bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 3519: High-throughput unbiased microwell-based single-cell CNV detection reveals tumor clonal subtypes in hepatocellular carcinoma
Authors: Wu Liang , Wang Yuzhou , Jiang Miaomiao , Liu Shiping 
Published: August 2019

Abstract
Single-cell genomics is critical for understanding cellular heterogeneity in cancer, but existing DNA amplification and library preparation methods are expensive and introduce coverage bias during sample pre-amplification. Based on a massively parallel 5184-microwell chip, we developed a high-throughput unbiased single-cell DNA library preparation approach up to 1800 cells for copy number variation (CNV) detection, which provides a comprehensive, scalable solution for revealing genome heterogeneity. Compared with existing methods, it is more efficient and cost-effective to construct a barcoded-single-cell library without pre-amplification in 2 days. Besides, it has significantly lower Median Absolute Pairwise Difference (MAPD:0.17±0.004) values which are used to evaluate the amplification biases and noises, than using MDA (multiple displacement amplification) method (MAPD: 0.79±0.23) or MALBAC (multiple annealing and looping-based amplification cycles) approach (MAPD : 0.24±0.002). We further successfully applied this approach to obtain single-cell CNV information of more than 500 cells from a tumor sample of hepatocellular carcinoma in one run. Using the unsupervised clustering, we identified at least two clonal subpopulations with a multitude of non-integer alterations across the genome. Overall, this is an unbiased, effective, high-throughput and low cost approach to figure out tumor heterogeneity and understand clonal evolution in tumor at the single-cell level.
Citation Format: Liang Wu, Yuzhou Wang, Miaomiao Jiang, Shiping Liu. High-throughput unbiased microwell-based single-cell CNV detection reveals tumor clonal subtypes in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3519.
Abstract 3523: A high-throughput screening strategy for the identification of novel lymphoproliferative elements
Authors: Jadin Laurence , Shaban Hiba , Kundu Anirban , Schreiber Gregory , Willis Scooter , Haerizadeh Farzad , Onuffer James , Frost Gregory 
Published: August 2019

Abstract
Introduction - Chimeric Antigen Receptor (CAR) T-cell therapy is effective against certain leukemias and lymphomas and shows promise for other incurable malignancies. Considerable challenges remain however to expand this platform technology beyond transplant-oriented hospital care. Centralized manufacturing of genetically modified T cells, lymphodepleting chemotherapy and patient management of current CAR-T therapies are associated with significant costs and treatment complexity. As a first step to reduce this treatment complexity, the present study describes a high throughput combinatorial domain library screening method to identify synthetic lymphoproliferative elements capable of driving in vivo expansion and survival of CAR-T cells in a lymphoreplete host without the homeostatic proliferation signals generated by lymphodepleting chemotherapy.
Methods - High-diversity semi-rationally-designed combinatorial libraries of putative lymphoproliferative protein subdomains were DNA barcoded and assembled into a lentiviral vector co-expressing a ROR2-targeted CAR. Human PBMC were transduced with the library and cultured in vitro for several days. Purified cells were injected into mice bearing xenograft tumors modified to express the ROR2 antigen and compared to unmodified xenograft controls. The expansion rate of integrated cells was monitored weekly by quantitative PCR and, after 21 days of exposure, genomic DNA was isolated from blood, spleen and xenograft tumor tissues. Enriched barcodes were amplified using PCR and amplicons were subjected to HiSeq Next-Generation Sequencing. Barcode decoding was achieved using PacBio long read sequencing analysis to align full-length construct sequences with barcode quantitation.
Results - Using this approach, putative CAR-T cell driver candidates and common key protein subdomains were identified that support selective in vivo expansion and survival of human lymphocytes in a tumor-bearing mouse model.
Conclusion - Taken together, these results demonstrate that a high throughput combinatorial screening strategy with quantitative bioinformatics is a viable method for identifying protein domain combinations capable of selectively driving human CAR-T cells in vivo. These small synthetic combinatorial protein domains may facilitate lymphodepleting chemotherapy-free regimens and lower CAR-T cell doses in the future.
Citation Format: Laurence Jadin, Hiba Shaban, Anirban Kundu, Gregory Schreiber, Scooter Willis, Farzad Haerizadeh, James Onuffer, Gregory Frost. A high-throughput screening strategy for the identification of novel lymphoproliferative elements [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3523.
Abstract 3524: Whole-exome sequencing of esophageal squamous cell carcinoma identifies recurrent mutations in Chinese subjects
Authors: Liu Meng , Feng Lin , An Hai yin 
Published: August 2019

Abstract
Background and Aims:
Epidemiological and etiological studies have shown that environmental and genetic factors play vital roles in esophageal carcinogenesis. However the genetic risk factors of esophageal squamous cell carcinoma (ESCC) still remain unclear. Here, we aimed to identify the recurrent mutations of ESCC in Chinese Uygur subjects from the China Xinjiang high-risk region by using a whole-exome sequencing method.
Materials and Methods:
We collected 50 Chinese Uygur patients with ESCC tumors and matched blood samples and adjacent normal oesophageal tissues (≥5cm from tumour site) at Xinjiang tumor hospital in China. Informed consent was obtained from each participant, and this study was approved by the Institutional Review Board of the Xinjiang tumor hospital. All the individuals underwent oesophagectomy and received no chemotherapy or radiotherapy before surgery. Whole-exome sequencing analysis (100bp) of DNA samples from 50 ESCC was performed on the Illumina HiSeq X Ten System.
Results:
We obtained 353,56 SNVs and 1157 indels in the exonic regions of 50 ESCC samples. Besides, we found the predominant mutations were the C&gt;T and C&gt;A transitions at TpCpW trinucleotide sites. And Three ESCC signatures were derived, which are highly similar to validated Signatrue-1, Signature-5 and Signature-13 in Cosmic. We identified four significantly mutated genes including TP53, CDKN2A, LGALS3 and C16orf3 (q&lt;0.5). Moreover, the aberrant pathways including the receptor tyrosine kinases (RTK/RAS/ phosphoinositide-3 kinase (PI3K)), NOTCH, WNT, Hippo, TP53 and cell cycle were identified.
Conclusions:
We provided mutated genes and disrupted pathways in ESCCs. These findings might be used to specific treatments for esophageal squamous cell carcinoma in Chinese Uygur subjects.
Acknowledgements: This work was supported by the Research Fund of Key Laboratory of Xinjiang oncology (No. 2017D04006) and Outstanding Youth Science and technology training project fund of Xinjiang (No. 2017Q058).
Citation Format: Meng Liu, Lin Feng, Hai yin An. Whole-exome sequencing of esophageal squamous cell carcinoma identifies recurrent mutations in Chinese subjects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3524.
Abstract 3529: Sample quality control of cell-free DNA
Authors: Graf Eva 
Published: August 2019

Abstract
Sequencing of cell-free DNA (cfDNA) is possible due to the establishment of low input library protocols for next-generation sequencing workflows. Accurate quantification of cfDNA samples is essential to determine suitable input amounts for cfDNA library preparation prior to sequencing. The main component of cfDNA samples is the mononucleosome with a size around 170 bp, sometimes with additional species representing nucleosome multimers. Further, cfDNA samples may contain larger DNA fragments dependent on pre-analytical sample treatment or extraction method. High molecular weight material can negatively influence library preparation and subsequently result in lower sequencing depth. Therefore, reliable quantification of cfDNA requires a method that separates DNA fragments by size, such as electrophoresis. This poster shows the use of an automated electrophoresis platform performing cfDNA quantification with region analysis. Moreover, the results include a score to qualify cfDNA samples according to their contamination level with high molecular weight material. This allows defining a threshold for objective sample qualification prior to library preparation. The analysis features are described with examples of typical sample patterns.
Citation Format: Eva Graf. Sample quality control of cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3529.
Abstract 3532: Tumor mutational burden (TMB) assessment and variant detection using an ultra-high multiplexed 20,000 amplicons NGS panel via a rapid 4 hour workflow
Authors: Pendleton Kathryn E. , Liu Yang L. , Lin Lifeng , Lee Lucie S. , Liu Jeffery , Liu Guoying , Liu Zhitong 
Published: August 2019

Abstract
Tumor mutational burden (TMB) is currently of high interest in the field of immuno-oncology due to its correlation with patient response to checkpoint inhibitor chemotherapy. Traditionally, TMB is calculated using whole exome sequencing; however, targeted sequencing approaches provide better coverage of the genetic regions of interest at lower costs. While hybrid-capture based target enrichment methods are well-established, the 2-5 day workflows are time consuming and require specialized equipment and highly trained operators. Here we present an ultrafast, 4-hour method for preparing target enriched NGS libraries for assessing TMB that would streamline and lower the cost of immuno-oncology studies. This multiplex-PCR-based technology provides a highly efficient, accurate and robust method for unbiased enrichment of tens of thousands of target regions while minimizing non-specific primer-primer interactions and GC bias and maximizing coverage uniformity. We demonstrate this using a highly-multiplexed prototype NGS panel that contains ~20,000 amplicons and covers 355 genes for assessment of TMB.
Sequencing-ready NGS libraries were prepared using the Paragon Genomics CleanPlex® target enrichment technology. The 3-step workflow combines target enrichment and NGS library preparation. The protocol includes an ultra-high multiplex PCR step to amplify regions of interest with target-specific primers, a background cleaning step to remove non-specific PCR products, and a final PCR to add Illumina sequencing adapter and sample indexes. Libraries were made using 20ng of genomic DNA and sequenced on an Illumina NextSeq® platform. Sequencing metrics such as on-target rates were calculated, and variants were identified using Paragon Genomics’ variant calling algorithm.
Using the CleanPlex technology, this prototype TMB panel exhibits &gt;95% uniformity at 0.2X mean, limited GC bias, and &gt;94% detection rate for mutants with 5% allele frequencies. The CleanPlex background cleaning step was essential for removing the undesirable byproducts of the multiplexed reaction step. CleanPlex technology demonstrates that it is capable of creating high quality amplicon libraries with high uniformity, low GC bias, and sensitive variant calling even in ultra-multiplexed libraries with ~20,000 amplicons with a workflow under 4-hours.
Citation Format: Kathryn E. Pendleton, Yang L. Liu, Lifeng Lin, Lucie S. Lee, Jeffery Liu, Guoying Liu, Zhitong Liu. Tumor mutational burden (TMB) assessment and variant detection using an ultra-high multiplexed 20,000 amplicons NGS panel via a rapid 4 hour workflow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3532.
Abstract 3538: Analysis of error profiles in deep next-generation sequencing data
Authors: Ma Xiaotu , Zhang Jinghui 
Published: August 2019

Abstract
Background
Sequencing errors are a key confounding factor for detecting low frequency genetic variants for cancer screening, testing, treatment and surveillance through deep next-generation sequencing (NGS). However, there is a lack of comprehensive understanding of errors introduced at various steps of a conventional NGS workflow, such as sample handling, library preparation, enrichment PCR, and sequencing. In this study we systematically investigated the above question by using the current NGS technology.
Results
We discovered that the substitution error rate can be computationally suppressed to 10-5~10-4, which is 10~100-fold lower than current reports. We then quantified substitution errors attributable to sample handling, library preparation, enrichment PCR, and sequencing using multiple deep sequencing datasets from multiple sequencing centers. We show that 1) error rate differs by nucleotide substitution types, ranging from 10-5 for A&gt;C/T&gt;G, C&gt;A/G&gt;T, and C&gt;G/G&gt;C changes to 10-4 for A&gt;G/T&gt;C changes; 2) C&gt;T/G&gt;A errors exhibit strong sequence context dependency; 3) sample-specific effects dominate elevated C&gt;A/G&gt;T errors; 4) target enrichment PCR lead to ~6-fold increase of overall error rate; 5) more than 70% of hotspot variants, such as BRAF V600E, can be detected at 0.1%~0.01% frequency with the current NGS technology by applying in-silico error suppression.
Conclusions
We present the first comprehensive analysis of error sources in conventional NGS workflows. The error profiles revealed by our study highlight new directions for further improving NGS accuracy both experimentally and computationally which will lead to enhanced precision for deep sequencing.
Citation Format: Xiaotu Ma, Jinghui Zhang. Analysis of error profiles in deep next-generation sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3538.
Abstract 3542: Turbolase: A radically streamlined high throughput sample prep to sequencing workflow
Authors: Masser Dustin , Stedtfeld Robert , Lenhart Justin , Makarov Vladimir , Kurihara Laurie 
Published: August 2019

Abstract
Normalase is a novel enzymatic library normalization method that eliminates library quantification and manual concentration adjustment of each sample prior to library pooling, and the uniform sample processing eliminates numerous error-prone pipetting steps. When combined with Swift 2S Turbo rapid library kits, a highly streamlined ‘Turbolase’ workflow is created that is readily automated on the Hamilton Star and other platforms, where simple bulk processing improves throughput and reduces cost for NGS laboratories. Swift 2S Turbo kits comprise two enzymatic steps and a single purification that completes DNA fragmentation, end repair and adapter ligation. This is followed by standard library amplification using Normalase PCR primers to condition the libraries and produce a required minimum yield in excess of the 4 nM final concentration. This is followed by a single purification and two 15-minute Normalase incubation steps that; 1) enzymatically select 4 nM of each library, and 2) enzymatically normalize each library to 4 nM within a single pool. The pools can then be directly sequenced without further purification. Normalase allows for greater than 10-fold variation in input quantity, while generating less than 10% variation in sample representation within a pool resulting in optimal cluster density and sample balance for Illumina sequencing. Because of the unique workflow, Normalase also eliminates index hopping for libraries sequenced on patterned flow cells. The ‘Turbolase’ workflow is compatible with full-length indexed adapters that have been added by ligation as well as workflows that require indexing PCR primers to complete library construction during library amplification. Normalase is also compatible with library preparation kits available from other vendors. Normalase for pooling of libraries for pre-hybridization capture is currently under development, which will make multiplexed hybridization capture protocols streamlined and robust from library generation to sequencing.
Citation Format: Dustin Masser, Robert Stedtfeld, Justin Lenhart, Vladimir Makarov, Laurie Kurihara. Turbolase: A radically streamlined high throughput sample prep to sequencing workflow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3542.
Abstract 3543: Clinical whole genome sequencing at scale
Authors: MacBeth Alyssa , Bowers Tera , Woolf Betty , Harden Maegan , Lennon Niall , Gabriel Stacey 
Published: August 2019

Abstract
Whole genome sequencing (WGS) offers the greatest potential to comprehensively and accurately identify all forms of human genetic variation. The accessibility of WGS data for diagnostic use has increased due to cost reductions in recent years, driving the need for a high quality, clinically validated offering. Our platform has completed an extensive benchmarking study of the Illumina PCR-free whole genome pipeline to establish clinical validity for the end-to-end laboratory, analytical, and computational processes. Our benchmarking study is comprehensive in nature, including measuring the precision, robustness, limits of detection for variant classes, and contamination estimates. A cohort of well-characterized reference controls and clinical samples with previously identified pathogenic variants were used to establish the performance characteristics of our clinical WGS test, which at 30X coverage has &gt;99% analytical sensitivity for SNVs and &gt;98% analytical sensitivity for small insertions and deletions. Our platform is capable of operating at a scale that supports applications from individual clinics (cancer and medical genetics related applications) as well as large scale ambitious collaborative projects such as the All Of Us program which aims to generate clinical grade whole genome variant calls for 1 million healthy research participants.
Citation Format: Alyssa MacBeth, Tera Bowers, Betty Woolf, Maegan Harden, Niall Lennon, Stacey Gabriel. Clinical whole genome sequencing at scale [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3543.
Abstract 3544: High performance multiplexed targeted enrichment sequencing from FFPE tissues
Authors: Consugar Mark , Arbiza Leonardo , Butcher Kristin , Chen Siyuan , Chilton Hutson , Gantt Richard , Hasin-Brumshtein Yehudit , Laugharn Jim , Simon Jayne , Thomann Ulrich , Thompson Christina , Zeitoun Ramsey 
Published: August 2019

Abstract
Library construction for Next-Generation Sequencing (NGS) using formalin-fixed paraffin-embedded (FFPE) samples offers unique challenges in acquiring high-quality sequencing data due to wide distribution of sample quality. Specifically, differences in formalin fixation methods lead to crosslinked and/or degraded nucleic acid and inconsistent extraction yields. Hence, FFPE extraction and library construction methods must be carefully considered for target enrichment applications. In collaboration, Covaris and Twist Bioscience demonstrate a complete library preparation and target enrichment solution that generates ready-to-sequence multiplexed libraries directly from FFPE tissue.This workflow leverages the Covaris truXTRAC FFPE total Nucleic Acid Plus Kit and oneTUBE-10 shearing with the world-class performance of Twist Bioscience’s Target Enrichment Solutions. Covaris, the Gold Standard for DNA shearing in NGS, also offers pre-analytical products that leverage Adaptive Focused Acoustics® (AFA)® technology. In this FFPE-specific application, the Covaris truXTRAC FFPE total Nucleic Acid Plus Kit and oneTUBE-10 shearing enable full emulsification of paraffin and disaggregation of tissue for highly efficient nucleic acid extraction and generation of size specific DNA libraries. With the Twist Bioscience Human Core Exome kit, the resulting libraries are indexed, pooled, and target enriched with uniquely optimized DNA probes to generate ready-to-sequence high quality multiplexed libraries.Using the aforementioned workflow, results from processing numerous FFPE tissue types are presented. Sequencing results demonstrate large improvements in general Picard metrics that include uniformity (Fold_80 &lt; 1.8), sequencing depth (20X coverage &gt;85% with 100X downsampling), and duplication rates (&lt;6%) when compared to similar published studies. These results demonstrate a validated solution for library preparation and targeted exome sequencing of FFPE samples that can be integrated into automated workflows. The truXTRAC kit and AFA® technology from Covaris generate size specific DNA libraries from FFPE samples which, when paired with Twist Bioscience’s superior target enrichment workflow, deliver multiplexed libraries for high performance targeted sequencing.
Citation Format: Mark Consugar, Leonardo Arbiza, Kristin Butcher, Siyuan Chen, Hutson Chilton, Richard Gantt, Yehudit Hasin-Brumshtein, Jim Laugharn, Jayne Simon, Ulrich Thomann, Christina Thompson, Ramsey Zeitoun. High performance multiplexed targeted enrichment sequencing from FFPE tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3544.