Abstract:
The present invention describes a method of measurement of pyrosequencing accuracy by directly calculating sequence errors from FLX Titanium pyrosequencing using mock community, according to the present invention, sequencing errors from FLX Titanium pyrosequencing in terms of microbial diversity and classification can be measured, resulting in possible effects of filtering.

Description:
TECHNICAL FIELD 
       [0001]    The present invention is related to mock community for measuring pyrosequencing accuracy and a method of measuring pyrosequencing accuracy using the same. More specifically, the present invention is related to an accuracy assessment of pyrosequencing by directly measuring the errors of FLX Titanium pyrosequencing using the amplicons of mock community. 
       BACKGROUND ART 
       [0002]    Massively parallel pyrosequencing system offers a high-throughput data of microbial diversity in environmental samples. It is now possible to generate hundreds of thousands of short (100-200 nucleotide) DNA sequence reads in a few hours without conventional cloning and sanger sequencing method. 
         [0003]    The 454/Roche Genome Sequencers are called pyrosequencers because their sequencing technology is based on the detection of pyrophosphates released during DNA synthesis. For analysis of multiple libraries, the currently available 454/Roche pyrosequencers can accommodate a certain number of independent samples, which have to be physically separated using manifolds on the sequencing medium. These separation manifolds occlude wells on the sequencing plate from accommodating bead-bound DNA template molecules, and thus restrict the number of output sequences. 
         [0004]    To overcome these limitations, barcodes or unique DNA sequence identifiers have been used in several experimental contexts. The barcodes allows independent samples to be pooled together before sequencing. And the barcodes as an identifier or type specifier help to separate barcoded samples from pyrosequenced results in subsequent bioinformatics. 
         [0005]    In addition, adapters which consist of forward and reverse sequences were ligated on PCR product including barcode and attach to beads on picoplate of 454 pyrosequencer for determination of sequencing direction. 
         [0006]    It was found a systematic error in metagenomes generated by 454-based pyrosequencing that leads to an overestimation of microbial diversity. In other words, an intrinsic artifact of the 454 pyrosequencing technique leads to the artificial amplification more than 15% of the original DNA sequencing templates. It has been suggested that multiple reading from a single template occur when amplified DNA attaches to empty beads during emulsion PCR. 
         [0007]    The analysis of 16S rRNA genes has announced an essential tool to evaluate microbial populations in diverse environment such as soil, ocean and human body for microbial ecologist. 
         [0008]    The high-sequence conservation of 16S rRNA genes allows to identify or/and classify bacteria in various phylogenetic levels. Therefore, bacterial diversity from environments were commonly assessed with 16S rRNA genes (16S). 
         [0009]    Bacterial diversity can be influenced by sample preparation, primer selection, and chimeric formation from PCR products. Chimeras are generated by production of hybrid sequences between multiple parent sequences. Chimeras were found to reproducibly form among independent amplifications and contributed to false perceptions of sample diversity and the false identification of novel taxa. 
         [0010]    There are few reports on pyrosequencing such as ‘Droplet-based pyrosequencing’(US Patent Application No. 20100291578), ‘Pyrosequencing Methods and Related Compositions’(US Patent Application No. 20090325154), and ‘Methods, Primers and Kits for Quantitative Detection of JAK2 V617F Mutants Using Pyrosequencing’ (WO patent Application No. 2008060090). However, these reports do not mention about mock community for accuracy assessment of pyrosequencing. In addition, there is another previous report on a method to improve accuracy and quality of pyrosequencing (Accuracy and quality of massively parallel DNA pyrosequencing, 2007 Huse et al., licensee BioMed Central Ltd.), it does not yet describe mock community for measuring pyrosequencing accuracy. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0011]      FIG. 1  shows a graph illustrating the raw sequences of region 1, 2, and 6 to 8. 
           [0012]      FIG. 2  shows a graph illustrating the initial process in pyrosequencing pipeline of RDP (Ribosomal Database Project). 
           [0013]      FIG. 3  shows a graph illustrating the error analysis by the dynamic programming. 
           [0014]      FIG. 4  shows a graph illustrating the overall error rate per read of 16S rRNA, bphA, and nifH. 
           [0015]      FIG. 5  shows a graph illustrating the substitution cumulative curve of 16S rRNA, bphA, and nifH. 
           [0016]      FIG. 6  shows a graph illustrating the error distribution of 16S rRNA. 
           [0017]      FIG. 7  shows a graph illustrating the error distribution of bphA. 
           [0018]      FIG. 8  shows a graph illustrating the error distribution of nifH. 
           [0019]      FIG. 9  shows strains, genome size, and target genes of mock community used in the present invention. 
           [0020]      FIG. 10  shows the PCR primer sequences without adapter used. 
           [0021]      FIG. 11  shows the PCR primer sequences with adapter used. 
           [0022]      FIGS. 12   a  to  12   f  show the DNA concentration and volume of PCR products gained from the present invention. 
       
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT 
     Technical Problem 
       [0023]    The aim of the present invention is to provide mock community for measuring pyrosequencing accuracy by directly measuring the sequencing error rate of the technology through comparisons between sequences of mock community generated from the invention and reference sequences and is to provide a method of measurement of pyrosequencing accuracy using the mock community. 
       Technical Solution 
       [0024]    In order to achieve the above mentioned aim to measure sequence errors from FLX Titanium pyrosequencing using metagenomic amplicons, the present invention provides a process of calculating sequence errors from pyrosequencing, a process of revealing parameters (primer, barcode, adapter) to influence the errors, a process of repeating artificial sequence, a process of revealing primer bias with regard to mismatch, barcode or adapter, and a process of determining chimeric extent and scope from mock community. 
         [0025]    The present invention provides mock community for measuring pyrosequencing accuracy characterized by comprising Rhodospillum rubrurn ATCC 11170 , Burkholderia vietarnensis  G4 , Burkholderia xenovorans  LB400 , Desulfitobacteruim hafniense  DCB-2, Nostoc. PCC 7120 , Polaromonas napthalenivorans  CJ2 , Rhodococcus  sp. RHA 1 , Pseudomonas putida  F1 , Neisseria sicca  ATCC 29256 , Ochrobactrum anthropi  ATCC 49188 , Chromobacterium violaceum  ATCC 12472,  Pseudomonas pickettii  PKO1 , Sphingobium yanoikuyae  B1 , Escherichia Coli  K-12 sub W3110 , Bacillus cents  ATCC 14579 , Corynebacterium glutamin  ATCC 13032 , Staphylococcus epidemidis  ATCC 12228 , Xanthomonas campestries  py. ATCC 33913 , Roseobacter denitrifican  Och 114, and  Rhodobacter sphaeroides  KD 131. 
         [0026]    In addition, the present invention provides a method for measuring pyrosequencing accuracy by comparing between sequences gained from pyrosequencing using above mock community and reference, i.e. “standard”, sequences generated from public database. To select best the standard sequences from mock community, the inventors determined the standard sequences using two methods; selection of three genes from the genome databases in NCBI and probe match using the specific primer sets with the genome databases in NCBI. All sequences were validated by HMM model of each gene to remove the unmatched sequences. 
       Advantageous Effects 
       [0027]    As described above, the present invention has effects on calculating accurate microbial diversity by providing mock community to measure directly pyrosequencing accuracy for reducing overestimated microbial diversity. 
       Best Mode for the Invention 
       [0028]    Hereinafter, the present invention will be described by the following examples in more detail. However, the purpose of these examples is only to illustrate the present invention, but not to limit the scope of the invention thereto in any way. 
         [0029]    Experimental materials used in the present invention are shown in Table 1. 
         [0000]    
       
         
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Material/Equipment 
                 Vendor 
                 Catalog number 
               
               
                   
               
             
             
               
                 AccuPrime ™ Taq DNA 
                 Invitrogen 
                 12346-086 
               
               
                 Plymerase High Fidelity 
               
               
                 AccuPrime ™ Pfx DNA Plymerase 
                 Invitrogen 
                 12344-024 
               
               
                 Forward and Reverse Primers 
                 Bioneer (Korea) 
                 Custom order 
               
               
                 premixed 
               
               
                 DNAse/RNAse free water 
               
               
                 Thermo Cycler 
                 Biorad 
                 C-1000 
               
               
                 Vortex 
               
               
                 Pipettes 
                 Eppendorf 
                 Custom order 
               
               
                 MinElute PCR Purification Kit 
                 Qiagen 
                 28004 
               
               
                 QIAquick PCR Purification Kit 
                 Qiagen 
                 28106 
               
               
                 Nanodrop Spectrophotometer 
                 Thermo Scientific 
                 ND-1000 
               
               
                 PowerSoil DNA Isolation Kit 
                 MoBio 
                 12888 
               
               
                 MinElute PCR Purification Kit 
               
               
                 Manual 
               
               
                   
               
             
          
         
       
     
         [0030]    Polymerase Chain Reaction (PCR) was performed with AccuPrime™ Taq DNA Polymerase High Fidelity or AccuPrime™ Pfx DNA Polymerase. The present invention tested nifH, bphA, and 16S rRNA gene(27F/518R) as functional target genes. 
       Example 1 
     Primer Setup for PCR 
       [0031]    As shown below, two kinds of primer sets are designed for PCR process and the primer sequences used are listed in  FIG. 10  and  FIG. 11 . 
         [0000]    
       
                 
         
             
             
         
       
     
         [0032]    Table 2 shows the sequences of specific primers of the target genes used in the present invention. 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Target 
                   
                   
                   
                   
               
               
                 Gene 
                 Primer name 
                 Sequences (5&#39; → 3&#39;) 
                 Reference 
                 Size 
               
               
                   
               
             
             
               
                 nifH 
                 Poly Forward 
                 TGCGAYCCSAARGCBGACTC 
                 Poly et al. Res. 
                 360 bps 
               
               
                   
                 Poly Reverse 
                 ATSGCCATCATYTCRCCGGA 
                 Microbiol. 2001 
                   
               
               
                   
               
               
                 bph4 
                 BPHD-f3 
                 AACTGGAARTTYGCNGCVGA 
                 Shoko et al. ISME J. 2009 
                 542 bps 
               
               
                   
                 BPHD-r1 
                 ACCCAGTTYTCNCCRTCGTC 
                   
                   
               
               
                   
               
               
                 nirK 
                 F1aCu 
                 ATCATGGTSCTGCCGCG 
                 Michotey et al. Appl. 
                 472 bps 
               
               
                   
                 R3Cu-GC 
                 GCCTCGATCAGRTTGTGGTT 
                 Environ. Microbiol. 2000 
                   
               
               
                   
               
               
                 16S rRNA 
                 27F 
                 GAGTTTGATCMTGGCTCAG 
                   
                 492 bps 
               
               
                   
                 518R 
                 WTTACCGCGGCTGCTGG 
               
               
                   
               
             
          
         
       
     
       Example 2 
     DNA Template Preparation 
       [0033]    The inventor used 20 bacterial strains of mock community for template preparation as shown on  FIG. 9 . Genomic DNA isolated from the bacterial strains was quantified by nanodrop spectrophotometers and the mock community was prepared by mixing equal amount of each genomic DNA (Table 3). 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE 3 
               
             
             
               
                   
                   
               
               
                   
                 (gram)            
                 #of 
                   
                 Mismatch 
                 (ng/ul)/ 
               
             
          
           
               
                 Gene 
                 
                           
                 
                 Prepared Strains (20 strains) 
                 16S 
                 GC % 
                 For 
                 Rev 
                 (260/280) 
               
               
                   
               
               
                 nifH 
                 (−)Cyano* 
                   Anabaena  sp. PCC 9109 
                 ? 
                   
                 1 
                 — 
                 157/2.00 
               
               
                 ** 
                 (−)Beta 
                   Burkholderia vietnamensis  G4 
                 6 
                 66 
                 0 
                 1 
                 158/1.91 
               
               
                   
                 (−)Beta 
                   Burkholderia xenovorans  LB400 
                 6 
                 62 
                 0 
                 0 
                 260/1.86 
               
               
                   
                 (−)Beta 
                   Polaromonas naphthalenivorans  CJ2 
                 2 
                 62 
                 0 
                 0 
                  61/1.85 
               
               
                   
                 (+)Firmicute 
                   Desulfitobacterium hafniense  DCB-2 
                 5 
                 47 
                 1 
                 2 
                  23/1.88 
               
               
                   
                 (−)Alpha 
                   Rhodospirillum rubrum  ATCC 11170 
                 4 
                 65 
                 1 
                 2 
                 128/2.07 
               
               
                 bphA 
                 (−)Beta 
                   Burkholderia xenovorans  LB400 
                 6 
                 62 
                 0 
                 0 
                 260/1.86 
               
               
                   
                 (−)Beta 
                   Polaromonas naphthalenivorans  CJ2 
                 2 
                 62 
                 1 
                 1 
                  61/1.85 
               
               
                 * 
                 (+)Actino 
                   Rhodococcus  sp. RHA1 
                 4 
                 67 
                 0 
                 0 
                 125/1.91 
               
               
                   
                 (−)Gamma 
                   Pseudomonas putida  F1 
                 6 
                 61 
                 0 
                 0 
                 158/2.02 
               
               
                 nirK 
                 (−)Beta 
                   Neisseria sicca  ATCC 29256 
                 7 
                 50 
                 — 
                 — 
                  51/1.86 
               
               
                   
                 (−)Alpha 
                   Ochrobactrum anthropi  ATCC 49188 
                 4 
                 56 
                 0 
                 0 
                 110/1.90 
               
               
                   
                 (−)Beta 
                   Chromobacterium violaceum  ATCC 12472 
                 8 
                 64 
                 — 
                 — 
                  58/1.78 
               
               
                   
                 (−)Beta 
                   Polaromonas naphthalenivorans  CJ2 
                 2 
                 62 
                 — 
                 — 
                  61/1.85 
               
               
                 16S 
                 (−)Gamma 
                   Pseudomonas pickettii  PKO1 
                 ? 
                   
                   
                   
                 272/1.9  
               
               
                 * 
                 (−)Alpha 
                   Sphingomonas yanoikuyae  B1 
                 ? 
                   
                   
                   
                  23/1.95 
               
               
                   
                 (−)Gamma 
                   Escherichia Coli  K-12 sub W3110 
                 7 
                 50 
                   
                   
                  56/1.81 
               
               
                   
                 (+)Firmicute 
                   Bacillus cereus  ATCC 14579 
                 13 
                 35 
                   
                   
                  17/1.77 
               
               
                   
                 (+)Actino 
                   Corynebacterium glutamine  ATCC 13032 
                 5 
                 53 
                   
                   
                 118/1.9  
               
               
                 * 
                 (+)Firmicute 
                   Staphylococcus epidermidis  ATCC 12228 
                 5 
                 32 
                   
                   
                  77/1.84 
               
               
                   
                 (−)Gamma 
                   Xanthomonas campestris  pv.  campestris  str. ATCC 
                 2 
                 65 
                   
                   
                  42/1.81 
               
               
                   
                   
                 33913 
               
               
                   
                 (−)Alpha 
                   Roseobacter denitrificans  OCh 114 
                 1 
                 58 
                   
                   
                  85/1.91 
               
               
                   
                 (−)Alpha 
                   Rhodobacter sphaeroides  KD131 
                 4 
                 69 
                   
                   
                 100/1.92 
               
               
                   
               
               
                 ?: not Genome sequencing 
               
               
                             indicates data missing or illegible when filed 
               
             
          
         
       
     
         [0034]    Table 4 represents samples used for natural community in the present invention. 
         [0000]    
       
         
               
               
               
             
           
               
                 TABLE 4 
               
               
                   
               
               
                 Sample Name 
                 Donation 
                 Note 
               
               
                   
               
             
             
               
                 Soil 1 
                 MSU 
                 Performed Illumina V4 16S 
               
               
                 River sediment 
                 Yonsei University 
                 River sediment (Wonju-stream) 
               
               
                 Tidal Flat 
                 Yonsei University 
                 Tidal Flat (Kangwha island) 
               
               
                 BioCathode 
                 Yonsei University 
                 Operation 1 month in anaerobic 
               
               
                   
                   
                 condition by Tuan 
               
               
                   
               
             
          
         
       
     
       Example 3 
     Preparation of 8 Regions for Pyrosequencing 
       [0035]    For DNA pyrosequencing, 8 plates shown in Table 5 were prepared with mock community, natural community, adapter, barcode, linker, and specific primer of target genes as mentioned above. Adapter primers consisted of forward annealing sequence (CGTATCGCCTCCCTCGCGCCATCAG) and reverse annealing sequence (CTATGCGCCTTGCCAGCCCGCTCAG) as provided in Roche 454 protocols ( FIG. 11 ). 
         [0000]    
       
         
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
             
               
             
               
             
           
               
                 TABLE 5 
               
               
                   
               
             
             
               
                 #1 Plate (region) 
               
             
          
           
               
                 DNA template 
                 Mixed same ratio of gDNA from each mock and 4 
               
               
                   
                 natural communities 
               
               
                 Primer composition 
                 Adapter + Barcode + Linker + Specific primer 
               
               
                 Target gene 
                 nifH, bphA, nirK, 16S 
               
             
          
           
               
                 #2 Plate 
               
             
          
           
               
                 DNA template 
                 Mixed same ratio of gDNA from each mock and 4 
               
               
                   
                 natural communities 
               
               
                 Primer composition 
                 Barcode + Linker + Specific primer 
               
               
                 Target gene 
                 nifH, bphA, nirK, 16S 
               
             
          
           
               
                 #3 Plate 
               
             
          
           
               
                 DNA template 
                 Mixed 5 different ratio of gDNA from mock 
               
               
                   
                 community 
               
               
                 Primer composition 
                 Adapter + Barcode + Linker + Specific primer 
               
               
                 Target gene 
                 nifH, bphA, nirK, 16S 
               
             
          
           
               
                 #4 Plate 
               
             
          
           
               
                 DNA template 
                 Mixed 5 different ratio of gDNA from mock 
               
               
                   
                 community 
               
               
                 Primer composition 
                 Barcode + Linker + Specific primer 
               
               
                 Target gene 
                 nifH, bphA, nirK, 16S 
               
             
          
           
               
                 #5 Plate 
               
             
          
           
               
                 DNA template 
                 Individual gDNA from the strains with those 
               
               
                   
                 particular genes 
               
               
                 Primer composition 
                 Adapter + Barcode + Linker + Specific primer 
               
               
                 Target gene 
                 nifH, bphA, nirK, 16S 
               
             
          
           
               
                 #6 Plate 
               
             
          
           
               
                 DNA template 
                 Mixed 5 different ratio of gDNA from mock 
               
               
                   
                 community 
               
               
                 Primer composition 
                 Adapter + Switched Barcode + Linker + Specific 
               
               
                   
                 primer 
               
               
                 Target gene 
                 nifH, bphA 
               
               
                 DNA template 
                 Mixed 6 different ratio of gDNA from mock and 
               
               
                   
                 natural community 
               
               
                 Primer composition 
                 Adapter + Barcode + Linker + Specific primer 
               
               
                 Target gene 
                 16S rRNA 
               
             
          
           
               
                 #7 Plate 
               
             
          
           
               
                 Replicate of #1 
               
             
          
           
               
                 #8 Plate 
               
             
          
           
               
                 Replicate of #2 
               
               
                   
               
             
          
         
       
     
         [0036]    The ratio of mock to natural community of plate #6 is shown in Table 6. 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
                 TABLE 6 
               
               
                   
                   
               
             
             
               
                   
                 Natural 
                 Ratio 1 
                 Ratio 2 
                 Ratio 3 
               
               
                   
                 Soil 1 
                 0.3:9 
                 1:9 
                 3:9 
               
               
                   
                   
                 Ratio 4 
                 Ratio 5 
                 Ratio 6 
               
               
                   
                 Biocathod 
                 0.3:9 
                 1:9 
                 3:9 
               
               
                   
                   
               
             
          
         
       
     
         [0037]    Table 7 represents barcoded primer set of plate #1, #2, #7, and #8. 
         [0000]    
       
         
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
               
                   
               
               
                 Target gene 
                 Mock Community 
                 Soil 1 
                 Soil 2 
                 Tidal flat 
                 BioCathode 
               
               
                   
               
             
             
               
                 nifH 
                 BC1 
                 BC2 
                 BC3 
                 BC4 
                 BC5 
               
               
                 bphA 
                 BC6 
                 BC7 
                 BC8 
                 BC9 
                 BC10 
               
               
                 nirK 
                 BC11 
                 BC12 
                 BC13 
                 BC14 
                 BC15 
               
               
                 16S rRNA 
                 BC16 
                 BC17 
                 BC18 
                 BC19 
                 BC20 
               
               
                   
               
             
          
         
       
     
         [0038]    Barcoded primer set of plate #3 and #4 is represented in Table 8. 
         [0000]    
       
         
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
               
                   
               
               
                 Target gene 
                 Ratio 1 
                 Ratio 2 
                 Ratio 3 
                 Ratio 4 
                 Ratio 5 
               
               
                   
               
             
             
               
                 nifH 
                 BC1 
                 BC2 
                 BC3 
                 BC4 
                 BC5 
               
               
                 bphA 
                 BC6 
                 BC7 
                 BC8 
                 BC9 
                 BC10 
               
               
                 nirK 
                 BC11 
                 BC12 
                 BC13 
                 BC14 
                 BC15 
               
               
                 16S rRNA 
                 BC16 
                 BC17 
                 BC18 
                 BC19 
                 BC20 
               
               
                   
               
             
          
         
       
     
         [0039]    Barcoded primer set of plate #5 is represented in Table 9. 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
             
           
               
                 TABLE 9 
               
               
                   
               
               
                 Target gene 
                 Strain 1 
                 Strain 2 
                 Strain 3 
                 Strain 4 
                 Strain 5 
               
               
                   
               
             
             
               
                 nifH 
                 BC1 
                 BC2 
                 BC3 
                 BC4 
                 BC5 
               
               
                 bphA 
                 BC6 
                 BC7 
                 BC8 
                 BC9 
                 BC10 
               
               
                 nirK 
                 BC11 
                 BC12 
                 BC13 
                 BC14 
                 BC15 
               
             
          
           
               
                 16S rRNA 
                 5 Representative strains will be amplified with BC16-BC20 
               
               
                   
               
             
          
         
       
     
         [0040]    Table 10 represents barcoded primer set for plate #6. 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
             
           
               
                 TABLE 10 
               
               
                   
               
             
             
               
                 Target gene 
                 Ratio 1 
                 Ratio 2 
                 Ratio 3 
                 Ratio 4 
                 Ratio 5 
               
               
                 nifH 
                 BC6 
                 BC7 
                 BC8 
                 BC9 
                 BC10 
               
               
                 bphA 
                 BC1 
                 BC2 
                 BC3 
                 BC4 
                 BC5 
               
               
                 Target gene 
                 Ratio 1 
                 Ratio 2 
                 Ratio 3 
                 Ratio 4 
                 Ratio 5 
               
               
                 16S rRNA 
                 BC16 
                 BC17 
                 BC18 
                 BC19 
                 BC20 
               
               
                   
                 Ratio 6 
               
             
          
           
               
                   
                 BC21 
                 More depth sequencing than others 
               
               
                   
                   
               
             
          
         
       
     
         [0041]    The barcoded primers used from Table 7 to Table 10 are 8 nucleotides long and the sequences are listed in Table 11. 
         [0000]    
       
         
               
               
               
               
               
               
             
           
               
                 TABLE 11 
               
               
                   
               
               
                 Barcode 
                 Sequence 
                 Barcode 
                 Sequence 
                 Barcode 
                 Sequence 
               
               
                   
               
             
             
               
                 BC1 
                 ACACGTCA 
                 BC8 
                 CTAGAGCT 
                 BC15 
                 TGCAGATC 
               
               
                   
               
               
                 BC2 
                 AGCTACGT 
                 BC9 
                 CTGTCAGA 
                 BC16 
                 ACACGACT 
               
               
                   
               
               
                 BC3 
                 AGCTGTAC 
                 BC10 
                 CTGAGTCA 
                 BC17 
                 ACAGTCAC 
               
               
                   
               
               
                 BC4 
                 ATATGCGC 
                 BC11 
                 TAGCTAGC 
                 BC18 
                 AGACGTCT 
               
               
                   
               
               
                 BC5 
                 ACACACTG 
                 BC12 
                 TCAGACTG 
                 BC19 
                 AGTCACTG 
               
               
                   
               
               
                 BC6 
                 CACTACAG 
                 BC13 
                 TCGACATG 
                 BC20 
                 ATCGTACG 
               
               
                   
               
               
                 BC7 
                 CATGACGT 
                 BC14 
                 TGAGTCAC 
                 BC21 
                 CACATGTG 
               
               
                   
               
             
          
         
       
     
         [0042]    Meanwhile,  FIG. 1  shows a graph illustrating raw data sequences obtained from the plate #1, #2, and #6 to #8. 
       Example 4 
     Master Mix Preparation for PCR 
       [0043]    Master mix contained 2.5 ul of 10×AccuPrime PCR Buffer II, 0.2 ul of Accuprime Taq Hifi, Mixed Primer, and 60 ng of genomic DNA template with RNAse/DNAse free water in a 25-ul total volumn. 
       Example 5 
     Polymerase Chain Reaction 
       [0044]    Reaction mixture obtained from the above was spin down briefly at 2000 rpm in a centrifuge, followed by PCR as shown in Table 12. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 12 
               
               
                   
               
               
                 Target 
                   
                   
                   
                 Target 
                   
                   
                   
               
               
                 gene 
                 Temp. 
                 Time 
                 Cycle 
                 gene 
                 Temp. 
                 Time 
                 Cycle 
               
               
                   
               
             
             
               
                 nifH 
                 94° C. 
                 1 min 
                   
                 bphA 
                 95° C. 
                  3 min 
                   
               
               
                   
                 94° C. 
                 1 min 
                 30 Cycle 
                   
                 95° C. 
                 45 sec 
                 30 Cycle 
               
               
                   
                 55° C. 
                 1 min 
                   
                   
                 60° C. 
                 45 sec 
               
               
                   
                 72° C. 
                 2 min 
                   
                   
                 72° C. 
                 40 sec 
               
               
                   
                 72° C. 
                 5 min 
                   
                   
                 72° C. 
                  4 min 
               
               
                   
                  4° C. 
                 forever 
                   
                   
                  4° C. 
                 forever 
               
               
                 nirK 
                 95° C. 
                 1 min 
                   
                 16S 
                 94° C. 
                  3 min 
               
               
                   
                 94° C. 
                 1 min 
                 30 Cycle 
                   
                 94° C. 
                 30 sec 
                 30 Cycle 
               
               
                   
                 51° C. 
                 1 min 
                   
                   
                 55° C. 
                 30 sec 
               
               
                   
                 72° C. 
                 1 min 
                   
                   
                 72° C. 
                  1 min 
               
               
                   
                 72° C. 
                 10 min  
                   
                   
                 72° C. 
                  5 min 
               
               
                   
                  4° C. 
                 forever 
                   
                   
                  4° C. 
                 forever 
               
               
                   
               
             
          
         
       
     
         [0045]    PCR products were purified using QIAquick PCR Purification Kit. 
       Example 6 
     Gel Analysis of PCR Products 
       [0046]    1 ul of PCR product was mixed with 1 ul of 10× loading dye and 8 ul of distilled water on parafilm by pipetting. 1% agarose gel with 1×TAE was prepared by SaFeview. After loading each PCR product into the gel, electrophoresis was run approximately 1 hour at 100V, followed by visualization on gel-doc and analysis. 
       Example 7 
     Quantification of PCR Products 
       [0047]    Quantification of PCR products was determined by nanodrop spectrophotometer according to the manufacture&#39;s instruction and the results obtained are shown as concentration in  FIGS. 12   a  to  12   f.    
       Example 8 
     PCR Pooling 
       [0048]    From the results of nanodrop spectrophotometer above, pooling amounts were calculated by pooling Calculator.xls or formula as follows: 
         [0000]      Amount (ul) of each sample=((vol/2)*(min))/sampleconc 
         [0049]    Where Vol is the total volume of each sample, Min is the concentration in ng/ul of the sample with the lowest concentration, and Sampleconc is the concentration in ng/ul of target sample. 
         [0050]    Samples were pooled by 1 ul of a minimum transfer volume. Sample was diluted if less than 1 ul was required. 
         [0051]    To purify the pool gained from the above, Qiagen minElute column was used according to the manufacturer&#39;s protocol. To increase the purity of samples, additional purification step was added and the results obtained were ≧1.8 at A 260/280 . 
       Example 9 
     Pyrosequencing 
       [0052]    Using the Genome Sequencing FLX Titanium pyrosequencing (Roche), according to the manufacture&#39;s instruction, sequencing was carried out at Macrogen Incorporation, Korea. 
       Example 10 
     Standard Sequencing Collection 
       [0053]    In order to collect the optimal DNA sequences from the mock community, the inventor selected standard sequences in two ways. Three target genes including nifH, bphA, and 16S rRNA Were selected from the NCBI genome database and their specific primers were used to identify probe match. To remove mismatch sequences, all of the DNA sequences were aligned to each target gene based on the Hidden Markov Model (HMM). 
       Example 11 
     Initial Processing 
       [0054]    In order to acquire good quality sequences, the sequences which showed ≧2 for forward primer mismatch or ≧0 for average exponential quality score were filtered through the RDP Pyro Initial Process tool [Cole, etc., 2009]. The bases prior to the forward primer were cut off from reads. Because of the long reads, the reverse primer was not validated for 16S and bphA. In the case of nifH reads, the reverse primer should be a perfect match and the reverse primer was cut off. After ambiguous bases or trimming process, read length less than 300 bps were cut off as well ( FIG. 2 ). 
       Example 12 
     Contaminated Sequence Detection 
       [0055]    Reads that passed the initial quality process were analyzed by specifically designed RDP tool ContaminateBot. Using RDP Seqmatch tool, reads were compared to the high-quality RDP public dataset and the mock community sequences. Reads that the difference of S_ab score was more than 0.2 and the sequences was closer to the RDP public dataset than the mock community were considered to be contaminated sequences, thus removed. 
       Example 13 
     Chimera Detection 
       [0056]    To discriminate potential chimera, reads that error rate was more than 3% were analyzed with specifically designed RDP tool ChimeraBot. This tool made partial alignments from 5′- or 3′-sequences relative to standard mock community sequences per individual read. Through the forward and the reverse alignment, the present invention acquired information such as maximum score of all possible combinations, mock community parents, and alignment breakpoint. As the total score was at least 10% higher than the score of optimal single-parent alignment and individual partial alignment was 95% identity, the reads were assumed to be potential chimera, therefore removed from the error calculation. 
       Example 14 
     Error Analysis 
       [0057]    The non-contaminant reads that passed the initial quality process were compared to the standard sequences of the mock community using RDP mock community analysis tool (http://pyro.cme.msu.edu/). Individual read calculated alignment between the standard sequences of the mock community and gained standard sequences with high similarity relative to the optimal alignment. Based on the optimal alignment, indel and mismatch errors were calculated ( FIG. 3 ). 
         [0058]    Overall error rates were measured by dividing the total results of the indel and mismatch to the sequence results of pyrosequencing of each gene (barcode+adapter/barcode/direction), and the difference for sequencing direction was identified by mismatch cumulative curve ( FIG. 5 ). 
         [0059]    In order to understand the distribution on the error number of each gene, the cumulative error distribution was illustrated in  FIG. 6  to  FIG. 8 .