCAAATTCAA 
                 16 
                 21054 
                 2105 
                 TSC2 
               
               
                   
                 TAGCTGTTACCTCGACGAGTACATCGCAAGGATGGTTCAGTAAG 
                   
                    36 
                  536 
                   
               
               
                   
                 AAAAGAATTGA 
                   
                   
                   
                   
               
               
                   
               
               
                  19 
                 GTTACCTCGACGAGTACATCGCAAGGATGGTTCAGTAAGAAAAG 
                 16 
                 21054 
                 2105 
                 TSC2 
               
               
                   
                 AATTGAGATCCTGTTCTGATAATGGTCCTAAGTTCAGCTCCGCA 
                   
                    86 
                  586 
                   
               
               
                   
                 GTGAATAAAGTT 
                   
                   
                   
                   
               
               
                   
               
               
                  20 
                 CTCGGCCATCCAGGCAGTGCTGCCGGGACTGAGCTCGGTGCTCC 
                 16 
                 21061 
                 2106 
                 TSC2 
               
               
                   
                 CTGCAGGATGATCTGTCTGCTGTGCGTCCGGACCGCGTCCTCTGT 
                   
                    46 
                  246 
                   
               
               
                   
                 GGACATAGAGG 
                   
                   
                   
                   
               
               
                   
               
               
                  21 
                 GATGATCTGTCTGCTGTGCGTCCGGACCGCGTCCTCTGTGGACAT 
                 16 
                 21061 
                 2106 
                 TSC2 
               
               
                   
                 AGAGGTCAGTGCCTCCCCTCCCCAGGGCCGGCCCATTTCACCCT 
                   
                    96 
                  296 
                   
               
               
                   
                 GGTTTCTGGGA 
                   
                   
                   
                   
               
               
                   
               
               
                  22 
                 TGTCCTCTCCTGTGGGGAGGAGCTGGGGTAGGACGGGCGTGAGC 
                 16 
                 21065 
                 2106 
                 TSC2 
               
               
                   
                 CGTCTCCCTCTCCACCAGGTCTCCCTGCAGGTGCTGGACGCCGTG 
                   
                    82 
                  682 
                   
               
               
                   
                 GTCTGCTACAA 
                   
                   
                   
                   
               
               
                   
               
               
                  23 
                 CCTCTCCACCAGGTCTCCCTGCAGGTGCTGGACGCCGTGGTCTG 
                 16 
                 21066 
                 2106 
                 TSC2 
               
               
                   
                 CTACAACTGCCTGCCGGCTGAGAGCCTCCCGCTGTTCATCGTTAC 
                   
                    32 
                  732 
                   
               
               
                   
                 CCTCTGTCGCA 
                   
                   
                   
                   
               
               
                   
               
               
                  24 
                 CTGCCTGCCGGCTGAGAGCCTCCCGCTGTTCATCGTTACCCTCTG 
                 16 
                 21066 
                 2106 
                 TSC2 
               
               
                   
                 TCGCACCATCAACGTCAAGGAGCTCTGCGAGCCTTGCTGGAAGG 
                   
                    82 
                  782 
                   
               
               
                   
                 TGGGGTTTCTG 
                   
                   
                   
                   
               
               
                   
               
               
                  25 
                 CCATCAACGTCAAGGAGCTCTGCGAGCCTTGCTGGAAGGTGGGG 
                 16 
                 21067 
                 2106 
                 TSC2 
               
               
                   
                 TTTCTGAAACTGCTCTGGAAGGTTCCTGAGAGCACATGGATGGG 
                   
                    32 
                  832 
                   
               
               
                   
                 ACAAGGGCCATC 
                   
                   
                   
                   
               
               
                   
               
               
                  26 
                 CGGGACTGGGGCTGGGGGCAGGGCTTATGCCTGCCAGCCCCTGA 
                 16 
                 21070 
                 2107 
                 TSC2 
               
               
                   
                 CACGCATTGTGTCTCGCAGCTGATGCGGAACCTCCTTGGCACCC 
                   
                    42 
                  142 
                   
               
               
                   
                 ACCTGGGCCACA 
                   
                   
                   
                   
               
               
                   
               
               
                  27 
                 TTGTGTCTCGCAGCTGATGCGGAACCTCCTTGGCACCCACCTGG 
                 16 
                 21070 
                 2107 
                 TSC2 
               
               
                   
                 GCCACAGCGCCATCTACAACATGTGCCACCTCATGGAGGACAGG 
                   
                    92 
                  192 
                   
               
               
                   
                 TGAGTGTGGTGG 
                   
                   
                   
                   
               
               
                   
               
               
                  28 
                 GCGCCATCTACAACATGTGCCACCTCATGGAGGACAGGTGAGTG 
                 16 
                 21071 
                 2107 
                 TSC2 
               
               
                   
                 TGGTGGGTGGGGCGCAGGGCAGTGGAGGCCAGCACAGCCCTCG 
                   
                    42 
                  242 
                   
               
               
                   
                 GGGCAGCTCCAGT 
                   
                   
                   
                   
               
               
                   
               
               
                  29 
                 CCATGGCGGACCCTGGGACAGGGCCCTGCTCACATTCCGTCTCT 
                 16 
                 21086 
                 2108 
                 TSC2 
               
               
                   
                 CTGGGGAACACTTTTAGAGCCTACATGGAGGACGCGCCCCTGCT 
                   
                    86 
                  786 
                   
               
               
                   
                 GAGAGGAGCCGT 
                   
                   
                   
                   
               
               
                   
               
               
                  30 
                 AACACTTTTAGAGCCTACATGGAGGACGCGCCCCTGCTGAGAGG 
                 16 
                 21087 
                 2108 
                 TSC2 
               
               
                   
                 AGCCGTGTTTTTTGTGGGCATGGCTCTCTGGGGAGCCCACCGGC 
                   
                    36 
                  836 
                   
               
               
                   
                 TCTATTCTCTCA 
                   
                   
                   
                   
               
               
                   
               
               
                  31 
                 GTTTTTTGTGGGCATGGCTCTCTGGGGAGCCCACCGGCTCTATTC 
                 16 
                 21087 
                 2108 
                 TSC2 
               
               
                   
                 TCTCAGGAACTCGCCGACATCTGTGTTGCCATCATTTTACCAGGT 
                   
                    86 
                  886 
                   
               
               
                   
                 AAGGCGGTTT 
                   
                   
                   
                   
               
               
                   
               
               
                  32 
                 GGAACTCGCCGACATCTGTGTTGCCATCATTTTACCAGGTAAGG 
                 16 
                 21088 
                 2108 
                 TSC2 
               
               
                   
                 CGGTTTCTGTGTGCAGTGAGCTGGCAGGAACGGGAGAGCTCCCC 
                   
                    36 
                  936 
                   
               
               
                   
                 TCACGCCTGCCC 
                   
                   
                   
                   
               
               
                   
               
               
                  33 
                 CACAGCAAGCAAGCAGCTCTGACCCTGTGTGCTGGCCGGGCTCG 
                 16 
                 21106 
                 2110 
                 TSC2 
               
               
                   
                 TGTTCCAGGCCATGGCATGTCCGAACGAGGTGGTGTCCTATGAG 
                   
                    18 
                  718 
                   
               
               
                   
                 ATCGTCCTGTCC 
                   
                   
                   
                   
               
               
                   
               
               
                  34 
                 AGGCCATGGCATGTCCGAACGAGGTGGTGTCCTATGAGATCGTC 
                 16 
                 21106 
                 2110 
                 TSC2 
               
               
                   
                 CTGTCCATCACCAGGCTCATCAAGAAGTATAGGAAGGAGCTCCA 
                   
                    68 
                  768 
                   
               
               
                   
                 GGTGGTGGCGTG 
                   
                   
                   
                   
               
               
                   
               
               
                  35 
                 ATCACCAGGCTCATCAAGAAGTATAGGAAGGAGCTCCAGGTGG 
                 16 
                 21107 
                 2110 
                 TSC2 
               
               
                   
                 TGGCGTGGGACATTCTGCTGAACATCATCGAACGGCTCCTTCAG 
                   
                    18 
                  818 
                   
               
               
                   
                 CAGCTCCAGGTGG 
                   
                   
                   
                   
               
               
                   
               
               
                  36 
                 GGACATTCTGCTGAACATCATCGAACGGCTCCTTCAGCAGCTCC 
                 16 
                 21107 
                 2110 
                 TSC2 
               
               
                   
                 AGGTGGGGTGGGGGCAGGAGCTCCGGGGAGCACCGGGAACCCA 
                   
                    68 
                  868 
                   
               
               
                   
                 GACAGGCAGGCTC 
                   
                   
                   
                   
               
               
                   
               
               
                  37 
                 GCCAAGTCCATGTGGGGAGTGGAAGTCAGCCTGTGTCATCGTGC 
                 16 
                 21118 
                 2111 
                 TSC2 
               
               
                   
                 CTGGTACTGCAGACCTTGGACAGCCCGGAGCTCAGGACCATCGT 
                   
                    15 
                  915 
                   
               
               
                   
                 CCATGACCTGTT 
                   
                   
                   
                   
               
               
                   
               
               
                  38 
                 CTGCAGACCTTGGACAGCCCGGAGCTCAGGACCATCGTCCATGA 
                 16 
                 21118 
                 2111 
                 TSC2 
               
               
                   
                 CCTGTTGACCACGGTGGAGGAGCTGTGTGACCAGAACGAGTTCC 
                   
                    65 
                  965 
                   
               
               
                   
                 ACGGGTCTCAGG 
                   
                   
                   
                   
               
               
                   
               
               
                  39 
                 GACCACGGTGGAGGAGCTGTGTGACCAGAACGAGTTCCACGGG 
                 16 
                 21119 
                 2112 
                 TSC2 
               
               
                   
                 TCTCAGGAGAGATACTTTGAACTGGTGGAGAGATGTGCGGACCA 
                   
                    15 
                  015 
                   
               
               
                   
                 GAGGCCTGTGAGA 
                   
                   
                   
                   
               
               
                   
               
               
                  40 
                 AGAGATACTTTGAACTGGTGGAGAGATGTGCGGACCAGAGGCC 
                 16 
                 21119 
                 2112 
                 TSC2 
               
               
                   
                 TGTGAGACCCCCTCCTGGGTGGGGCCTTTGGGCTTTGGCTGGTG 
                   
                    65 
                  065 
                   
               
               
                   
                 GGGAGGGGCCGGG 
                   
                   
                   
                   
               
               
                   
               
               
                  41 
                 GACCAGCAGCCCAGTGTGGAGAAGGAGAGCGCCGGAGGGGCAG 
                 16 
                 21124 
                 2112 
                 TSC2 
               
               
                   
                 AGGGGCAACACCGGCTCTTCTTTTGACAGGAGTCCTCCCTCCTG 
                   
                    25 
                  525 
                   
               
               
                   
                 AACCTGATCTCCT 
                   
                   
                   
                   
               
               
                   
               
               
                  42 
                 ACACCGGCTCTTCTTTTGACAGGAGTCCTCCCTCCTGAACCTGAT 
                 16 
                 21124 
                 2112 
                 TSC2 
               
               
                   
                 CTCCTATAGAGCGCAGTCCATCCACCCGGCCAAGGACGGCTGGA 
                   
                    75 
                  575 
                   
               
               
                   
                 TTCAGAACCTG 
                   
                   
                   
                   
               
               
                   
               
               
                  43 
                 ATAGAGCGCAGTCCATCCACCCGGCCAAGGACGGCTGGATTCAG 
                 16 
                 21125 
                 2112 
                 TSC2 
               
               
                   
                 AACCTGCAGGCGCTGATGGAGAGATTCTTCAGGTAGGGGGTCCT 
                   
                    25 
                  625 
                   
               
               
                   
                 CTGTAGCCTTGC 
                   
                   
                   
                   
               
               
                   
               
               
                  44 
                 CAGGCGCTGATGGAGAGATTCTTCAGGTAGGGGGTCCTCTGTAG 
                 16 
                 21125 
                 2112 
                 TSC2 
               
               
                   
                 CCTTGCCTGGCACCTGGAGCCTGGCCCTGTCTCTGTCTGGGGCCC 
                   
                    75 
                  675 
                   
               
               
                   
                 ACCCGGGCTGG 
                   
                   
                   
                   
               
               
                   
               
               
                  45 
                 CTGGTGTGGGGCTGTGGCCGGGCACTCCCCACCCGCCCCAGCAG 
                 16 
                 21129 
                 2113 
                 TSC2 
               
               
                   
                 GCTGCCGTCCCGCAGGAGCGAGTCCCGAGGCGCCGTGCGCATCA 
                   
                    13 
                  013 
                   
               
               
                   
                 AGGTGCTGGACG 
                   
                   
                   
                   
               
               
                   
               
               
                  46 
                 GTCCCGCAGGAGCGAGTCCCGAGGCGCCGTGCGCATCAAGGTG 
                 16 
                 21129 
                 2113 
                 TSC2 
               
               
                   
                 CTGGACGTGCTGTCCTTTGTGCTGCTCATCAACAGGCAGTTCTAT 
                   
                    63 
                  063 
                   
               
               
                   
                 GAGGTGCGTGTC 
                   
                   
                   
                   
               
               
                   
               
               
                  47 
                 TGCTGTCCTTTGTGCTGCTCATCAACAGGCAGTTCTATGAGGTGC 
                 16 
                 21130 
                 2113 
                 TSC2 
               
               
                   
                 GTGTCCAGGCGGCCGCAGCTGGGGGCTCAGGGCTATTTCTCCGT 
                   
                    13 
                  113 
                   
               
               
                   
                 GGGCGGGCTGT 
                   
                   
                   
                   
               
               
                   
               
               
                  48 
                 GGAATTGGAAGTGTCACGAGATGTGGCCCTCGTTGGGCTGGCGC 
                 16 
                 21142 
                 2114 
                 TSC2 
               
               
                   
                 TCATTGGCCTCCCTTGTGCCTGTGCAGGAGGAGCTGATTAACTC 
                   
                    01 
                  301 
                   
               
               
                   
                 AGTGGTCATCTC 
                   
                   
                   
                   
               
               
                   
               
               
                  49 
                 GCCTCCCTTGTGCCTGTGCAGGAGGAGCTGATTAACTCAGTGGT 
                 16 
                 21142 
                 2114 
                 TSC2 
               
               
                   
                 CATCTCGCAGCTCTCCCACATCCCCGAGGATAAAGACCACCAGG 
                   
                    51 
                  351 
                   
               
               
                   
                 TCCGAAAGCTGG 
                   
                   
                   
                   
               
               
                   
               
               
                  50 
                 GCAGCTCTCCCACATCCCCGAGGATAAAGACCACCAGGTCCGAA 
                 16 
                 21143 
                 2114 
                 TSC2 
               
               
                   
                 AGCTGGCCACCCAGTTGCTGGTGGACCTGGCAGAGGGCTGCCAC 
                   
                    01 
                  401 
                   
               
               
                   
                 ACACACCACTTC 
                   
                   
                   
                   
               
               
                   
               
               
                  51 
                 CCACCCAGTTGCTGGTGGACCTGGCAGAGGGCTGCCACACACAC 
                 16 
                 21143 
                 2114 
                 TSC2 
               
               
                   
                 CACTTCAACAGCCTGCTGGACATCATCGAGAAGGTGAGAGCCGT 
                   
                    51 
                  451 
                   
               
               
                   
                 TGTACCCGGGGC 
                   
                   
                   
                   
               
               
                   
               
               
                  52 
                 AACAGCCTGCTGGACATCATCGAGAAGGTGAGAGCCGTTGTACC 
                 16 
                 21144 
                 2114 
                 TSC2 
               
               
                   
                 CGGGGCCGGGTGCTAGCGTGCCAGAGCTCCGTGGGCAGCAATG 
                   
                    01 
                  501 
                   
               
               
                   
                 GCCTCTGGGCCCT 
                   
                   
                   
                   
               
               
                   
               
               
                  53 
                 AGTGTTCTCACGGCTGCTGACTCAGAACCATGAGCCTGTGTGTA 
                 16 
                 21154 
                 2115 
                 TSC2 
               
               
                   
                 AGTCCTGGCCTTCTCTTCAAAGGTGATGGCCCGCTCCCTCTCCCC 
                   
                    53 
                  553 
                   
               
               
                   
                 ACCCCCGGAGC 
                   
                   
                   
                   
               
               
                   
               
               
                  54 
                 GGCCTTCTCTTCAAAGGTGATGGCCCGCTCCCTCTCCCCACCCCC 
                 16 
                 21155 
                 2115 
                 TSC2 
               
               
                   
                 GGAGCTGGAAGAAAGGGATGTGGCCGCATACTCGGCCTCCTTGG 
                   
                    03 
                  603 
                   
               
               
                   
                 AGGATGTGAAG 
                   
                   
                   
                   
               
               
                   
               
               
                  55 
                 TGGAAGAAAGGGATGTGGCCGCATACTCGGCCTCCTTGGAGGAT 
                 16 
                 21155 
                 2115 
                 TSC2 
               
               
                   
                 GTGAAGACAGCCGTCCTGGGGCTTCTGGTCATCCTTCAGGTGGG 
                   
                    53 
                  653 
                   
               
               
                   
                 TGTTCTGCACGA 
                   
                   
                   
                   
               
               
                   
               
               
                  56 
                 ACAGCCGTCCTGGGGCTTCTGGTCATCCTTCAGGTGGGTGTTCTG 
                 16 
                 21156 
                 2115 
                 TSC2 
               
               
                   
                 CACGAGGCCTCTGCTCCCGGGGCGCGCATGGCTAGCGTCCACCA 
                   
                    03 
                  703 
                   
               
               
                   
                 GCTGCATCTGC 
                   
                   
                   
                   
               
               
                   
               
               
                  57 
                 GTGCTGTCTTAGGACTGCGTTTTCACCTCCTGCGCCGTGGTGAGC 
                 16 
                 21203 
                 2120 
                 TSC2 
               
               
                   
                 TGCGTCCTCTCTCTGCAGACCAAGCTGTACACCCTGCCTGCAAG 
                   
                    93 
                  493 
                   
               
               
                   
                 CCACGCCACGC 
                   
                   
                   
                   
               
               
                   
               
               
                  58 
                 CCTCTCTCTGCAGACCAAGCTGTACACCCTGCCTGCAAGCCACG 
                 16 
                 21204 
                 2120 
                 TSC2 
               
               
                   
                 CCACGCGTGTGTATGAGATGCTGGTCAGCCACATTCAGCTCCAC 
                   
                    43 
                  543 
                   
               
               
                   
                 TACAAGCACAGC 
                   
                   
                   
                   
               
               
                   
               
               
                  59 
                 GTGTGTATGAGATGCTGGTCAGCCACATTCAGCTCCACTACAAG 
                 16 
                 21204 
                 2120 
                 TSC2 
               
               
                   
                 CACAGCTACACCCTGCCAATCGCGAGCAGCATCCGGCTGCAGGT 
                   
                    93 
                  593 
                   
               
               
                   
                 ATGGTGGCTGGG 
                   
                   
                   
                   
               
               
                   
               
               
                  60 
                 TACACCCTGCCAATCGCGAGCAGCATCCGGCTGCAGGTATGGTG 
                 16 
                 21205 
                 2120 
                 TSC2 
               
               
                   
                 GCTGGGGTTGCGCAGCCAGTTCCTGGGGGCCCAGCCAGGTATCC 
                   
                    43 
                  643 
                   
               
               
                   
                 CCGTCTCGGCAG 
                   
                   
                   
                   
               
               
                   
               
               
                  61 
                 GCAGGTGGGACGCCGCCTGTCCTGGGCCTGCACGAGCTTGGCTC 
                 16 
                 21214 
                 2121 
                 TSC2 
               
               
                   
                 TGGCTTTCACCATCCTCTTCCTGACAGGCCTTTGACTTCCTGTTG 
                   
                    39 
                  539 
                   
               
               
                   
                 CTGCTGCGGGC 
                   
                   
                   
                   
               
               
                   
               
               
                  62 
                 TCACCATCCTCTTCCTGACAGGCCTTTGACTTCCTGTTGCTGCTG 
                 16 
                 21214 
                 2121 
                 TSC2 
               
               
                   
                 CGGGCCGACTCACTGCACCGCCTGGGCCTGCCCAACAAGGATGG 
                   
                    89 
                  589 
                   
               
               
                   
                 AGTCGTGCGGT 
                   
                   
                   
                   
               
               
                   
               
               
                  63 
                 CGACTCACTGCACCGCCTGGGCCTGCCCAACAAGGATGGAGTCG 
                 16 
                 21215 
                 2121 
                 TSC2 
               
               
                   
                 TGCGGTTCAGCCCCTACTGCGTCTGCGACTACATGTACGCGGGA 
                   
                    39 
                  639 
                   
               
               
                   
                 CCTCGCCCACGG 
                   
                   
                   
                   
               
               
                   
               
               
                  64 
                 TCAGCCCCTACTGCGTCTGCGACTACATGTACGCGGGACCTCGC 
                 16 
                 21215 
                 2121 
                 TSC2 
               
               
                   
                 CCACGGCCCATGAGGCTCAGGGCGTCAGAGGCGCTGGGGCTGT 
                   
                    89 
                  689 
                   
               
               
                   
                 GGTGGCGCTGTTT 
                   
                   
                   
                   
               
               
                   
               
               
                  65 
                 GGGTTGGGAAGAGCCAAGTCTGTTCCGTTCCTGCTGCGGGGACT 
                 16 
                 21217 
                 2121 
                 TSC2 
               
               
                   
                 TGGCCTCAGCTGCTTCTCTTGCTTCTGCAGGGAGCCAGAGAGAG 
                   
                    10 
                  810 
                   
               
               
                   
                 GCTCTGAGAAGA 
                   
                   
                   
                   
               
               
                   
               
               
                  66 
                 CAGCTGCTTCTCTTGCTTCTGCAGGGAGCCAGAGAGAGGCTCTG 
                 16 
                 21217 
                 2121 
                 TSC2 
               
               
                   
                 AGAAGAAGACCAGCGGCCCCCTTTCTCCTCCCACAGGGCCTCCT 
                   
                    60 
                  860 
                   
               
               
                   
                 GGCCCGGCGCCT 
                   
                   
                   
                   
               
               
                   
               
               
                  67 
                 AGACCAGCGGCCCCCTTTCTCCTCCCACAGGGCCTCCTGGCCCG 
                 16 
                 21218 
                 2121 
                 TSC2 
               
               
                   
                 GCGCCTGCAGGCCCCGCCGTGCGGCTGGGGTCCGTGCCCTACTC 
                   
                    10 
                  910 
                   
               
               
                   
                 CCTGCTCTTCCG 
                   
                   
                   
                   
               
               
                   
               
               
                  68 
                 GCAGGCCCCGCCGTGCGGCTGGGGTCCGTGCCCTACTCCCTGCT 
                 16 
                 21218 
                 2121 
                 TSC2 
               
               
                   
                 CTTCCGCGTCCTGCTGCAGTGCTTGAAGCAGGTGAGTGGGGCCG 
                   
                    60 
                  960 
                   
               
               
                   
                 GGCAGGGACCAT 
                   
                   
                   
                   
               
               
                   
               
               
                  69 
                 CGTCCTGCTGCAGTGCTTGAAGCAGGTGAGTGGGGCCGGGCAGG 
                 16 
                 21219 
                 2122 
                 TSC2 
               
               
                   
                 GACCATCCGTCCCACGTTGGGCCAGGAGGACAGGGAGCTGCCA 
                   
                    10 
                  010 
                   
               
               
                   
                 CCTGCCTGCTGGG 
                   
                   
                   
                   
               
               
                   
               
               
                  70 
                 CTAGCTTCCGCCTCTGTCTCTAGGGTCCAGAAGGCCCTGTCCTGA 
                 16 
                 21221 
                 2122 
                 TSC2 
               
               
                   
                 CGCCTCCTCTCCTCGCAGGAGTCTGACTGGAAGGTGCTGAAGCT 
                   
                    78 
                  278 
                   
               
               
                   
                 GGTTCTGGGCA 
                   
                   
                   
                   
               
               
                   
               
               
                  71 
                 CCTCTCCTCGCAGGAGTCTGACTGGAAGGTGCTGAAGCTGGTTC 
                 16 
                 21222 
                 2122 
                 TSC2 
               
               
                   
                 TGGGCAGGCTGCCTGAGTCCCTGCGCTATAAAGTGCTCATCTTT 
                   
                    28 
                  328 
                   
               
               
                   
                 ACTTCCCCTTGC 
                   
                   
                   
                   
               
               
                   
               
               
                  72 
                 GGCTGCCTGAGTCCCTGCGCTATAAAGTGCTCATCTTTACTTCCC 
                 16 
                 21222 
                 2122 
                 TSC2 
               
               
                   
                 CTTGCAGTGTGGACCAGCTGTGCTCTGCTCTCTGCTCCATGGTAC 
                   
                    78 
                  378 
                   
               
               
                   
                 CATGGCCGGC 
                   
                   
                   
                   
               
               
                   
               
               
                  73 
                 AGTGTGGACCAGCTGTGCTCTGCTCTCTGCTCCATGGTACCATGG 
                 16 
                 21223 
                 2122 
                 TSC2 
               
               
                   
                 CCGGCCTGGGGTTGGGGTGGGGGACCCAGTAGGGTTTTTCCCCA 
                   
                    28 
                  428 
                   
               
               
                   
                 AAAGACTGCGA 
                   
                   
                   
                   
               
               
                   
               
               
                  74 
                 GGCTACCCCGTGACCTGGCCGCTGGGGAGAGGTTTCATGCCTGG 
                 16 
                 21227 
                 2122 
                 TSC2 
               
               
                   
                 ATTTGGTCATCAGCTTTCAGGCCCAAAGACACTGGAGCGGCTCC 
                   
                    92 
                  892 
                   
               
               
                   
                 GAGGCGCCCCAG 
                   
                   
                   
                   
               
               
                   
               
               
                  75 
                 TCATCAGCTTTCAGGCCCAAAGACACTGGAGCGGCTCCGAGGCG 
                 16 
                 21228 
                 2122 
                 TSC2 
               
               
                   
                 CCCCAGAAGGCTTCTCCAGAACTGACTTGCACCTGGCCGTGGTT 
                   
                    42 
                  942 
                   
               
               
                   
                 CCAGTGCTGACA 
                   
                   
                   
                   
               
               
                   
               
               
                  76 
                 AAGGCTTCTCCAGAACTGACTTGCACCTGGCCGTGGTTCCAGTG 
                 16 
                 21228 
                 2122 
                 TSC2 
               
               
                   
                 CTGACAGCATTAATCTCTTACCATAACTACCTGGACAAAACCAA 
                   
                    92 
                  992 
                   
               
               
                   
                 ACAGGTAGGAGG 
                   
                   
                   
                   
               
               
                   
               
               
                  77 
                 GCATTAATCTCTTACCATAACTACCTGGACAAAACCAAACAGGT 
                 16 
                 21229 
                 2123 
                 TSC2 
               
               
                   
                 AGGAGGTCAGAGCAGGACAGGCGAGCTTGATGGGGCCTGGGAT 
                   
                    42 
                  042 
                   
               
               
                   
                 TCGAGGGCCTGGC 
                   
                   
                   
                   
               
               
                   
               
               
                  78 
                 GCGAGGCTGCCTCTGCTGCAAGCGGGTGGGGCCTGAGGTGTCCT 
                 16 
                 21241 
                 2124 
                 TSC2 
               
               
                   
                 GTCTCCTGCAGCGCGAGATGGTCTACTGCCTGGAGCAGGGCCTC 
                   
                    45 
                  245 
                   
               
               
                   
                 ATCCACCGCTGT 
                   
                   
                   
                   
               
               
                   
               
               
                  79 
                 TGCAGCGCGAGATGGTCTACTGCCTGGAGCAGGGCCTCATCCAC 
                 16 
                 21241 
                 2124 
                 TSC2 
               
               
                   
                 CGCTGTGCCAGCCAGTGCGTCGTGGCCTTGTCCATCTGCAGCGT 
                   
                    95 
                  295 
                   
               
               
                   
                 GGAGATGCCTGA 
                   
                   
                   
                   
               
               
                   
               
               
                  80 
                 GCCAGCCAGTGCGTCGTGGCCTTGTCCATCTGCAGCGTGGAGAT 
                 16 
                 21242 
                 2124 
                 TSC2 
               
               
                   
                 GCCTGACATCATCATCAAGGCGCTGCCTGTTCTGGTGGTGAAGC 
                   
                    45 
                  345 
                   
               
               
                   
                 TCACGCACATCT 
                   
                   
                   
                   
               
               
                   
               
               
                  81 
                 CATCATCATCAAGGCGCTGCCTGTTCTGGTGGTGAAGCTCACGC 
                 16 
                 21242 
                 2124 
                 TSC2 
               
               
                   
                 ACATCTCAGCCACAGCCAGCATGGCCGTCCCACTGCTGGAGTTC 
                   
                    95 
                  395 
                   
               
               
                   
                 CTGTCCAGTGAG 
                   
                   
                   
                   
               
               
                   
               
               
                  82 
                 CAGCCACAGCCAGCATGGCCGTCCCACTGCTGGAGTTCCTGTCC 
                 16 
                 21243 
                 2124 
                 TSC2 
               
               
                   
                 AGTGAGTCCCCGCCCTGCCTGCGCATGCACCCGAGAGGTTCGGG 
                   
                    45 
                  445 
                   
               
               
                   
                 CTGTGTAACCTG 
                   
                   
                   
                   
               
               
                   
               
               
                  83 
                 AGAGGCGCTGCACGGGACCCCGGCTCCCCTGACCACCCTCTCCA 
                 16 
                 21257 
                 2125 
                 TSC2 
               
               
                   
                 TTACCGCAGCTCTGGCCAGGCTGCCGCACCTCTACAGGAACTTT 
                   
                    46 
                  846 
                   
               
               
                   
                 GCCGCGGAGCAG 
                   
                   
                   
                   
               
               
                   
               
               
                  84 
                 CAGCTCTGGCCAGGCTGCCGCACCTCTACAGGAACTTTGCCGCG 
                 16 
                 21257 
                 2125 
                 TSC2 
               
               
                   
                 GAGCAGTATGCCAGTGTGTTCGCCATCTCCCTGCCGTACACCAA 
                   
                    96 
                  896 
                   
               
               
                   
                 CCCCTCCAAGTG 
                   
                   
                   
                   
               
               
                   
               
               
                  85 
                 TATGCCAGTGTGTTCGCCATCTCCCTGCCGTACACCAACCCCTCC 
                 16 
                 21258 
                 2125 
                 TSC2 
               
               
                   
                 AAGTGAGTGGTCGCCCCAGGCCCTGTGCCTCCCAGCCGTGGCCC 
                   
                    46 
                  946 
                   
               
               
                   
                 CCGCTAGGCCT 
                   
                   
                   
                   
               
               
                   
               
               
                  86 
                 GTTTTTTGCACTTCATGCCCTGGGGATGTTTCCCTGCTGCCAGGA 
                 16 
                 21259 
                 2126 
                 TSC2 
               
               
                   
                 TGGAGTGCCAGCCCCCTTCTCATCTCAGGTTTAATCAGTACATCG 
                   
                    95 
                  095 
                   
               
               
                   
                 TGTGTCTGGC 
                   
                   
                   
                   
               
               
                   
               
               
                  87 
                 TGCCAGCCCCCTTCTCATCTCAGGTTTAATCAGTACATCGTGTGT 
                 16 
                 21260 
                 2126 
                 TSC2 
               
               
                   
                 CTGGCCCATCACGTCATAGCCATGTGGTTCATCAGGTGCCGCCT 
                   
                    45 
                  145 
                   
               
               
                   
                 GCCCTTCCGGA 
                   
                   
                   
                   
               
               
                   
               
               
                  88 
                 CCATCACGTCATAGCCATGTGGTTCATCAGGTGCCGCCTGCCCTT 
                 16 
                 21260 
                 2126 
                 TSC2 
               
               
                   
                 CCGGAAGGATTTTGTCCCTTTCATCACTAAGGTGGGCTCAGGGC 
                   
                    95 
                  195 
                   
               
               
                   
                 CGGTGAAGGCT 
                   
                   
                   
                   
               
               
                   
               
               
                  89 
                 AGGATTTTGTCCCTTTCATCACTAAGGTGGGCTCAGGGCCGGTG 
                 16 
                 21261 
                 2126 
                 TSC2 
               
               
                   
                 AAGGCTGTGTCTCTCGGTAGGCCAGGGCTTGCTTTGCCCTTGGCT 
                   
                    45 
                  245 
                   
               
               
                   
                 GTCCATGGTCG 
                   
                   
                   
                   
               
               
                   
               
               
                  90 
                 CCTCCAGCCCCCATTGCCACCCCTCACTGTCTGGGTGTGCTCACT 
                 16 
                 21264 
                 2126 
                 TSC2 
               
               
                   
                 CTGCCAGGGCCTGCGGTCCAATGTCCTCTTGTCTTTTGATGACAC 
                   
                    39 
                  539 
                   
               
               
                   
                 CCCCGAGAAG 
                   
                   
                   
                   
               
               
                   
               
               
                  91 
                 AGGGCCTGCGGTCCAATGTCCTCTTGTCTTTTGATGACACCCCCG 
                 16 
                 21264 
                 2126 
                 TSC2 
               
               
                   
                 AGAAGGACAGCTTCAGGGCCCGGAGTACTAGTCTCAACGAGAG 
                   
                    89 
                  589 
                   
               
               
                   
                 ACCCAAGAGGTA 
                   
                   
                   
                   
               
               
                   
               
               
                  92 
                 GACAGCTTCAGGGCCCGGAGTACTAGTCTCAACGAGAGACCCA 
                 16 
                 21265 
                 2126 
                 TSC2 
               
               
                   
                 AGAGGTACGGCCTGCGGGGGTGTGCCTGGAGTCGGTGTGGGGT 
                   
                    39 
                  639 
                   
               
               
                   
                 GGGGAAGGACATGG 
                   
                   
                   
                   
               
               
                   
               
               
                  93 
                 TTCTCCCCTTCCCGGGAGCTGGGCTCTCTGGGGCGTTGGGGCTCC 
                 16 
                 21275 
                 2127 
                 TSC2 
               
               
                   
                 TTCCTCACCCGATAGTCTGAGGATAGCCAGACCCCCCAAACAAG 
                   
                    38 
                  638 
                   
               
               
                   
                 GCTTGAATAAC 
                   
                   
                   
                   
               
               
                   
               
               
                  94 
                 CACCCGATAGTCTGAGGATAGCCAGACCCCCCAAACAAGGCTTG 
                 16 
                 21275 
                 2127 
                 TSC2 
               
               
                   
                 AATAACTCTCCACCCGTGAAAGAATTCAAGGAGAGCTCTGCAGC 
                   
                    88 
                  688 
                   
               
               
                   
                 CGAGGCCTTCCG 
                   
                   
                   
                   
               
               
                   
               
               
                  95 
                 TCTCCACCCGTGAAAGAATTCAAGGAGAGCTCTGCAGCCGAGGC 
                 16 
                 21276 
                 2127 
                 TSC2 
               
               
                   
                 CTTCCGGTGCCGCAGCATCAGTGTGTCTGAACATGTGGTCCGCA 
                   
                    38 
                  738 
                   
               
               
                   
                 GGTAGCGGGACT 
                   
                   
                   
                   
               
               
                   
               
               
                  96 
                 GTGCCGCAGCATCAGTGTGTCTGAACATGTGGTCCGCAGGTAGC 
                 16 
                 21276 
                 2127 
                 TSC2 
               
               
                   
                 GGGACTGTCGGGTGGGGGGCACGGACCCTGGAGCTTGGCCCCGT 
                   
                    88 
                  788 
                   
               
               
                   
                 GAGCACCTGGGT 
                   
                   
                   
                   
               
               
                   
               
               
                  97 
                 CTTGGTGATAGGTGGCTCGGCCCGCCCTACCTGGCACCCTGACC 
                 16 
                 21289 
                 2129 
                 TSC2 
               
               
                   
                 CTGGTCACGGCCTCTCCCTCCAGCAGGATACAGACGTCCCTCAC 
                   
                    65 
                  065 
                   
               
               
                   
                 CAGTGCCAGCTT 
                   
                   
                   
                   
               
               
                   
               
               
                  98 
                 ACGGCCTCTCCCTCCAGCAGGATACAGACGTCCCTCACCAGTGC 
                 16 
                 21290 
                 2129 
                 TSC2 
               
               
                   
                 CAGCTTGGGGTCTGCAGATGAGAACTCCGTGGCCCAGGCTGACG 
                   
                    15 
                  115 
                   
               
               
                   
                 ATAGCCTGAAAA 
                   
                   
                   
                   
               
               
                   
               
               
                  99 
                 GGGGTCTGCAGATGAGAACTCCGTGGCCCAGGCTGACGATAGCC 
                 16 
                 21290 
                 2129 
                 TSC2 
               
               
                   
                 TGAAAAACCTCCACCTGGAGCTCACGGAAACCTGTCTGGACATG 
                   
                    65 
                  165 
                   
               
               
                   
                 ATGGCTCGATAC 
                   
                   
                   
                   
               
               
                   
               
               
                 100 
                 ACCTCCACCTGGAGCTCACGGAAACCTGTCTGGACATGATGGCT 
                 16 
                 21291 
                 2129 
                 TSC2 
               
               
                   
                 CGATACGTCTTCTCCAACTTCACGGCTGTCCCGAAGAGGTCCAG 
                   
                    15 
                  215 
                   
               
               
                   
                 GCGGCACTACAG 
                   
                   
                   
                   
               
               
                   
               
               
                 101 
                 GTCTTCTCCAACTTCACGGCTGTCCCGAAGAGGTCCAGGCGGCA 
                 16 
                 21291 
                 2129 
                 TSC2 
               
               
                   
                 CTACAGGGCTGGGCGGGCCTGCGGGAGCTCCACGGGCAAGCTG 
                   
                    65 
                  265 
                   
               
               
                   
                 GGTTTCACGCTCC 
                   
                   
                   
                   
               
               
                   
               
               
                 102 
                 GCGGCACTACAGGGCTGGGCGGGCCTGCGGGAGCTCCACGGGC 
                 16 
                 21292 
                 2129 
                 TSC2 
               
               
                   
                 AAGCTGGGTTTCACGCTCCCTGTCTTCTAGGTCTCCTGTGGGCGA 
                   
                    03 
                  303 
                   
               
               
                   
                 GTTCCTCCTAGC 
                   
                   
                   
                   
               
               
                   
               
               
                 103 
                 GTTTCACGCTCCCTGTCTTCTAGGTCTCCTGTGGGCGAGTTCCTC 
                 16 
                 21292 
                 2129 
                 TSC2 
               
               
                   
                 CTAGCGGGTGGCAGGACCAAAACCTGGCTGGTTGGGAACAAGC 
                   
                    53 
                  353 
                   
               
               
                   
                 TTGTCACTGTGA 
                   
                   
                   
                   
               
               
                   
               
               
                 104 
                 GGGTGGCAGGACCAAAACCTGGCTGGTTGGGAACAAGCTTGTC 
                 16 
                 21293 
                 2129 
                 TSC2 
               
               
                   
                 ACTGTGACGACAAGCGTGGGAACCGGGACCCGGTCGTTACTAG 
                   
                    03 
                  403 
                   
               
               
                   
                 GCCTGGACTCGGGG 
                   
                   
                   
                   
               
               
                   
               
               
                 105 
                 CGACAAGCGTGGGAACCGGGACCCGGTCGTTACTAGGCCTGGA 
                 16 
                 21293 
                 2129 
                 TSC2 
               
               
                   
                 CTCGGGGGAGCTGCAGTCCGGCCCGGAGTCGAGGTGACTGCACC 
                   
                    53 
                  453 
                   
               
               
                   
                 TTCCTTTCCTCCG 
                   
                   
                   
                   
               
               
                   
               
               
                 106 
                 GAGCTGCAGTCCGGCCCGGAGTCGAGGTGACTGCACCTTCCTTT 
                 16 
                 21294 
                 2129 
                 TSC2 
               
               
                   
                 CCTCCGCGCCTGCCAGCCTCGACACCGGCTGTCCCGAGCCCAGG 
                   
                    03 
                  503 
                   
               
               
                   
                 CCCACGTGGCAC 
                   
                   
                   
                   
               
               
                   
               
               
                 107 
                 GGCCCACGTGGCACCCTCGTACCAGCCTGGGGACTAAGTCCACC 
                 16 
                 21294 
                 2129 
                 TSC2 
               
               
                   
                 CTGTGCGTGGGATTCTCTTCTCAGCTCCAGCCCCGGGGTGCATGT 
                   
                    89 
                  589 
                   
               
               
                   
                 GAGACAGACCA 
                   
                   
                   
                   
               
               
                   
               
               
                 108 
                 GTGGGATTCTCTTCTCAGCTCCAGCCCCGGGGTGCATGTGAGAC 
                 16 
                 21295 
                 2129 
                 TSC2 
               
               
                   
                 AGACCAAGGAGGCGCCGGCCAAGCTGGAGTCCCAGGCTGGGCA 
                   
                    39 
                  639 
                   
               
               
                   
                 GCAGGTGTCCCGT 
                   
                   
                   
                   
               
               
                   
               
               
                 109 
                 AGGAGGCGCCGGCCAAGCTGGAGTCCCAGGCTGGGCAGCAGGT 
                 16 
                 21295 
                 2129 
                 TSC2 
               
               
                   
                 GTCCCGTGGGGCCCGGGATCGGGTCCGTTCCATGTCGGGTGAGC 
                   
                    89 
                  689 
                   
               
               
                   
                 CTTGGCCCCAGCC 
                   
                   
                   
                   
               
               
                   
               
               
                 110 
                 GGGGCCCGGGATCGGGTCCGTTCCATGTCGGGTGAGCCTTGGCC 
                 16 
                 21296 
                 2129 
                 TSC2 
               
               
                   
                 CCAGCCACCTCCACACAGGCACCGGGGCTCCCTCAGTTGCTGCT 
                   
                    39 
                  739 
                   
               
               
                   
                 GGTCCCAGTGTT 
                   
                   
                   
                   
               
               
                   
               
               
                 111 
                 TTCAGCTTGAGGCTGGTGGTTTTGCATCAGGTAAGTGGTGGTCA 
                 16 
                 21300 
                 2130 
                 TSC2 
               
               
                   
                 CCAGTCCTCTGCCCTCTTCTTCAGGGGGCCATGGTCTTCGAGTTG 
                   
                    97 
                  197 
                   
               
               
                   
                 GCGCCCTGGAC 
                   
                   
                   
                   
               
               
                   
               
               
                 112 
                 CTCTGCCCTCTTCTTCAGGGGGCCATGGTCTTCGAGTTGGCGCCC 
                 16 
                 21301 
                 2130 
                 TSC2 
               
               
                   
                 TGGACGTGCCGGCCTCCCAGTTCCTGGGCAGTGCCACTTCTCCA 
                   
                    47 
                  247 
                   
               
               
                   
                 GGACCACGGAC 
                   
                   
                   
                   
               
               
                   
               
               
                 113 
                 GTGCCGGCCTCCCAGTTCCTGGGCAGTGCCACTTCTCCAGGACC 
                 16 
                 21301 
                 2130 
                 TSC2 
               
               
                   
                 ACGGACTGCACCAGCCGCGAAACCTGAGAAGGCCTCAGCTGGC 
                   
                    97 
                  297 
                   
               
               
                   
                 ACCCGGGTTCCTG 
                   
                   
                   
                   
               
               
                   
               
               
                 114 
                 TGCACCAGCCGCGAAACCTGAGAAGGCCTCAGCTGGCACCCGG 
                 16 
                 21302 
                 2130 
                 TSC2 
               
               
                   
                 GTTCCTGTGCAGGAGAAGACGAACCTGGCGGCCTATGTGCCCCT 
                   
                    47 
                  347 
                   
               
               
                   
                 GCTGACCCAGGGC 
                   
                   
                   
                   
               
               
                   
               
               
                 115 
                 TGCAGGAGAAGACGAACCTGGCGGCCTATGTGCCCCTGCTGACC 
                 16 
                 21302 
                 2130 
                 TSC2 
               
               
                   
                 CAGGGCTGGGCGGAGATCCTGGTCCGGAGGCCCACAGGTACTG 
                   
                    97 
                  397 
                   
               
               
                   
                 GGCGGGGCTGGCC 
                   
                   
                   
                   
               
               
                   
               
               
                 116 
                 TGGGCGGAGATCCTGGTCCGGAGGCCCACAGGTACTGGGCGGG 
                 16 
                 21303 
                 2130 
                 TSC2 
               
               
                   
                 GCTGGCCTGAGCGCCATCTTTCTGCCAGTCACCCACAGAGCTGT 
                   
                    47 
                  447 
                   
               
               
                   
                 GGACACTCAGGGG 
                   
                   
                   
                   
               
               
                   
               
               
                 117 
                 AGGCCCCTGGGGGGCCAGAGATGGGTAAGGGGAGGTACTGGCC 
                 16 
                 21315 
                 2131 
                 TSC2 
               
               
                   
                 TCAGGCCAAAGGTGCTGCCGCCTCCGCAGGGAACACCAGCTGGC 
                   
                    23 
                  623 
                   
               
               
                   
                 TGATGAGCCTGGA 
                   
                   
                   
                   
               
               
                   
               
               
                 118 
                 AAAGGTGCTGCCGCCTCCGCAGGGAACACCAGCTGGCTGATGA 
                 16 
                 21315 
                 2131 
                 TSC2 
               
               
                   
                 GCCTGGAGAACCCGCTCAGCCCTTTCTCCTCGGACATCAACAAC 
                   
                    73 
                  673 
                   
               
               
                   
                 ATGCCCCTGCAGG 
                   
                   
                   
                   
               
               
                   
               
               
                 119 
                 GAACCCGCTCAGCCCTTTCTCCTCGGACATCAACAACATGCCCC 
                 16 
                 21316 
                 2131 
                 TSC2 
               
               
                   
                 TGCAGGAGCTGTCTAACGCCCTCATGGCGGCTGAGCGCTTCAAG 
                   
                    23 
                  723 
                   
               
               
                   
                 GAGCACCGGGAC 
                   
                   
                   
                   
               
               
                   
               
               
                 120 
                 AGCTGTCTAACGCCCTCATGGCGGCTGAGCGCTTCAAGGAGCAC 
                 16 
                 21316 
                 2131 
                 TSC2 
               
               
                   
                 CGGGACACAGCCCTGTACAAGTCACTGTCGGTGCCGGCAGCCAG 
                   
                    73 
                  773 
                   
               
               
                   
                 CACGGCCAAACC 
                   
                   
                   
                   
               
               
                   
               
               
                 121 
                 ACAGCCCTGTACAAGTCACTGTCGGTGCCGGCAGCCAGCACGGC 
                 16 
                 21317 
                 2131 
                 TSC2 
               
               
                   
                 CAAACCCCCTCCTCTGCCTCGCTCCAACACAGGTGAGTGGCATG 
                   
                    23 
                  823 
                   
               
               
                   
                 GCGGGCCTTGGC 
                   
                   
                   
                   
               
               
                   
               
               
                 122 
                 CCCTCCTCTGCCTCGCTCCAACACAGGTGAGTGGCATGGCGGGC 
                 16 
                 21317 
                 2131 
                 TSC2 
               
               
                   
                 CTTGGCACGGGCTCTGCTCCCACTGGCCTGGTGCTCCCGGTGAC 
                   
                    73 
                  873 
                   
               
               
                   
                 GGCAATGTGGCT 
                   
                   
                   
                   
               
               
                   
               
               
                 123 
                 CTCTGCTCGACCTGTGTGTAGCCCCTCCTCCTGCTGACGTGGCCG 
                 16 
                 21323 
                 2132 
                 TSC2 
               
               
                   
                 CACACGGCCTTCCCTTGCAGTGGCCTCTTTCTCCTCCCTGTACCA 
                   
                    71 
                  471 
                   
               
               
                   
                 GTCCAGCTGC 
                   
                   
                   
                   
               
               
                   
               
               
                 124 
                 GGCCTTCCCTTGCAGTGGCCTCTTTCTCCTCCCTGTACCAGTCCA 
                 16 
                 21324 
                 2132 
                 TSC2 
               
               
                   
                 GCTGCCAAGGACAGCTGCACAGGAGCGTTTCCTGGGCAGGTATC 
                   
                    21 
                  521 
                   
               
               
                   
                 GCCTCTCAGAG 
                   
                   
                   
                   
               
               
                   
               
               
                 125 
                 CAAGGACAGCTGCACAGGAGCGTTTCCTGGGCAGGTATCGCCTC 
                 16 
                 21324 
                 2132 
                 TSC2 
               
               
                   
                 TCAGAGGGAAGCGGTTGGCTGCAGAGCGCCACTCTGCCTCATAG 
                   
                    71 
                  571 
                   
               
               
                   
                 GTGCTGTGCTCG 
                   
                   
                   
                   
               
               
                   
               
               
                 126 
                 GGCCACGTCAGGGCCAGGGCCTGGCCCAGCCCCACATCCAGCA 
                 16 
                 21336 
                 2133 
                 TSC2 
               
               
                   
                 GCCCCGTCTGTGTCCTCCCAGACTCCGCCGTGGTCATGGAGGAG 
                   
                    31 
                  731 
                   
               
               
                   
                 GGAAGTCCGGGCG 
                   
                   
                   
                   
               
               
                   
               
               
                 127 
                 CTGTGTCCTCCCAGACTCCGCCGTGGTCATGGAGGAGGGAAGTC 
                 16 
                 21336 
                 2133 
                 TSC2 
               
               
                   
                 CGGGCGAGGTTCCTGTGCTGGTGGAGCCCCCAGGGTTGGAGGAC 
                   
                    81 
                  781 
                   
               
               
                   
                 GTTGAGGCAGCG 
                   
                   
                   
                   
               
               
                   
               
               
                 128 
                 AGGTTCCTGTGCTGGTGGAGCCCCCAGGGTTGGAGGACGTTGAG 
                 16 
                 21337 
                 2133 
                 TSC2 
               
               
                   
                 GCAGCGCTAGGCATGGACAGGCGCACGGATGCCTACAGCAGGG 
                   
                    31 
                  831 
                   
               
               
                   
                 TGAGTGTGGCTCA 
                   
                   
                   
                   
               
               
                   
               
               
                 129 
                 CTAGGCATGGACAGGCGCACGGATGCCTACAGCAGGGTGAGTG 
                 16 
                 21337 
                 2133 
                 TSC2 
               
               
                   
                 TGGCTCAGAGCCTGGACCCTGCTGACCTCGGGGGGCTCCTTAGG 
                   
                    81 
                  881 
                   
               
               
                   
                 GGAGGCAGGGCTC 
                   
                   
                   
                   
               
               
                   
               
               
                 130 
                 GGTGGGCTCGAGGGTGCCTGCTGACAGGGGTTCTCTTTGGGATG 
                 16 
                 21341 
                 2134 
                 TSC2 
               
               
                   
                 GTCCTTTCTAGTCGTCCTCAGTCTCCAGCCAGGAGGAGAAGTCG 
                   
                    73 
                  273 
                   
               
               
                   
                 CTCCACGCGGAG 
                   
                   
                   
                   
               
               
                   
               
               
                 131 
                 TCTAGTCGTCCTCAGTCTCCAGCCAGGAGGAGAAGTCGCTCCAC 
                 16 
                 21342 
                 2134 
                 TSC2 
               
               
                   
                 GCGGAGGAGCTGGTTGGCAGGGGCATCCCCATCGAGCGAGTCG 
                   
                    23 
                  323 
                   
               
               
                   
                 TCTCCTCGGAGGG 
                   
                   
                   
                   
               
               
                   
               
               
                 132 
                 GAGCTGGTTGGCAGGGGCATCCCCATCGAGCGAGTCGTCTCCTC 
                 16 
                 21342 
                 2134 
                 TSC2 
               
               
                   
                 GGAGGGTGGCCGGCCCTCTGTGGACCTCTCCTTCCAGCCCTCGC 
                   
                    73 
                  373 
                   
               
               
                   
                 AGCCCCTGAGCA 
                   
                   
                   
                   
               
               
                   
               
               
                 133 
                 TGGCCGGCCCTCTGTGGACCTCTCCTTCCAGCCCTCGCAGCCCCT 
                 16 
                 21343 
                 2134 
                 TSC2 
               
               
                   
                 GAGCAAGTCCAGCTCCTCTCCCGAGCTGCAGACTCTGCAGGACA 
                   
                    23 
                  423 
                   
               
               
                   
                 TCCTCGGGGAC 
                   
                   
                   
                   
               
               
                   
               
               
                 134 
                 AGTCCAGCTCCTCTCCCGAGCTGCAGACTCTGCAGGACATCCTC 
                 16 
                 21343 
                 2134 
                 TSC2 
               
               
                   
                 GGGGACCCTGGGGACAAGGCCGACGTGGGCCGGCTGAGCCCTG 
                   
                    73 
                  473 
                   
               
               
                   
                 AGGTTAAGGCCCG 
                   
                   
                   
                   
               
               
                   
               
               
                 135 
                 CCTGGGGACAAGGCCGACGTGGGCCGGCTGAGCCCTGAGGTTA 
                 16 
                 21344 
                 2134 
                 TSC2 
               
               
                   
                 AGGCCCGGTCACAGTCAGGGACCCTGGACGGGGAAAGTGCTGC 
                   
                    23 
                  523 
                   
               
               
                   
                 CTGGTCGGCCTCGG 
                   
                   
                   
                   
               
               
                   
               
               
                 136 
                 GTCACAGTCAGGGACCCTGGACGGGGAAAGTGCTGCCTGGTCG 
                 16 
                 21344 
                 2134 
                 TSC2 
               
               
                   
                 GCCTCGGGCGAAGACAGTCGGGGCCAGCCCGAGGGTCCCTTGCC 
                   
                    73 
                  573 
                   
               
               
                   
                 TTCCAGCTCCCCC 
                   
                   
                   
                   
               
               
                   
               
               
                 137 
                 GCGAAGACAGTCGGGGCCAGCCCGAGGGTCCCTTGCCTTCCAGC 
                 16 
                 21345 
                 2134 
                 TSC2 
               
               
                   
                 TCCCCCCGCTCGCCCAGTGGCCTCCGGCCCCGAGGTTACACCAT 
                   
                    23 
                  623 
                   
               
               
                   
                 CTCCGACTCGGC 
                   
                   
                   
                   
               
               
                   
               
               
                 138 
                 CGCTCGCCCAGTGGCCTCCGGCCCCGAGGTTACACCATCTCCGA 
                 16 
                 21345 
                 2134 
                 TSC2 
               
               
                   
                 CTCGGCCCCATCACGCAGGGGCAAGAGAGTAGAGAGGGACGCC 
                   
                    73 
                  673 
                   
               
               
                   
                 TTAAAGAGCAGAG 
                   
                   
                   
                   
               
               
                   
               
               
                 139 
                 CCCATCACGCAGGGGCAAGAGAGTAGAGAGGGACGCCTTAAAG 
                 16 
                 21346 
                 2134 
                 TSC2 
               
               
                   
                 AGCAGAGCCACAGCCTCCAATGCAGAGAAAGTGCCAGGCATCA 
                   
                    23 
                  723 
                   
               
               
                   
                 ACCCCAGGTGGGCC 
                   
                   
                   
                   
               
               
                   
               
               
                 140 
                 CCACAGCCTCCAATGCAGAGAAAGTGCCAGGCATCAACCCCAG 
                 16 
                 21346 
                 2134 
                 TSC2 
               
               
                   
                 GTGGGCCTCTTGCTTCCGGGCGGGGCTCCTGACACCTCTCCTGCG 
                   
                    73 
                  773 
                   
               
               
                   
                 GGAACCTGGTGC 
                   
                   
                   
                   
               
               
                   
               
               
                 141 
                 TGGGCTGTGGCTGCCCTGGCCAGGCCCTCACCTGGGTGCCCACC 
                 16 
                 21348 
                 2134 
                 TSC2 
               
               
                   
                 ATCCCCTCCCTGTGCAGTTTCGTGTTCCTGCAGCTCTACCATTCC 
                   
                    90 
                  990 
                   
               
               
                   
                 CCCTTCTTTGG 
                   
                   
                   
                   
               
               
                   
               
               
                 142 
                 TCCCTGTGCAGTTTCGTGTTCCTGCAGCTCTACCATTCCCCCTTCT 
                 16 
                 21349 
                 2135 
                 TSC2 
               
               
                   
                 TTGGCGACGAGTCAAACAAGCCAATCCTGCTGCCCAATGAGGTA 
                   
                    40 
                  040 
                   
               
               
                   
                 GGCGTGGCCT 
                   
                   
                   
                   
               
               
                   
               
               
                 143 
                 CGACGAGTCAAACAAGCCAATCCTGCTGCCCAATGAGGTAGGC 
                 16 
                 21349 
                 2135 
                 TSC2 
               
               
                   
                 GTGGCCTCCCTCTCCTGCATCCGCTGGAGCTGTGTGGCTCGGGTG 
                   
                    90 
                  090 
                   
               
               
                   
                 AATGGTGGGGGG 
                   
                   
                   
                   
               
               
                   
               
               
                 144 
                 CGGCCTCCTGTGGACGGGCGTCTGGGGCTCAGGCAGGGCTCTGT 
                 16 
                 21351 
                 2135 
                 TSC2 
               
               
                   
                 GTGCCACAGTCACAGTCCTTTGAGCGGTCGGTGCAGCTCCTCGA 
                   
                    77 
                  277 
                   
               
               
                   
                 CCAGATCCCATC 
                   
                   
                   
                   
               
               
                   
               
               
                 145 
                 CAGTCACAGTCCTTTGAGCGGTCGGTGCAGCTCCTCGACCAGAT 
                 16 
                 21352 
                 2135 
                 TSC2 
               
               
                   
                 CCCATCATACGACACCCACAAGATCGCCGTCCTGTATGTTGGAG 
                   
                    27 
                  327 
                   
               
               
                   
                 AAGGCCAGGTGA 
                   
                   
                   
                   
               
               
                   
               
               
                 146 
                 ATACGACACCCACAAGATCGCCGTCCTGTATGTTGGAGAAGGCC 
                 16 
                 21352 
                 2135 
                 TSC2 
               
               
                   
                 AGGTGAGGCTGCGGGGCCGGCCTAGGTGCCTGGACAGGGCCAG 
                   
                    77 
                  377 
                   
               
               
                   
                 CTGGGCCTCAGCC 
                   
                   
                   
                   
               
               
                   
               
               
                 147 
                 CGGGGCAGGGCCCGGCCCGGGAGTGATGCCACCCTGCCTCTCCC 
                 16 
                 21361 
                 2136 
                 TSC2 
               
               
                   
                 CTCTCCCCACAGAGCAACAGCGAGCTCGCCATCCTGTCCAATGA 
                   
                    37 
                  237 
                   
               
               
                   
                 GCATGGCTCCTA 
                   
                   
                   
                   
               
               
                   
               
               
                 148 
                 CCACAGAGCAACAGCGAGCTCGCCATCCTGTCCAATGAGCATGG 
                 16 
                 21361 
                 2136 
                 TSC2 
               
               
                   
                 CTCCTACAGGTACACGGAGTTCCTGACGGGCCTGGGCCGGCTCA 
                   
                    87 
                  287 
                   
               
               
                   
                 TCGAGCTGAAGG 
                   
                   
                   
                   
               
               
                   
               
               
                 149 
                 CAGGTACACGGAGTTCCTGACGGGCCTGGGCCGGCTCATCGAGC 
                 16 
                 21362 
                 2136 
                 TSC2 
               
               
                   
                 TGAAGGACTGCCAGCCGGACAAGGTGTACCTGGGAGGCCTGGA 
                   
                    37 
                  337 
                   
               
               
                   
                 CGTGTGTGGTGAG 
                   
                   
                   
                   
               
               
                   
               
               
                 150 
                 ACTGCCAGCCGGACAAGGTGTACCTGGGAGGCCTGGACGTGTGT 
                 16 
                 21362 
                 2136 
                 TSC2 
               
               
                   
                 GGTGAGGACGGCCAGTTCACCTACTGCTGGCACGATGACATCAT 
                   
                    87 
                  387 
                   
               
               
                   
                 GCAAGGTACGGC 
                   
                   
                   
                   
               
               
                   
               
               
                 151 
                 GACGGCCAGTTCACCTACTGCTGGCACGATGACATCATGCAAGG 
                 16 
                 21363 
                 2136 
                 TSC2 
               
               
                   
                 TACGGCCTGGCGCCTACCCGCTCCTGCTGCCCCAGGCCTCAGGG 
                   
                    37 
                  437 
                   
               
               
                   
                 CACGGCTCCCAT 
                   
                   
                   
                   
               
               
                   
               
               
                 152 
                 ACAGAGGGCCTCAGCACTGGCCCCACAAACCCATCCGGCCCTGC 
                 16 
                 21366 
                 2136 
                 TSC2 
               
               
                   
                 TCACCCTCAGCCGTCTTCCACATCGCCACCCTGATGCCCACCAA 
                   
                 7   8 
                  778 
                   
               
               
                   
                 GGACGTGGACAA 
                   
                   
                   
                   
               
               
                   
               
               
                 153 
                 TCAGCCGTCTTCCACATCGCCACCCTGATGCCCACCAAGGACGT 
                 16 
                 21367 
                 2136 
                 TSC2 
               
               
                   
                 GGACAAGCACCGCTGCGACAAGAAGCGCCACCTGGGCAACGAC 
                   
                    28 
                  828 
                   
               
               
                   
                 TTTGTGTCCATTG 
                   
                   
                   
                   
               
               
                   
               
               
                 154 
                 GCACCGCTGCGACAAGAAGCGCCACCTGGGCAACGACTTTGTGT 
                 16 
                 21367 
                 2136 
                 TSC2 
               
               
                   
                 CCATTGTCTACAATGACTCCGGTGAGGACTTCAAGCTTGGCACC 
                   
                    78 
                  878 
                   
               
               
                   
                 ATCAAGGTGAGT 
                   
                   
                   
                   
               
               
                   
               
               
                 155 
                 TCTACAATGACTCCGGTGAGGACTTCAAGCTTGGCACCATCAAG 
                 16 
                 21368 
                 2136 
                 TSC2 
               
               
                   
                 GTGAGTGAGGGGCCGTCAGTGAGGCTGGGCCCCAGGCAGGTGC 
                   
                    28 
                  928 
                   
               
               
                   
                 CCACTGCTGTGTC 
                   
                   
                   
                   
               
               
                   
               
               
                 156 
                 CCGAGATCAGCCTTCAGCACACGCTGTGTGCGGGGATGACCCTT 
                 16 
                 21378 
                 2137 
                 TSC2 
               
               
                   
                 TCTCTTGTCCGGGCAGGGCCAGTTCAACTTTGTCCACGTGATCGT 
                   
                    03 
                  903 
                   
               
               
                   
                 CACCCCGCTGG 
                   
                   
                   
                   
               
               
                   
               
               
                 157 
                 GTCCGGGCAGGGCCAGTTCAACTTTGTCCACGTGATCGTCACCC 
                 16 
                 21378 
                 2137 
                 TSC2 
               
               
                   
                 CGCTGGACTACGAGTGCAACCTGGTGTCCCTGCAGTGCAGGAAA 
                   
                    53 
                  953 
                   
               
               
                   
                 GGTAGGGCCGGG 
                   
                   
                   
                   
               
               
                   
               
               
                 158 
                 ACTACGAGTGCAACCTGGTGTCCCTGCAGTGCAGGAAAGGTAGG 
                 16 
                 21379 
                 2138 
                 TSC2 
               
               
                   
                 GCCGGGTGGGGCCCTGCAGTGCAGGAAAGGTAGGGCCGGGTGG 
                   
                    03 
                  003 
                   
               
               
                   
                 GGCCCTGCAGTGT 
                   
                   
                   
                   
               
               
                   
               
               
                 159 
                 TGCAGTGTGGCGCCAAGAGCCCTGGGCCTGGCGTGACCACCAAG 
                 16 
                 21379 
                 2138 
                 TSC2 
               
               
                   
                 TCTCCCCAGACATGGAGGGCCTTGTGGACACCAGCGTGGCCAAG 
                   
                    95 
                  095 
                   
               
               
                   
                 ATCGTGTCTGAC 
                   
                   
                   
                   
               
               
                   
               
               
                 160 
                 CAGACATGGAGGGCCTTGTGGACACCAGCGTGGCCAAGATCGT 
                 16 
                 21380 
                 2138 
                 TSC2 
               
               
                   
                 GTCTGACCGCAACCTGCCCTTCGTGGCCCGCCAGATGGCCCTGC 
                   
                    45 
                  145 
                   
               
               
                   
                 ACGCAAATGTGAG 
                   
                   
                   
                   
               
               
                   
               
               
                 161 
                 CGCAACCTGCCCTTCGTGGCCCGCCAGATGGCCCTGCACGCAAA 
                 16 
                 21380 
                 2138 
                 TSC2 
               
               
                   
                 TGTGAGTGGGGGTGGGTCCAGGCGTGAGCTGGTGGGACAGGCC 
                   
                    95 
                  195 
                   
               
               
                   
                 CAGGTGCCACCTG 
                   
                   
                   
                   
               
               
                   
               
               
                 162 
                 AGGCCCAGGTGCCACCTGATAGTGAGCTCACCCCCTGCCTACGT 
                 16 
                 21381 
                 2138 
                 TSC2 
               
               
                   
                 CCCCAGATGGCCTCACAGGTGCATCATAGCCGCTCCAACCCCAC 
                   
                    77 
                  277 
                   
               
               
                   
                 CGATATCTACCC 
                   
                   
                   
                   
               
               
                   
               
               
                 163 
                 ATGGCCTCACAGGTGCATCATAGCCGCTCCAACCCCACCGATAT 
                 16 
                 21382 
                 2138 
                 TSC2 
               
               
                   
                 CTACCCCTCCAAGTGGATTGCCCGGCTCCGCCACATCAAGCGGC 
                   
                    27 
                  327 
                   
               
               
                   
                 TCCGCCAGCGGG 
                   
                   
                   
                   
               
               
                   
               
               
                 164 
                 CTCCAAGTGGATTGCCCGGCTCCGCCACATCAAGCGGCTCCGCC 
                 16 
                 21382 
                 2138 
                 TSC2 
               
               
                   
                 AGCGGGTAGGGAATATGGGGCTCCCTCAGCGGGGTGTGCTGGCT 
                   
                    77 
                  377 
                   
               
               
                   
                 GCCCAAGCTGTG 
                   
                   
                   
                   
               
               
                   
               
               
                 165 
                 GCGGGTGTGTGGGCAGAGCGGTTGCCACGCCTCCCAGACTTACT 
                 16 
                 21383 
                 2138 
                 TSC2 
               
               
                   
                 GCCCAAGCCGCCTCTGCCTTCAGATCTGCGAGGAAGCCGCCTAC 
                   
                    79 
                  479 
                   
               
               
                   
                 TCCAACCCCAGC 
                   
                   
                   
                   
               
               
                   
               
               
                 166 
                 GCCGCCTCTGCCTTCAGATCTGCGAGGAAGCCGCCTACTCCAAC 
                 16 
                 21384 
                 2138 
                 TSC2 
               
               
                   
                 CCCAGCCTACCTCTGGTGCACCCTCCGTCCCATAGCAAAGCCCC 
                   
                    29 
                  529 
                   
               
               
                   
                 TGCACAGACTCC 
                   
                   
                   
                   
               
               
                   
               
               
                 167 
                 CTACCTCTGGTGCACCCTCCGTCCCATAGCAAAGCCCCTGCACA 
                 16 
                 21384 
                 2138 
                 TSC2 
               
               
                   
                 GACTCCAGCCGAGCCCACACCTGGCTATGAGGTGGGCCAGCGG 
                   
                    79 
                  579 
                   
               
               
                   
                 AAGCGCCTCATCT 
                   
                   
                   
                   
               
               
                   
               
               
                 168 
                 AGCCGAGCCCACACCTGGCTATGAGGTGGGCCAGCGGAAGCGC 
                 16 
                 21385 
                 2138 
                 TSC2 
               
               
                   
                 CTCATCTCCTCGGTGGAGGACTTCACCGAGTTTGTGTGAGGCCG 
                   
                    29 
                  629 
                   
               
               
                   
                 GGGCCCTCCCTCC 
                   
                   
                   
                   
               
               
                   
               
               
                 169 
                 CCTCGGTGGAGGACTTCACCGAGTTTGTGTGAGGCCGGGGCCCT 
                 16 
                 21385 
                 2138 
                 TSC2 
               
               
                   
                 CCCTCCTGCACTGGCCTTGGACGGTATTGCCTGTCAGTGAAATA 
                   
                    79 
                  679 
                   
               
               
                   
                 AATAAAGTCCTG 
                   
                   
                   
                   
               
               
                   
               
               
                 170 
                 GTGCATTCACACCTCCTGTTCTGTGCCAACAATATGCAAGTTAAC 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 ACTGATTGACCATCATTCCTTAGCTGTGTTCATGATGAGTCTCAT 
                   
                  1557 
                 7165 
                   
               
               
                   
                 TGTAGTCCAT 
                   
                   
                    7 
                   
               
               
                   
               
               
                 171 
                 TTGACCATCATTCCTTAGCTGTGTTCATGATGAGTCTCATTGTAG 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 TCCATGATATGTAGCTGTCCAACACTGTCCGGGGTCGGGGGAGA 
                   
                  1607 
                 7170 
                   
               
               
                   
                 CGGGTGAGGGC 
                   
                   
                    7 
                   
               
               
                   
               
               
                 172 
                 GATATGTAGCTGTCCAACACTGTCCGGGGTCGGGGGAGACGGGT 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 GAGGGCCATCTAGGTTCAGGGGAATCTTGGCTTCCACACCCAAG 
                   
                  1657 
                 7175 
                   
               
               
                   
                 TCTTTGCCCAGT 
                   
                   
                    7 
                   
               
               
                   
               
               
                 173 
                 CATCTAGGTTCAGGGGAATCTTGGCTTCCACACCCAAGTCTTTGC 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 CCAGTTCTGTCTTTAGGCTCTCAGAAAGGCTACTGGTCATGCCGT 
                   
                  1707 
                 7180 
                   
               
               
                   
                 CCTCATCACA 
                   
                   
                    7 
                   
               
               
                   
               
               
                 174 
                 TCTGTCTTTAGGCTCTCAGAAAGGCTACTGGTCATGCCGTCCTCA 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 TCACACTGGCTCTCGCTCTTATTACGAAATAACTCTCGAGCCTTC 
                   
                  1757 
                 7185 
                   
               
               
                   
                 ATACCCAGGA 
                   
                   
                    7 
                   
               
               
                   
               
               
                 175 
                 CTGGCTCTCGCTCTTATTACGAAATAACTCTCGAGCCTTCATACC 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 CAGGAAGCTTTTTGAACTGGGAAGTGAGCCCACAGTGGTGGGG 
                   
                  1807 
                 7190 
                   
               
               
                   
                 ATGCTGGCAGAC 
                   
                   
                    7 
                   
               
               
                   
               
               
                 176 
                 AGCTTTTTGAACTGGGAAGTGAGCCCACAGTGGTGGGGATGCTG 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 GCAGACGCTTCTCCCATAGTCGTCTCCCACCGACTGCTGAATGG 
                   
                  1857 
                 7195 
                   
               
               
                   
                 GCCTGCCCTCTG 
                   
                   
                    7 
                   
               
               
                   
               
               
                 177 
                 GCTTCTCCCATAGTCGTCTCCCACCGACTGCTGAATGGGCCTGCC 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 CTCTGGTGTGGGGGTTTCTCTGGGGTAGAAAGCTCGCTGCTGCT 
                   
                  1907 
                 7200 
                   
               
               
                   
                 GCTGCTGCTGC 
                   
                   
                    7 
                   
               
               
                   
               
               
                 178 
                 GTGTGGGGGTTTCTCTGGGGTAGAAAGCTCGCTGCTGCTGCTGC 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 TGCTGCCTCCACCACCTCTGCTTCCACTACTGCCCCGGGCGCTGC 
                   
                  1957 
                 7205 
                   
               
               
                   
                 TGGGCCTGGGG 
                   
                   
                    7 
                   
               
               
                   
               
               
                 179 
                 CTCCACCACCTCTGCTTCCACTACTGCCCCGGGCGCTGCTGGGCC 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 TGGGGGTCTTGGTCTCACCGTTGTGGCCAGATGCCTCTTCATTGT 
                   
                  2007 
                 7210 
                   
               
               
                   
                 GCCCTACCAT 
                   
                   
                    7 
                   
               
               
                   
               
               
                 180 
                 GTCTTGGTCTCACCGTTGTGGCCAGATGCCTCTTCATTGTGCCCT 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 ACCATGGAATCTGAGCACCCGTCATTACAACAGTCAAGCCTGTA 
                   
                  2057 
                 7215 
                   
               
               
                   
                 AGAAAGCCGGG 
                   
                   
                    7 
                   
               
               
                   
               
               
                 181 
                 GGAATCTGAGCACCCGTCATTACAACAGTCAAGCCTGTAAGAAA 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 GCCGGGGAGGAAAAAAGGAGCTGGTGATTGGACTGTCCACATT 
                   
                  2107 
                 7220 
                   
               
               
                   
                 CGGAGGATGTGGA 
                   
                   
                    7 
                   
               
               
                   
               
               
                 182 
                 CGTGACACAGTCCTTATGCTGGAATTGGCAGCTTAGTCCCAAGG 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 TCATGAATCAGTTCTTTGTTCCTACCTTTCTTCTGCTGCTTCAGCT 
                   
                  2501 
                 7260 
                   
               
               
                   
                 GCTTCTGCTT 
                   
                   
                    1 
                   
               
               
                   
               
               
                 183 
                 ATCAGTTCTTTGTTCCTACCTTTCTTCTGCTGCTTCAGCTGCTTCT 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 GCTTTTTCTTCTTCAAGTTTTTTCAGGAGGCCATCTTTCTCCAACC 
                   
                  2551 
                 7265 
                   
               
               
                   
                 TGCCATAT 
                   
                   
                    1 
                   
               
               
                   
               
               
                 184 
                 TTTCTTCTTCAAGTTTTTTCAGGAGGCCATCTTTCTCCAACCTGCC 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 ATATAAATCTAAGATCTCCAATTCAAACACCTGGGTTATCCTTTT 
                   
                  2601 
                 7270 
                   
               
               
                   
                 CTGAGCCTC 
                   
                   
                    1 
                   
               
               
                   
               
               
                 185 
                 AAATCTAAGATCTCCAATTCAAACACCTGGGTTATCCTTTTCTGA 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 GCCTCATACCTGCTCTCTGCGGCCTGCAGCTGTCCTCTGAAAGAT 
                   
                  2651 
                 7275 
                   
               
               
                   
                 ACAGACCAGC 
                   
                   
                    1 
                   
               
               
                   
               
               
                 186 
                 ATACCTGCTCTCTGCGGCCTGCAGCTGTCCTCTGAAAGATACAG 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 ACCAGCCAGAATATAGGAAGTTCCACTTAATAAAAACACAAAA 
                   
                  2701 
                 7280 
                   
               
               
                   
                 GCCTTTCCTGATG 
                   
                   
                    1 
                   
               
               
                   
               
               
                 187 
                 AATATAGGAAGTTCCACTTAATAAAAACACAAAAGCCTTTCCTG 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 ATGAAAGTTACCTTGCCTGGAGTTTGACATCCTCTAGATATTTCT 
                   
                  2754 
                 7285 
                   
               
               
                   
                 TCTGTTCCAAA 
                   
                   
                    4 
                   
               
               
                   
               
               
                 188 
                 GTTACCTTGCCTGGAGTTTGACATCCTCTAGATATTTCTTCTGTT 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 CCAAAAGAAGGTGGTCTTTCTTGGCCAGGTGAGATTCCAGTTCC 
                   
                  2804 
                 7290 
                   
               
               
                   
                 AAAATCCGTTT 
                   
                   
                    4 
                   
               
               
                   
               
               
                 189 
                 AGAAGGTGGTCTTTCTTGGCCAGGTGAGATTCCAGTTCCAAAAT 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 CCGTTTTTGGGAGGTATCAAGCCTCTGAGTCTGCTGGAGAACAT 
                   
                  2854 
                 7295 
                   
               
               
                   
                 GGCTTCTGTTTT 
                   
                   
                    4 
                   
               
               
                   
               
               
                 190 
                 TTGGGAGGTATCAAGCCTCTGAGTCTGCTGGAGAACATGGCTTC 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 TGTTTTTTTCTAGCTCTTTCCGATAGGCGGCTTTCATCATTTCTAC 
                   
                  2904 
                 7300 
                   
               
               
                   
                 TTCCTGAAAA 
                   
                   
                    4 
                   
               
               
                   
               
               
                 191 
                 TTTCTAGCTCTTTCCGATAGGCGGCTTTCATCATTTCTACTTCCTG 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 AAAAAAAAAAAAAAAAAAGACTGGAATTAGTACTTATAAAAAA 
                   
                  2954 
                 7305 
                   
               
               
                   
                 TAAACATGCTG 
                   
                   
                    4 
                   
               
               
                   
               
               
                 192 
                 GACATACTGTCTGGGTCTGAAACGCTTTCCCCACTAAGGTCTGG 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 CTCCCGAGCCCTGGCATACCTTTGTGGTATCTGAGTGCTTGTTCT 
                   
                  6038 
                 7613 
                   
               
               
                   
                 GCAGTTGTTCC 
                   
                   
                    8 
                   
               
               
                   
               
               
                 193 
                 AGCCCTGGCATACCTTTGTGGTATCTGAGTGCTTGTTCTGCAGTT 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 GTTCCAAATAGAGCTCGTTGACCTCCCCAAGAACCAACAGCTGC 
                   
                  6088 
                 7618 
                   
               
               
                   
                 CTGTTCAAGAA 
                   
                   
                    8 
                   
               
               
                   
               
               
                 194 
                 AAATAGAGCTCGTTGACCTCCCCAAGAACCAACAGCTGCCTGTT 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 CAAGAACTCCATCTGCTGCTGGACCGACTCACTGTTTGAGAGCT 
                   
                  6138 
                 7623 
                   
               
               
                   
                 AACCAAAAAACA 
                   
                   
                    8 
                   
               
               
                   
               
               
                 195 
                 CTCCATCTGCTGCTGGACCGACTCACTGTTTGAGAGCTAACCAA 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 AAAACATGAGCAAAGTGAAAAATCCGACGACATAAAACTAGCA 
                   
                  6188 
                 7628 
                   
               
               
                   
                 CATAGACGTCATT 
                   
                   
                    8 
                   
               
               
                   
               
               
                 196 
                 ACCTGTCTGAAGGAAGAATGTTAGCAAATGGTGTTTCAGCAGAT 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 TCAGGTCTGCCTCATTTCTTCTTACCTTTTGGGAAACCTGACTGA 
                   
                  6906 
                 7700 
                   
               
               
                   
                 GCAGCAGCTCA 
                   
                   
                    6 
                   
               
               
                   
               
               
                 197 
                 CTGCCTCATTTCTTCTTACCTTTTGGGAAACCTGACTGAGCAGCA 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 GCTCAGTGTGACACACCTTGTTGTTGGCCTTCTTCAGTTCTATCC 
                   
                  6956 
                 7705 
                   
               
               
                   
                 GCAGCTCCGC 
                   
                   
                    6 
                   
               
               
                   
               
               
                 198 
                 GTGTGACACACCTTGTTGTTGGCCTTCTTCAGTTCTATCCGCAGC 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 TCCGCAATCATGTTCCTGCAGTCCTCCAGCTTCGTCTGCCCAAAG 
                   
                  7006 
                 7710 
                   
               
               
                   
                 AGACGTGGAC 
                   
                   
                    6 
                   
               
               
                   
               
               
                 199 
                 AATCATGTTCCTGCAGTCCTCCAGCTTCGTCTGCCCAAAGAGAC 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 GTGGACATGAAGTTTGAGGAACACCAACAGGCCAGATCACAGG 
                   
                  7056 
                 7715 
                   
               
               
                   
                 CCTACCTAGCCAC 
                   
                   
                    6 
                   
               
               
                   
               
               
                 200 
                 CTCCCCACTGCTCTCCGGCATTCTCGCAGTTGGCTTTGCCTGGTG 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 CTGCAGTTTATACCTGTAATTCCTGGCTCTGGTTGTAGAATTCCT 
                   
                  7933 
                 7803 
                   
               
               
                   
                 CTCGGTCATG 
                   
                   
                    3 
                   
               
               
                   
               
               
                 201 
                 GTTTATACCTGTAATTCCTGGCTCTGGTTGTAGAATTCCTCTCGG 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 TCATGCTGCAGCTGTCTGATCTGGCTGTGGAGCTTGGTTACCATA 
                   
                  7983 
                 7808 
                   
               
               
                   
                 GTGTCACGCT 
                   
                   
                    3 
                   
               
               
                   
               
               
                 202 
                 CTGCAGCTGTCTGATCTGGCTGTGGAGCTTGGTTACCATAGTGTC 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 ACGCTGCTCCTGGAGCTGATTGTATCTAGCTTGTTCTTTCTGCAG 
                   
                  8033 
                 7813 
                   
               
               
                   
                 ACTAACCTTC 
                   
                   
                    3 
                   
               
               
                   
               
               
                 203 
                 GCTCCTGGAGCTGATTGTATCTAGCTTGTTCTTTCTGCAGACTAA 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 CCTTCCACATCTGGATGTCCTTCTCTTGTAACTTCAACTGATCTTT 
                   
                  8083 
                 7818 
                   
               
               
                   
                 CTAGCAGAG 
                   
                   
                    3 
                   
               
               
                   
               
               
                 204  
                 CACATCTGGATGTCCTTCTCTTGTAACTTCAACTGATCTTTCTAG 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 CAGAGACCAGAAATGTCATCATTTTAGCTGTCTTCCAACACAGG 
                   
                  8133 
                 7823 
                   
               
               
                   
                 CAATTTAACAC 
                   
                   
                    3 
                   
               
               
                   
               
               
                 205 
                 AAGCTATCATGCTGACCCAAAACAAAACAAAAAGCAAGCTCCA 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 CCTGTCCCCTCCCCAGTCCTCACCATGGCAGCATTATGTTCCTCC 
                   
                  8971 
                 7907 
                   
               
               
                   
                 AGAGCTGCTGCT 
                   
                   
                    1 
                   
               
               
                   
               
               
                 206 
                 CCTCCCCAGTCCTCACCATGGCAGCATTATGTTCCTCCAGAGCTG 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 CTGCTTTGATCACCTTGCGGAGGAGCCGCCTGTTCCGGAGGGCA 
                   
                  9021 
                 7912 
                   
               
               
                   
                 TGCTGCTGCCT 
                   
                   
                    1 
                   
               
               
                   
               
               
                 207 
                 TTGATCACCTTGCGGAGGAGCCGCCTGTTCCGGAGGGCATGCTG 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 CTGCCTCTTAAAACGCTCATAGAGTAACTGGTTGTGCAGTAAAA 
                   
                  9071 
                 7917 
                   
               
               
                   
                 GCAACTGGTCTC 
                   
                   
                    1 
                   
               
               
                   
               
               
                 208 
                 CTTAAAACGCTCATAGAGTAACTGGTTGTGCAGTAAAAGCAACT 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 GGTCTCGGAGGGTGCGGATCTCATCTGAAGGAGGAGAGCCTGAT 
                   
                  9121 
                 7922 
                   
               
               
                   
                 TGTAAAGCAGAG 
                   
                   
                    1 
                   
               
               
                   
               
               
                 209 
                 GGAGGGTGCGGATCTCATCTGAAGGAGGAGAGCCTGATTGTAA 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 AGCAGAGGGAGGGTGGCAGAAATGCCTTTTACAGATGGTTCAAT 
                   
                  9171 
                 7927 
                   
               
               
                   
                 CAAGCCCCCTTCC 
                   
                   
                    1 
                   
               
               
                   
               
               
                 210 
                 AAGCAAGCAGGAACCATGTGGGCTGGATTTGGAGCTAAAGTAA 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 CAACTTTACCTCCAAAGTGGGTCCAGTCGACAGACTTGCTGGGT 
                   
                  9745 
                 7984 
                   
               
               
                   
                 AAAGGCAACCTAG 
                   
                   
                    5 
                   
               
               
                   
               
               
                 211 
                 ACCTCCAAAGTGGGTCCAGTCGACAGACTTGCTGGGTAAAGGCA 
                  9 
                 13577 
                 1357 
                 TSC1 
               
               
                   
                 ACCTAGGAAGAAAGTTTTTGAGTAACAAAGTTACCGATCTTACC 
                   
                  9795 
                 7989 
                   
               
               
                   
                 AAGAAAAAAACG 
                   
                   
                    5 
                   
               
               
                   
               
               
                 212 
                 CTCTTACACTTTCTGTACTTCACAATAAAATGGACCATTTAACAC 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 AGAAGAGAGTGCCCCAGTCCCTTACTTGTTCAGCTCCTTGCTGTG 
                   
                  0897 
                 8099 
                   
               
               
                   
                 CGCGTCTGCT 
                   
                   
                    7 
                   
               
               
                   
               
               
                 213 
                 AGAGTGCCCCAGTCCCTTACTTGTTCAGCTCCTTGCTGTGCGCGT 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 CTGCTCCCTGCTGTATCAGTCTGTCCAGCACTTCCATTGGGGAGG 
                   
                  0947 
                 8104 
                   
               
               
                   
                 TAGAGGGCAC 
                   
                   
                    7 
                   
               
               
                   
               
               
                 214 
                 CCCTGCTGTATCAGTCTGTCCAGCACTTCCATTGGGGAGGTAGA 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 GGGCACACCATCTTCCTCTGTGTTTCCTTTTGCTTTCTTTAACAGC 
                   
                  0997 
                 8109 
                   
               
               
                   
                 TCCTCAGTCT 
                   
                   
                    7 
                   
               
               
                   
               
               
                 215 
                 ACCATCTTCCTCTGTGTTTCCTTTTGCTTTCTTTAACAGCTCCTCA 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 GTCTTCCTGATGACAAAATGATGGGCTGTCTTTGGCAATGCCAC 
                   
                  1047 
                 8114 
                   
               
               
                   
                 CTCAAAAAGA 
                   
                   
                    7 
                   
               
               
                   
               
               
                 216 
                 TCCTGATGACAAAATGATGGGCTGTCTTTGGCAATGCCACCTCA 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 AAAAGATGATCATACGGGGGAGGCTGCCCGCTTCCAAAGCCCA 
                   
                  1097 
                 8119 
                   
               
               
                   
                 CTCTCGTCGGAGG 
                   
                   
                    7 
                   
               
               
                   
               
               
                 217 
                 TGATCATACGGGGGAGGCTGCCCGCTTCCAAAGCCCACTCTCGT 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 CGGAGGTGGAATTTTACAAGGACTGGGAGTGAAGATACTGGTCT 
                   
                  1147 
                 8124 
                   
               
               
                   
                 CCAAAGAAGTCT 
                   
                   
                    7 
                   
               
               
                   
               
               
                 218 
                 TGGAATTTTACAAGGACTGGGAGTGAAGATACTGGTCTCCAAAG 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 AAGTCTGGCATTCCCTGTCTCCCGCAGGGCTTTCATCAGCACTGC 
                   
                  1197 
                 8129 
                   
               
               
                   
                 CGCAGGGCAGG 
                   
                   
                    7 
                   
               
               
                   
               
               
                 219 
                 GGCATTCCCTGTCTCCCGCAGGGCTTTCATCAGCACTGCCGCAG 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 GGCAGGTCTATGGGAGTAAAGGCTTGCTTTGGTGTGTCAGGCCC 
                   
                  1247 
                 8134 
                   
               
               
                   
                 AAGCTTGTCCAG 
                   
                   
                    7 
                   
               
               
                   
               
               
                 220 
                 TCTATGGGAGTAAAGGCTTGCTTTGGTGTGTCAGGCCCAAGCTT 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 GTCCAGGGAGGAGTGTAAAGGCTCAGGGTTCACGCTGGCGCCCT 
                   
                  1297 
                 8139 
                   
               
               
                   
                 GAGAACTGGAGG 
                   
                   
                    7 
                   
               
               
                   
               
               
                 221 
                 GGAGGAGTGTAAAGGCTCAGGGTTCACGCTGGCGCCCTGAGAA 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 CTGGAGGCTGCCGAGTGGGTCTTCCGCTGAGAACCTGGGAGACT 
                   
                  1347 
                 8144 
                   
               
               
                   
                 GTCTCGGTAAAAG 
                   
                   
                    7 
                   
               
               
                   
               
               
                 222 
                 CTGCCGAGTGGGTCTTCCGCTGAGAACCTGGGAGACTGTCTCGG 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 TAAAAGGGAGAGTCAAAGCCTCCTCGAGGAACCACAGGCTCTG 
                   
                  1397 
                 8149 
                   
               
               
                   
                 CCTCTGCTGTGGT 
                   
                   
                    7 
                   
               
               
                   
               
               
                 223 
                 GGAGAGTCAAAGCCTCCTCGAGGAACCACAGGCTCTGCCTCTGC 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 TGTGGTGATCTCAGAAAGTTCTCTAGATATTGCAGCTGAGAGGA 
                   
                  1447 
                 8154 
                   
               
               
                   
                 AGAGAGGAAACA 
                   
                   
                    7 
                   
               
               
                   
               
               
                 224 
                 GATCTCAGAAAGTTCTCTAGATATTGCAGCTGAGAGGAAGAGAG 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 GAAACAAAAGAAATGGCAGTCGGTATTCCACCTGGGAAAGACT 
                   
                  1497 
                 8159 
                   
               
               
                   
                 AGGCAGTTTGGGT 
                   
                   
                    7 
                   
               
               
                   
               
               
                 225 
                 GCACAAAATCCCAGATTTATAGCAGAGCGAGGGTCAGGTTTTAT 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 CAACTCATAGCAATCCCACATACATTACCTTCTTCTTTATCTTTTT 
                   
                  2045 
                 8214 
                   
               
               
                   
                 CAATACTATC 
                   
                   
                    5 
                   
               
               
                   
               
               
                 226 
                 ATAGCAATCCCACATACATTACCTTCTTCTTTATCTTTTTCAATA 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 CTATCTTCTTCAGAGGCCAGATCACCTAAAAACCCTGGAAGATC 
                   
                  2095 
                 8219 
                   
               
               
                   
                 ACTTAGAGTGA 
                   
                   
                    5 
                   
               
               
                   
               
               
                 227 
                 TTCTTCAGAGGCCAGATCACCTAAAAACCCTGGAAGATCACTTA 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 GAGTGACAGAACCTTTGCTGCCAGGTGGCTCTTCTGAAGAGAAA 
                   
                  2145 
                 8224 
                   
               
               
                   
                 CAAAGACAACTG 
                   
                   
                    5 
                   
               
               
                   
               
               
                 228 
                 CAGAACCTTTGCTGCCAGGTGGCTCTTCTGAAGAGAAACAAAGA 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 CAACTGAAGTCAAAGAAATACAGTGTAATCCCTGTAAGTGTAAA 
                   
                  2195 
                 8229 
                   
               
               
                   
                 ACTGCTTACACT 
                   
                   
                    5 
                   
               
               
                   
               
               
                 229 
                 TTCTTAAACACATATAACCCAATTAGAAGAGGCAAGCAAGGCCT 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 GTAGTAACGCAGAAATTTTACCTGATCCTCTGTCATTCAGAAGA 
                   
                  2622 
                 8272 
                   
               
               
                   
                 TGGTGTTGTCTG 
                   
                   
                    2 
                   
               
               
                   
               
               
                 230 
                 ACGCAGAAATTTTACCTGATCCTCTGTCATTCAGAAGATGGTGTT 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 GTCTGTGTAGACATGGTCTTGCAGAATCCATTCTCTCTTCCTGAA 
                   
                  2672 
                 8277 
                   
               
               
                   
                 AAGATAAGTA 
                   
                   
                    2 
                   
               
               
                   
               
               
                 231 
                 TGTAGACATGGTCTTGCAGAATCCATTCTCTCTTCCTGAAAAGAT 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 AAGTATCATTTATATCACAAGACGAAAAATGTTGCACATGTTCT 
                   
                  2722 
                 8282 
                   
               
               
                   
                 CGAGCATATTG 
                   
                   
                    2 
                   
               
               
                   
               
               
                 232 
                 ATCACACCTTGAGAGCAGCTTGTTAGTCCATTTTCAATTATTCTG 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 ATTCAAACCCATTGCATTTTAGGTCAGAATTCTATCTGGCATAAT 
                   
                  5792 
                 8589 
                   
               
               
                   
                 TAGGCTTCTC 
                   
                   
                    2 
                   
               
               
                   
               
               
                 233 
                 AACCCATTGCATTTTAGGTCAGAATTCTATCTGGCATAATTAGGC 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 TTCTCAAAGTGAGGCTTGCAAGTGAGTCACTGTGCCTGGGCAGA 
                   
                  5842 
                 8594 
                   
               
               
                   
                 GGGATAGCAGA 
                   
                   
                    2 
                   
               
               
                   
               
               
                 234 
                 AAAGTGAGGCTTGCAAGTGAGTCACTGTGCCTGGGCAGAGGGA 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 TAGCAGACGAGCTGGATCGCACCTTCCTGGGGGGTGTGACTGTG 
                   
                  5892 
                 8599 
                   
               
               
                   
                 GCCTGGGGGAGTG 
                   
                   
                    2 
                   
               
               
                   
               
               
                 235 
                 CGAGCTGGATCGCACCTTCCTGGGGGGTGTGACTGTGGCCTGGG 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 GGAGTGAAATGTGCACGTAGTCATCCGAATGACAGAGTGGGGC 
                   
                  5942 
                 8604 
                   
               
               
                   
                 TGGAGGAGGAGAG 
                   
                   
                    2 
                   
               
               
                   
               
               
                 236 
                 AAATGTGCACGTAGTCATCCGAATGACAGAGTGGGGCTGGAGG 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 AGGAGAGGTTGCTGGGGTTCCCAGAGGAGTTCCTTTTCCACCTG 
                   
                  5992 
                 8609 
                   
               
               
                   
                 CTTAGAGACAAGG 
                   
                   
                    2 
                   
               
               
                   
               
               
                 237 
                 GTTGCTGGGGTTCCCAGAGGAGTTCCTTTTCCACCTGCTTAGAGA 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 CAAGGGCAGAACATATATGAACACTGAGCCCAACTATTAGAAA 
                   
                  6042 
                 8614 
                   
               
               
                   
                 AACTGCCGATTT 
                   
                   
                    2 
                   
               
               
                   
               
               
                 238 
                 GAGAGCTCCTCCTGCCATTAAAGGCAGGCCAAAACCAACTAATC 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 AAATCCAACCTAAGACATACATACCAGTTGTACCAAAGACTTTA 
                   
                  6320 
                 8642 
                   
               
               
                   
                 CTGTAAGGGTGT 
                   
                   
                    0 
                   
               
               
                   
               
               
                 239 
                 AACCTAAGACATACATACCAGTTGTACCAAAGACTTTACTGTAA 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 GGGTGTGACAGATCAGGTGGGACATTTCCAGGAGAAGTTGGAG 
                   
                  6370 
                 8647 
                   
               
               
                   
                 GAGTGGTCATACC 
                   
                   
                    0 
                   
               
               
                   
               
               
                 240 
                 GACAGATCAGGTGGGACATTTCCAGGAGAAGTTGGAGGAGTGG 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 TCATACCACAAACCATAGATGGGCTCCAAAGAGTAGCCTGGGA 
                   
                  6420 
                 8652 
                   
               
               
                   
                 AGTTAATAAAGTAC 
                   
                   
                    0 
                   
               
               
                   
               
               
                 241 
                 ACAAACCATAGATGGGCTCCAAAGAGTAGCCTGGGAAGTTAAT 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 AAAGTACATCAGCAGTGGCAAAGGAATGCTAAGTCATCCACGA 
                   
                  6470 
                 8657 
                   
               
               
                   
                 GGTTTATATCCATG 
                   
                   
                    0 
                   
               
               
                   
               
               
                 242 
                 GGATCCTTAAAAGTGACTCCTGAAATGAGCAGTGTGAAATTTTC 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 CCAACCACATACTAAATCTGACCCAAAGGGTCAGCTTCACCAGA 
                   
                  6712 
                 8681 
                   
               
               
                   
                 AAGCAGAGGAGA 
                   
                   
                    2 
                   
               
               
                   
               
               
                 243 
                 ACATACTAAATCTGACCCAAAGGGTCAGCTTCACCAGAAAGCAG 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 AGGAGAGAGCAGGCACACTAGTTGACACCATACTTGTGGTGGTT 
                   
                  6762 
                 8686 
                   
               
               
                   
                 CAGTTATCAGCC 
                   
                   
                    2 
                   
               
               
                   
               
               
                 244 
                 GAGCAGGCACACTAGTTGACACCATACTTGTGGTGGTTCAGTTA 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 TCAGCCGTGTCGATGGGGAACTCAGAGTCTGAGGTAGCTGCCCT 
                   
                  6812 
                 8691 
                   
               
               
                   
                 GGCATATTTAAC 
                   
                   
                    2 
                   
               
               
                   
               
               
                 245 
                 GTGTCGATGGGGAACTCAGAGTCTGAGGTAGCTGCCCTGGCATA 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 TTTAACAACATCAGCCGAGACGTGGAGTAAGGGGTAGAAGTAG 
                   
                  6862 
                 8696 
                   
               
               
                   
                 CACACCCTAAAAT 
                   
                   
                    2 
                   
               
               
                   
               
               
                 246 
                 AACATCAGCCGAGACGTGGAGTAAGGGGTAGAAGTAGCACACC 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 CTAAAATGGAAGAGAAGAACACAGGGGGTTAGTGTGTGGTTTT 
                   
                  6912 
                 8701 
                   
               
               
                   
                 AGGTTATTCTGGTT 
                   
                   
                    2 
                   
               
               
                   
               
               
                 247 
                 ACAAATAATGTTTTCCAGAGACAAAGTTGCAAAACAGATAAGTA 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 CCAAAGACACTTTTTACCATAGCTATTCTGTGTGTCAGCATAAG 
                   
                  7607 
                 8770 
                   
               
               
                   
                 GGCTGGTGGTGA 
                   
                   
                    7 
                   
               
               
                   
               
               
                 248 
                 ACACTTTTTACCATAGCTATTCTGTGTGTCAGCATAAGGGCTGGT 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 GGTGACATCGGCTGAACGATGAGGAAAGCGGGCTGAGATTTGG 
                   
                  7657 
                 8775 
                   
               
               
                   
                 TGAGACACAGAA 
                   
                   
                    7 
                   
               
               
                   
               
               
                 249 
                 CATCGGCTGAACGATGAGGAAAGCGGGCTGAGATTTGGTGAGA 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 CACAGAATAGCCATCTTCATATGAGGCTTCTGTGGGATCCAGAG 
                   
                  7707 
                 8780 
                   
               
               
                   
                 AGATTTTGGCACA 
                   
                   
                    7 
                   
               
               
                   
               
               
                 250 
                 TAGCCATCTTCATATGAGGCTTCTGTGGGATCCAGAGAGATTTT 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 GGCACACTCGATCACAACATCATGAGTTTCTAATCTCTTCCACCT 
                   
                  7757 
                 8785 
                   
               
               
                   
                 GTAAAATGCAA 
                   
                   
                    7 
                   
               
               
                   
               
               
                 251 
                 CTCGATCACAACATCATGAGTTTCTAATCTCTTCCACCTGTAAAA 
                  9 
                 13578 
                 1357 
                 TSC1 
               
               
                   
                 TGCAATGAAAGTCAAGAAATGCAAACTGTAATCAACTGAATTAA 
                   
                  7807 
                 8790 
                   
               
               
                   
                 ATACTTCAGAG 
                   
                   
                    7 
                   
               
               
                   
               
               
                 252 
                 CTCCTAGATCACATTTTCAATCTCTCGAAAGATTCTTTAAAATTT 
                  9 
                 13579 
                 1357 
                 TSC1 
               
               
                   
                 TGACACTAGTTTCTATACCTTCGAGGGTCCAGTTCATGGTCCTTG 
                   
                  6686 
                 9678 
                   
               
               
                   
                 GATCCAGTCA 
                   
                   
                    6 
                   
               
               
                   
               
               
                 253 
                 CTAGTTTCTATACCTTCGAGGGTCCAGTTCATGGTCCTTGGATCC 
                  9 
                 13579 
                 1357 
                 TSC1 
               
               
                   
                 AGTCACTAATTCCGGATGAATTCGCACATGCTCCATCATTGGCT 
                   
                  6736 
                 9683 
                   
               
               
                   
                 AGAAGAGTTGG 
                   
                   
                    6 
                   
               
               
                   
               
               
                 254 
                 CTAATTCCGGATGAATTCGCACATGCTCCATCATTGGCTAGAAG 
                  9 
                 13579 
                 1357 
                 TSC1 
               
               
                   
                 AGTTGGGTTGACAAATTATAAAGGGCTGAATGTTTGTGGAACAT 
                   
                  6786 
                 9688 
                   
               
               
                   
                 CCAAATGATGGA 
                   
                   
                    6 
                   
               
               
                   
               
               
                 255 
                 ATACAAAAGGTATAAATGCAGCCTATCTAAACAGTATACTAAGT 
                  9 
                 13579 
                 1357 
                 TSC1 
               
               
                   
                 AGCAAACAAACAAGCAGTTTCAATTTACCTTGACCACTTCTTCA 
                   
                  7133 
                 9723 
                   
               
               
                   
                 AAAGTCTCCAGG 
                   
                   
                    3 
                   
               
               
                   
               
               
                 256 
                 CAAACAAGCAGTTTCAATTTACCTTGACCACTTCTTCAAAAGTCT 
                  9 
                 13579 
                 1357 
                 TSC1 
               
               
                   
                 CCAGGTTTTCTTTCATACTGTAATGAGAACGCAAAAAGGAGACG 
                   
                  7183 
                 9728 
                   
               
               
                   
                 AAGTTGCAAGG 
                   
                   
                    3 
                   
               
               
                   
               
               
                 257 
                 TTTTCTTTCATACTGTAATGAGAACGCAAAAAGGAGACGAAGTT 
                  9 
                 13579 
                 1357 
                 TSC1 
               
               
                   
                 GCAAGGGTACATTCCATAAAGGCGATGAAAGAGTGCGTACACA 
                   
                  7233 
                 9733 
                   
               
               
                   
                 CTGGCATGGAGAT 
                   
                   
                    3 
                   
               
               
                   
               
               
                 258 
                 GTACATTCCATAAAGGCGATGAAAGAGTGCGTACACACTGGCAT 
                  9 
                 13579 
                 1357 
                 TSC1 
               
               
                   
                 GGAGATGGACGAGATAGACTTCCGCCACGTGGCCTAGAAAAGG 
                   
                  7283 
                 9738 
                   
               
               
                   
                 AACCCGTTGAGAA 
                   
                   
                    3 
                   
               
               
                   
               
               
                 259 
                 GGACGAGATAGACTTCCGCCACGTGGCCTAGAAAAGGAACCCG 
                  9 
                 13579 
                 1357 
                 TSC1 
               
               
                   
                 TTGAGAAGAGCCTCTTAGTTGGAGACAGATTGAGGAGTGCAAA 
                   
                  7333 
                 9743 
                   
               
               
                   
                 ACAGCTATAAACAA 
                   
                   
                    3 
                   
               
               
                   
               
               
                 260 
                 AATGAAAGCATTCACCTCACAGGGCCCAACAGGTATATGAGGA 
                  9 
                 13579 
                 1357 
                 TSC1 
               
               
                   
                 GATCTGTACCTGGTTTCTTCAGGCACCATGATGACAGACGGCCA 
                   
                  8682 
                 9878 
                   
               
               
                   
                 AAAATGTCAAAGA 
                   
                   
                    2 
                   
               
               
                   
               
               
                 261 
                 ACCTGGTTTCTTCAGGCACCATGATGACAGACGGCCAAAAATGT 
                  9 
                 13579 
                 1357 
                 TSC1 
               
               
                   
                 CAAAGAAATCAAGAAGATGCTGTTTCCCAGACTGTGGAATCATT 
                   
                  8732 
                 9883 
                   
               
               
                   
                 GGTAGCATGGTT 
                   
                   
                    2 
                   
               
               
                   
               
               
                 262 
                 AATCAAGAAGATGCTGTTTCCCAGACTGTGGAATCATTGGTAGC 
                  9 
                 13579 
                 1357 
                 TSC1 
               
               
                   
                 ATGGTTATCAACACCAAGACGCCTGTTGTGAGGACAACGACGTC 
                   
                  8782 
                 9888 
                   
               
               
                   
                 AGTGTCCATCTG 
                   
                   
                    2 
                   
               
               
                   
               
               
                 263 
                 ATCAACACCAAGACGCCTGTTGTGAGGACAACGACGTCAGTGTC 
                  9 
                 13579 
                 1357 
                 TSC1 
               
               
                   
                 CATCTGCAGGAGAAAAGGTCAAACAGGAAACGTCTGTCAGGCA 
                   
                  8832 
                 9893 
                   
               
               
                   
                 CTGGCACCAGGAT 
                   
                   
                    2 
                   
               
               
                   
               
               
                 264 
                 GCTTTAAGTTGCCTAAAATTTCAGAAACTATACTCATAAAACCA 
                  9 
                 13580 
                 1358 
                 TSC1 
               
               
                   
                 TTTCATTCAAATCCTTACAAACATCCTACCTTGAGACATTTTAGT 
                   
                  0900 
                 0100 
                   
               
               
                   
                 AAAGAAGGCAA 
                   
                   
                    0 
                   
               
               
                   
               
               
                 265 
                 TCAAATCCTTACAAACATCCTACCTTGAGACATTTTAGTAAAGA 
                  9 
                 13580 
                 1358 
                 TSC1 
               
               
                   
                 AGGCAAAAGAGGTGCTTGAGAGAGCTTATGCTTCCAAGATGGCT 
                   
                  0950 
                 0105 
                   
               
               
                   
                 GCAGTCTTATGA 
                   
                   
                    0 
                   
               
               
                   
               
               
                 266 
                 AAGAGGTGCTTGAGAGAGCTTATGCTTCCAAGATGGCTGCAGTC 
                  9 
                 13580 
                 1358 
                 TSC1 
               
               
                   
                 TTATGACATGACCCAGTAACGAGAGGATGGATAAACGAGTGGC 
                   
                  1000 
                 0110 
                   
               
               
                   
                 GGCTTTGCCCACA 
                   
                   
                    0 
                   
               
               
                   
               
               
                 267 
                 CATGACCCAGTAACGAGAGGATGGATAAACGAGTGGCGGCTTT 
                  9 
                 13580 
                 1358 
                 TSC1 
               
               
                   
                 GCCCACATATTCGTTAATCCTGTCCAAGAGGTGCTGAAAATGTA 
                   
                  1050 
                 0115 
                   
               
               
                   
                 AAAGAACAAGGGC 
                   
                   
                    0 
                   
               
               
                   
               
               
                 268 
                 TATTCGTTAATCCTGTCCAAGAGGTGCTGAAAATGTAAAAGAAC 
                  9 
                 13580 
                 1358 
                 TSC1 
               
               
                   
                 AAGGGCAGTCCTCACATGAATGTATGAAGTTAACACAAATAAA 
                   
                  1100 
                 0120 
                   
               
               
                   
                 GACAGCAATGATG 
                   
                   
                    0 
                   
               
               
                   
               
               
                 269 
                 ACAGTGGCCGTGCACAGAAGCTGTTGTACTCATGAAGAACATAT 
                  9 
                 13580 
                 1358 
                 TSC1 
               
               
                   
                 GAAATGCCTATGATATTTCAGCCATTACCTTGTCATGTGGCTCTT 
                   
                  2515 
                 0261 
                   
               
               
                   
                 GCAAGGTGGTC 
                   
                   
                    5 
                   
               
               
                   
               
               
                 270 
                 CCTATGATATTTCAGCCATTACCTTGTCATGTGGCTCTTGCAAGG 
                  9 
                 13580 
                 1358 
                 TSC1 
               
               
                   
                 TGGTCAGGATGTGCAATGCCGGCTGAGAGCTGGTTTCCAGGTAA 
                   
                  2565 
                 0266 
                   
               
               
                   
                 TAATCCACCAA 
                   
                   
                    5 
                   
               
               
                   
               
               
                 271 
                 AGGATGTGCAATGCCGGCTGAGAGCTGGTTTCCAGGTAATAATC 
                  9 
                 13580 
                 1358 
                 TSC1 
               
               
                   
                 CACCAAGGTGTTTACAAGCATAGGGCCACGGTCTAAATCAAGAA 
                   
                  2615 
                 0271 
                   
               
               
                   
                 AAGGGCAATGGA 
                   
                   
                    5 
                   
               
               
                   
               
               
                 272 
                 GGTGTTTACAAGCATAGGGCCACGGTCTAAATCAAGAAAAGGG 
                  9 
                 13580 
                 1358 
                 TSC1 
               
               
                   
                 CAATGGATGATACTTATTCCCCTTAACATCCTAAATTTACCTTAC 
                   
                  2665 
                 0276 
                   
               
               
                   
                 ACAGCTTCCTGT 
                   
                   
                    5 
                   
               
               
                   
               
               
                 273 
                 AGGATTCTAGTGGCTCTAAAGTCAATCTCTTCTTTCTAGAAGATA 
                  9 
                 13580 
                 1358 
                 TSC1 
               
               
                   
                 AGCTAAAAAGGATATTATTTTGCTAACCAGAATTGAGGTTCTCT 
                   
                  4081 
                 0418 
                   
               
               
                   
                 TTAAAGACAGC 
                   
                   
                    1 
                   
               
               
                   
               
               
                 274 
                 AAAAGGATATTATTTTGCTAACCAGAATTGAGGTTCTCTTTAAA 
                  9 
                 13580 
                 1358 
                 TSC1 
               
               
                   
                 GACAGCTGTCACGTCGTCCCGCACACCCAGCATGGGGGAGTCCA 
                   
                  4131 
                 0423 
                   
               
               
                   
                 GCATGGCAAGAA 
                   
                   
                    1 
                   
               
               
                   
               
               
                 275 
                 TGTCACGTCGTCCCGCACACCCAGCATGGGGGAGTCCAGCATGG 
                  9 
                 13580 
                 1358 
                 TSC1 
               
               
                   
                 CAAGAAGCTCCCCGACATTTGCTTGTTGGGCCATTCTCTCGCTCG 
                   
                  4181 
                 0428 
                   
               
               
                   
                 AAGGCGCTGTG 
                   
                   
                    1 
                   
               
               
                   
               
               
                 276 
                 GCTCCCCGACATTTGCTTGTTGGGCCATTCTCTCGCTCGAAGGCG 
                  9 
                 13580 
                 1358 
                 TSC1 
               
               
                   
                 CTGTGCTGGCTCCAGGACGTGTGCTACAGGTTCTGAAGGTTCTTC 
                   
                  4231 
                 0433 
                   
               
               
                   
                 ATTGGGGCCA 
                   
                   
                    1 
               
               
                   
               
            
           
         
       
     
     2. Construction of gDNA Library 
     The library was constructed according to the conventional method: 
     1) Sampling. 
     a) DNA concentration of each sample was detected and recorded by Qubit2.0; 
     b) 50 ng of the sample was taken and placed in a 0.2 ml PCR tube, 
     2) DNA Fragmentation 
     a) Samples in the PCR tubes were fragmented according to the following table 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Components 
                 volume (μL) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Input Double-stranded DNA 
                 35 
               
               
                   
                 KAPA Frag Buffer (10X) 
                 5 
               
               
                   
                 KAPA Frag Enzyme 
                 10 
               
               
                   
                 Total 
                 50 
               
               
                   
                   
               
            
           
         
       
     
     b) Fragmentation reaction was performed according to the following procedures: 
     
       
         
           
               
               
               
               
               
             
               
                   
                   
               
               
                   
                 Temperature 
                   
                 Time 
                 Heated-lid temperature 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 4° C. 
                 30 
                 S 
                 50° C. 
               
               
                   
                 37° C.  
                 20 
                 min* 
               
            
           
           
               
               
               
               
            
               
                   
                 4° C. 
                 Hold 
               
               
                   
                   
               
            
           
         
       
     
     3) End Repair and A-Tailing 
     a) The fragmented samples in the PCR tubes were processed according to the following table 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Components 
                 volume (μL) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Fragmented DNA 
                 50 
               
               
                   
                 End Repair &amp; A-Tailing Buffer 
                 7 
               
               
                   
                 End Repair &amp; A-Tailing Enzyme Mix 
                 3 
               
               
                   
                 Total 
                 60 
               
               
                   
                   
               
            
           
         
       
     
     b) The reaction was performed according to the following procedures: 
     
       
         
           
               
               
               
             
               
                   
               
               
                 Temperature 
                 Time 
                 Heated-lid temperature 
               
               
                   
               
             
            
               
                 65° C. 
                 30 min 
                 85° C. 
               
               
                  4° C. 
                 Hold 
               
               
                   
               
            
           
         
       
     
     4) Adapter Connection 
     a) The repaired and a-tailed samples in the PCR tubes were processed according to the following table: 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Components 
                 volume (μL) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 End repair and A-tailing product 
                 60 
               
               
                   
                 Index Adapter 
                 2.5 
               
               
                   
                 PCR-grade water 
                 7.5 
               
               
                   
                 Ligation Buffer 
                 30 
               
               
                   
                 DNA Ligase 
                 10 
               
               
                   
                 Total 
                 110 
               
               
                   
                   
               
            
           
         
       
     
     b) The reaction was performed according to following procedures: 
     
       
         
           
               
               
               
             
               
                   
               
               
                 Temperature 
                 Time 
                 Heated-lid temperature 
               
               
                   
               
             
            
               
                 20° C. 
                 20 min 
                 85° C. 
               
               
                  4° C. 
                 Hold 
               
               
                   
               
            
           
         
       
     
     5) Purification of Ligation Product and Screening Fragment 
     a) The ligation product was taken out, transferred to a 1.5 mL EP tube containing 88 μL of Ampure xp Beads, mixed well, micro-centrifuged, placed at room temperature for 5 min, and then it was placed on a magnetic stand until the solution became clear to remove the supernatant; 
     b) 200 μL of freshly prepared 80% ethanol was added to the EP tube which was then rotated several times and the supernatant was removed after the solution became clear, and the tube was slightly dried at room temperature; 
     c) 50.0 μL of ddH2O was added to the tube and mixed well, micro-centrifuged, placed at room temperature for 5 minutes, and then the tube was placed on a magnetic stand until the solution became clear. Then, the supernatant was transferred to a 1.5 mL EP tube containing 50 μL of Ampure xp Beads, mixed well, micro-centrifuged, and placed at room temperature for 5 minutes, followed by putting the EP tube on the magnetic stand until the solution was clear to remove the supernatant; 
     d) 200 μL of freshly prepared 80% ethanol was added to the EP tube which was then rotated several times, and the supernatant was removed after the solution was clear; 
     e) 200 μL of freshly prepared 80% ethanol was added to the EP tube which was then several times, and the supernatant was removed after the solution was clear, and the tube was slightly dried at room temperature; 
     f) 21 μL ddH2O Elute was used for eluting, and then for later use in the next PCR step. 
     6) PCR Reaction 
     a) A new PCR tube was taken to prepare a PCR system according to the following table: 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Components 
                 volume (μL) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Adapter-ligated library 
                 20 
               
               
                   
                 KAPA HiFi HotStart ReadyMix (2X) 
                 25 
               
               
                   
                 Library Amplification Primer Mix (10X) 
                 5 
               
               
                   
                 Total 
                 50 
               
               
                   
                   
               
            
           
         
       
     
     b) Amplification was done according to the following reaction procedure: 
     98° C., 45 s→(98° C., 15 s, 60° C., 30 s, 72° C., 30 s) 8 cycles→72° C., 1 min→4° C., ∞. 
     7) Purification of PCR Product 
     a) The PCR product was taken out, transferred to a 1.5 mL EP tube containing 50 μL of Ampure xp Beads, mixed well, micro-centrifuged, placed at room temperature for 10 to 15 min, and then the tube was placed on a magnetic stand until the solution became clear to remove the supernatant; 
     b) 200 μL of freshly prepared 80% ethanol was added to the EP tube which was then rotated several times, and the supernatant was removed after the solution was clear; 
     c) 200 μL of freshly prepared 80% ethanol was added to the EP tube which was then rotated several times, and the supernatant was removed after the solution was clear, and the tube was slightly dried at room temperature; 
     d) 21 μL ddH2O was added to elute the library; 
     e) The library concentration of each sample was detected and recorded by Qubit2.0; 
     f) The library fragment size of each sample (Optional) was detected by a 2100 chip analyzer. 
     3. Hybrid Capture 
     1) Probe Hybridization 
     a) DNA library Pooling: According to the concentration measured by Qubit2.0, samples, with an amount of 100 ng for each sample, were mixed in a PCR tube, and 5 samples were subjected to one hybridization reaction; 
     b) Blocking Oligos were added to the PCR tube of the pooled library, 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Components 
                 volume (μL) 
               
               
                   
                   
               
             
            
               
                   
                 Pooled Library (Illumina) 
                 500 ng 
               
               
                   
                 Cot-1 DNA 
                  5 μg 
               
               
                   
                 xGen ® Universal Blockers-TS Mix 
                 2 
               
               
                   
                   
               
            
           
         
       
     
     c) After well mixing by repetitive pipetting, the mixed solution was dried by a vacuum filtration system (the temperature was set to 60° C.); 
     d) The following hybridization buffer was added to the dried PCR tube, mixed well by repetitive pipetting, and the tube was placed at room temperature for 5 to 10 minutes: 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Components 
                 Volume (μL) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 xGen 2X Hybridization Buffer 
                 8.5 
               
               
                   
                 xGen Hybridization Buffer Enhancer 
                 2.7 
               
               
                   
                 Nuclease-Free Water 
                 1.8 
               
               
                   
                   
               
            
           
         
       
     
     e) The PCR tube was placed on a PCR machine, incubated at 95° C. for 10 minutes to denature; 
     f) The PCR tube was taken out immediately after the denaturation, and then it was placed on a pre-cooled metal plate, and 4 μL of xGen Lockdown Probe pool (probe) was immediately added; 
     g) Hybridization was performed according to the following procedure: 
     
       
         
           
               
               
               
             
               
                   
               
               
                 Temperature 
                 Time 
                 Heated-lid temperature 
               
               
                   
               
             
            
               
                 65° C. 
                 &gt;14 h 
                 75° C. 
               
               
                   
               
            
           
         
       
     
     2) Capture Elution 
     a) Each wash Buffer was diluted in the following proportions (each one is satisfied with the amount of one capture elution): 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                   
                 volume(μl) 
                   
                 total volume 
               
               
                   
                 of Stock 
                 volume(μl) 
                 of 1X 
               
               
                 Reagent Name 
                 solution 
                 of ddH 2 O 
                 Buffer (μl) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 xGen 10X Wash Buffer I 
                 30 
                 270 
                 300 
               
               
                 xGen 10X Wash Buffer II 
                 20 
                 180 
                 200 
               
               
                 xGen 10X Wash Buffer III 
                 20 
                 180 
                 200 
               
               
                 xGen 10X Stringent Wash 
                 40 
                 360 
                 400 
               
               
                 Buffer 
               
               
                 xGen 2X Bead Wash Buffer 
                 250 
                 250 
                 500 
               
               
                   
               
            
           
         
       
     
     b) 400 μl of 1×Stringent Wash Buffer was pre-heated in a 65° C. metal warm bath; 
     c) 100 μl of 1×Wash Buffer I was aliquoted and placed in the 65° C. warm bath to preheat, and the remaining 20010 of 1×Wash Buffer I was placed at room temperature for use; 
     d) Dynabeads® M-270 Streptavidin beads were taken out from 4° C. and warmed to room temperature. After enough shaking, 100 μl of the solution was pipetted into a 1.5 ml centrifuge tube, which was then placed on a magnetic stand to remove the supernatant after the solution was clear; 
     e) The centrifuge tube was taken out from the magnetic stand, 20010 of 1×Bead Wash Buffer was added into the centrifuge tube, which was shaken for 10 s, put back on the magnetic stand after microcentrifugation, and then the supernatant was removed after the solution was clear; 
     f) The above steps were repeated once; 
     g) 100 μl of 1×Bead Wash Buffer was added for resuspending Dynabeads® M-270 Streptavidin beads, transferred to a new 20010 PCR tube, the tube was placed on the magnetic stand, the supernatant was removed for later use after the solution became clear; 
     h) The samples that were hybridized overnight (the program of the PCR instrument was maintained at 65° C. and the heated lid was kept at 75° C.) were taken out, all the liquid was transferred to the Dynabeads®M-270 Streptavidin beads washed in the previous step, mixed well by repetitive pipetting, and placed back to the PCR machine again after micro-centrifugation; 
     i) After a 12 min-reaction, the samples were taken out and mixed 3 times; 
     j) After the reaction was completed, the PCR tube was taken out, 10010 of 1×Wash Buffer I (preheated at 65° C.) was added, fully shaken for 10 s, and then the liquid was transferred to a 1.5 ml centrifuge tube, which was placed on the magnetic stand, and the supernatant was removed after the solution became clear; 
     k) The centrifuge tube was taken out from the magnetic stand, 20010 of 1×Stringent Buffer (preheated at 65° C.) was added and mixed well, and the tube was quickly put back into the 65° C. water bath, and warmed for 5 minutes; 
     l) The step k) was repeated once; 
     m) After warming in the warm bath, the tube was placed on the magnetic stand, and the supernatant was removed after the solution became clear; 
     n) The centrifuge tube was taken out from the magnetic stand, 20010 of 1×Wash Buffer I (room temperature) was added, shaken for 2 minutes, placed on the magnetic frame after microcentrifugation, and the supernatant was removed after the solution became clear; 
     o) The centrifuge tube was taken out from the magnetic stand, 20010 of 1×Wash Buffer II was added, shaken for 1 min, placed on the magnetic stand after microcentrifugation, and the supernatant was removed after the solution became clear; 
     p) The centrifuge tube was taken out from the magnetic stand, 20010 of 1×Wash Buffer III was added, shaken for 30 s, and placed on the magnetic stand after microcentrifugation, and the supernatant was removed after the solution became clear; 
     q) 20 μl of ddH2O was added to resuspend and elute Dynabeads® M-270 Streptavidin beads for PCR. 
     3) Second PCR (Post-PCR) 
     a) A new PCR tube was taken to prepare a PCR system according to the following table: 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 System Components 
                 Volume (μl) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Capture elution product from the previous step 
                 20 
               
               
                   
                 KAPA HiFi HotStart ReadyMix 
                 25 
               
               
                   
                 10 μM Illumina P5 primer 
                 2.5 
               
               
                   
                 10 μM Illumina P7 primer 
                 2.5 
               
               
                   
                 Total volume 
                 50 
               
               
                   
                   
               
            
           
         
       
     
     b) The library was amplified according to the following reaction procedure: 
     98° C., 45 s→(98° C., 15 s, 60° C., 30 s, 72° C., 30 s) 11 cycles→72° C., 1 min→4° C., ∞. 
     4) Purification for PCR Product 
     a) The PCR product was taken out, transferred to a 1.5 mL EP tube containing 750, of Ampure xp Beads, mixed well, micro-centrifuged, and placed at room temperature for 10 to 15 min, and then the EP tube was placed on the magnetic stand until the solution became clear to remove the supernatant; 
     b) 200 μL of freshly prepared 80% ethanol was added to the EP tube, rotated several times, and the supernatant was removed after the solution became clear; 
     c) 200 μL of freshly prepared 80% ethanol was added to the EP tube, rotated several times, and the supernatant was removed after the solution became clear, and the tube was dried at room temperature; 
     d) 21.0 μL of ddH2O was added to elute the library. 
     5) Quality Control for Library 
     a) The concentration of the final library was measured by Qubit 2.0; 
     b) Library fragment sizes (Optional) were detected by Agilent 2100 Bioanalyzer; 
     6) Sequencing on the machine 
     Illumina Nextseq500 was used. 
     II. Sorting. 
     The above sequencing data were processed and analyzed by bioinformatics. If TSC1 and TSC2 genes 0 were detected to be negative or there was only one-hit mutated locus, a supplementary detection was performed by the chromosomal microarray analysis and the Multiplex ligation-dependent probe amplification. When a locus was detected to be an undefined locus originated from either somatic mutation or the germline mutation, the locus can be verified by Sanger sequencing. 
     III. Performing Chromosomal Microarray Analysis and Multiplex Ligation-Dependent Probe Amplification. 
     If TSC1 and TSC2 genes were detected to be negative or there was only a one-hit mutated locus (that is, gene mutation, or fragment deletion/insertion, or copy number variations, etc. only occurred in one of the TSC1 gene and TSC2 gene), a supplementary detection was performed in the chromosome microarray analysis and Multiplex ligation-dependent probe amplification. The agents used were as follows. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Analysis 
                   
                   
                 Article 
               
               
                 method 
                 Name of Agents 
                 manufacture 
                 Number 
               
               
                   
               
             
            
               
                 CMA 
                 OncoScan ® CNV FFPE 
                 Affymetrix 
                 902695 
               
               
                   
                 Assay Kit 
               
               
                 MLPA 
                 TSC1&amp;TSC2 Probe 
                 MRC-Holland 
                 P124&amp;P046 
               
               
                   
               
            
           
         
       
     
     IV. Performing Sanger Sequencing. 
     If a locus was detected to be an undefined locus derived from either a somatic mutation or a germline mutation, specifically, the undefined locus derived from either a somatic mutation or a germline mutation was referred to a variant with a mutation frequency of about 50%. The patients leukocyte specimen (ABI 3730XL) was verified by Sanger sequencing, and it can be identified whether the patient belonged to S-LAM or TSC-LAM through the validation results. 
     If the leukocytes of the patient also had the above mutations, it can be indicated that the patient belongs to TSC-LAM; otherwise, it can be indicated that the patient belongs to S-LAM. 
     Example 2 
     The joint detection for LAM was studied with the method of Example 1. 
     1. Comparison of Single Detection Method and Joint Detection Method. 
     In this study, a total of 61 LAM patients were employed in a group, and a single detection method and the method of Example 1 were used for detection at the same time. 
     The single detection method was a method that only used NGS, a Target Sequencing based Hybridization capture. 
     The results were shown in  FIGS.  3  to  4   .  FIG.  3    showed the detection ratio of variants by using different detection methods, wherein NGS meant Target Sequencing based Hybridization capture, CMA meant chromosome microarray analysis, and MLPA meant multiplex ligation-dependent probe amplification detection. The above results showed that, SNV, INDEL, CNV, LOH and other variant types 0 can be comprehensively detected for LAM patient samples by the multi-method joint detection method, while 12.8% of the variants would be lost when only single NGS detection method was used. 
     When a single detection method was used, the overall positive detection rates for TSC1 and TSC2 genes were 72.13%, and there were 15 patients detected with 1-hit, and 29 patients with 2-hits, respectively. When the joint detection scheme was used, the overall positive detection rate was 75.41%, and 1-hit was 5 detected in 8 patients, and 2-hits were detected in 38 patients, respectively. The results were shown in  FIG.  4   . 
     The joint detection scheme not only improves the detection rate of positive mutations in LAM patients, but more importantly, the detection results are more in line with Knudson&#39;s “double hit” theory. 
     2. Research Findings. 
     When the joint detection method of Example 1 was used, 30 new mutations were found in 61 LAM patients employed in the group, as follows: 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                   
                   
                   
                 Mutation 
               
               
                 Gene HGVS(cDNA) 
                 HGVS(protein) 
                 Exon 
                 type 
               
               
                   
               
             
            
               
                 TSC2 NM_000548.4: c.2220 + 2_2220 + 9del 
                 N/A 
                 splice 
                 Deletion 
               
               
                 TSC1 NM_000368.4: c.979C &gt; T 
                 p.Pro327Ser 
                 exon10 
                 SNV 
               
               
                 TSC2 NM_000548.4: c.3059_3063del 
                 p.Leu1020Profs*146 
                 exon27 
                 Deletion 
               
               
                 TSC2 NM_000548.4: c.430A &gt; T 
                 p.Lys144* 
                 exon5 
                 SNV 
               
               
                 TSC2 NM_000548.4: c.1808C &gt; G 
                 p.Thr603Ser 
                 exon17 
                 SNV 
               
               
                 TSC2 NM_000548.4: c.1221C &gt; G 
                 p.Tyr407* 
                 exon12 
                 SNV 
               
               
                 TSC2 NM_000548.4: c.5106delC 
                 p.Ile1702Metfs*124 
                 exon40 
                 Deletion 
               
               
                 TSC2 NM_000548.4: c.781C &gt; G 
                 p.Arg261Gly 
                 exon9 
                 SNV 
               
               
                 TSC2 NM_000548.4: c.5004delT 
                 p.Phe1668Leufs*4 
                 exon39 
                 Deletion 
               
               
                 TSC2 NM_000548.4: c.1118A &gt; G 
                 p.Gln373Arg 
                 exon11 
                 SNV 
               
               
                 TSC2 NM_000548.4: c.4933_4937delinsACAATGTCTACAATGTCTACA 
                 p.Phe1645Thrfs*13 
                 exon38 
                 Insertion 
               
               
                 TSC2 NM_000548.4: c.2220 + 2_2220 + 9del 
                 N/A 
                 splice 
                 Deletion 
               
               
                 TSC2 NM_000548.4: c.3755C &gt; T 
                 p.Ser1252Leu 
                 exon31 
                 SNV 
               
               
                 TSC2 NM_000548.4: c.151delG 
                 p.Glu51Asnfs*10 
                 exon3 
                 Deletion 
               
               
                 TSC2 NM_000548.4: c.l714delC 
                 p.Gln572Argfs*126 
                 exon16 
                 Deletion 
               
               
                 TSC2 NM_000548.4: c.460_461del 
                 p.Thr154Leufs*8 
                 exon5 
                 Deletion 
               
               
                 TSC2 NM_000548.4: c.2098-1G &gt; C 
                 N/A 
                 splice 
                 SNV 
               
               
                 TSC2 NM_000548.4: c.25T &gt; C 
                 p.Ser9Pro 
                 exon2 
                 SNV 
               
               
                 TSC2 NM_000548.4: c.400G &gt; T 
                 p.Glu134* 
                 exon5 
                 SNV 
               
               
                 TSC2 NM_000548.4: c.219_223del 
                 p.Phe73Leufs*51 
                 exon3 
                 Deletion 
               
               
                 TSC2 NM_000548.4: c.4682delT 
                 p.Ile1561Thrfs*15 
                 exon37 
                 Deletion 
               
               
                 TSC2 NM_000548.4: c.3500_3507del 
                 p.Glu1167Valfs*64 
                 exon30 
                 Deletion 
               
               
                 TSC2 NM_000548.4: c.788delT 
                 p.Leu263Profs*30 
                 exon9 
                 Deletion 
               
               
                 TSC2 NM_000548.4: c.2267delG 
                 p.Gly756Alafs*15 
                 exon21 
                 Deletion 
               
               
                 TSC2 NM_000548.4: c.3132delG 
                 p.Arg1044Serfs*9 
                 exon28 
                 Deletion 
               
               
                 TSC2 NM_000548.4: c.1829T &gt; A 
                 p.Ile610Asn 
                 exon17 
                 SNV 
               
               
                 TSC2 NM_000548.4: c.5116_5119dupCGCA 
                 p.Asn1707Thrfs*23 
                 exon40 
                 Insertion 
               
               
                 TSC2 NM_000548.4: c.2153_2154del 
                 p.Arg718Leufs*2 
                 exon20 
                 Deletion 
               
               
                 TSC2 NM_000548.4: c.2018C &gt; G 
                 p.Ala673Gly 
                 exon19 
                 SNV 
               
               
                 TSC1 NM_000368.4: c.1450A &gt; G 
                 p.Arg484Gly 
                 exon15 
                 SNV 
               
               
                   
               
            
           
         
       
     
     These new mutations will greatly enrich the genetic database of LAM, a rare disease, and have important clinical value in promoting the research progress and treatment guidance of the disease. 
     Example 3 
     An example of the joint detection for LAM was conducted with the method of Example 1. 
     I. Sample Source 
     The samples were obtained from fixed tissue samples from the Department of Respiratory Medicine of a tertiary hospital in Guangzhou, and they were clinically diagnosed as S-LAM. 
     II. Detection Methods and Results 
     1. Target Sequencing Based Hybridization Capture (Target Capture Sequencing, a NGS) was Performed with Probes 
     The sequencing results showed that the sample had a single mutated locus, which is TSC2: NM_000548.4:c.3412C&gt;T (p.Arg1138*), and the variant frequency was 50.9%. 
     2. Chromosome Microarray Analysis and Multiplex Ligation-Dependent Probe Amplification 
     According to the method of Example 1, a supplementary detection was performed by the chromosome microarray analysis. The results showed that there was a loss of heterozygosity (LOH) variation in the TSC2 gene of the patient: arr&lt;GRCh37&gt;16p13.3p11.2(83886_30809063)x2 mos hmz. The size of the variant was 30.73 Mb, and its LOH fragment was shown in  FIG.  5   ; the detection result was negative when the multiplex ligation probe amplification was used. 
     3. Sanger Sequencing 
     Since the variant frequency of the locus detected by NGS was 50.9%, it was impossible to determine whether the locus was derived from a somatic mutation or a germline mutation. Therefore, primers were designed for such locus and the leukocyte samples of the patient were sequenced. The verification results showed that there was no above-mentioned mutation in leukocytes of the patient, and therefore the mutation was a somatic mutation, that is, sporadic LAM (S-LAM). 
     III. Conclusion 
     The detection results of the patient showed that a premature stop codon was created at codon 1138 of the TSC2 gene, with a mutation frequency of 50.9%. It was determined by the Sanger method that the mutation did not exist in leukocytes and was a somatic mutation, leading to normal protein disfunction. In the past, this mutation had been reported for more than 50 times in individuals with LAM, and it was a pathogenic mutation. The detection results of the supplementary detection showed that the patient also had the LOH phenomenon of the TSC2 gene. Studies have shown that LOH can lead to the inactivation of the suppressor genes, thereby affecting the occurrence and development of the tumor. The pathogenesis of the patient can be completely known through this joint detection scheme, and according to the results, sporadic LAM is diagnosed, without hereditary. 
     Although the present invention has been disclosed in the form of preferred embodiments and variations thereon, it will be understood that numerous additional modifications and variations could be made thereto without departing from the scope of the invention. 
     For the sake of clarity, it is to be understood that the use of ‘a’ or ‘an’ throughout this application does not exclude a plurality, and ‘comprising’ does not exclude other steps or elements.