Next-generation Sequencing Libraries Research Articles

Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR/Cas systems.

The CRISPR/Cas system has been extensively applied to make precise genetic modifications in various organisms. Despite its importance and widespread use, large-scale mutation screening remains time-consuming, labour-intensive and costly. Here, we developed Hi-TOM (available at https://doi.org/www.hi-tom.net/hi-tom/ ), an online tool to track the mutations with precise percentage for multiple samples and multiple target sites. We also described a corresponding next-generation sequencing (NGS) library construction strategy by fixing the bridge sequences and barcoding primers. Analysis of the samples from rice, hexaploid wheat and human cells reveals that the Hi-TOM tool has high reliability and sensitivity in tracking various mutations, especially complex chimeric mutations frequently induced by genome editing. Hi-TOM does not require special design of barcode primers, cumbersome parameter configuration or additional data analysis. Thus, the streamlined NGS library construction and comprehensive result output make Hi-TOM particularly suitable for high-throughput identification of all types of mutations induced by CRISPR/Cas systems.

BRCA-analyzer: Automatic workflow for processing NGS reads of BRCA1 and BRCA2 genes

The use of targeted next-generation sequencing (NGS) provides great new opportunities for molecular and medical genetics. However, in order to take advantage of these opportunities, we need to have reliable tools for extracting the necessary information from the huge amount of data generated by NGS. Here we present our automatic multithreaded workflow for processing NGS data of BRCA1 and BRCA2 genes obtained with NGS technology named BRCA-analyzer. Optimizing it on the sequencing data of 899 samples from 693 patients, we were able to find the most reliable tools and adjust their parameters in such a way that all pathogenic variants found were confirmed by Sanger’s sequencing. For 82 and 24 DNA samples from blood and formalin-fixed paraffin-embedded blocks, NGS libraries were prepared with GeneRead BRCA panel v2 (Qiagen). The reads obtained were processed with BRCA-analyzer and Qiagen GeneRead Data analysis workflow. In total 27 pathogenic variants were found and confirmed by Sanger’s sequencing, with all of them determined with BRCA-analyzer. Qiagen GeneRead Data analysis discarded 5 true pathogenic variants due to their location in homopolymeric sequence stretches. For other 793 samples, libraries were prepared by the in-house method, and NGS data were analyzed by BRCA-analyzer in comparison to another free automatic amplicon NGS workflow Canary. From total 137 pathogenic variations, BRCA-analyzer found 135 and Canary 123. Mutations were missed by BRCA-analyzer due to the trimming primer sequences from reads before mapping to be fixed in the next version. On the freely available NGS data, we showed that BRCA-analyzer could also be used for hybrid capture gene panels, although it needs more extensive testing on such library preparation methods. Thus, BRCA-analyzer is an automatic workflow for processing NGS data of BRCA1/2 genes with variant filters adapted to amplicon-based targeted NGS data. BRCA-analyzer can be used to identify germline as well as somatic mutations. BRCA-analyzer is freely available at https://github.com/aakechin/BRCA-analyzer.

Universal Target Capture of HIV Sequences From NGS Libraries

Background: Global surveillance of viral sequence diversity is needed to keep pace with the constant evolution of HIV. Recent next generation sequencing (NGS) methods have realized the goal of sequencing circulating virus directly from patient specimens. Yet, a simple, universal approach that maximizes sensitivity and sequencing capacity remains elusive. Here we present a novel HIV enrichment strategy to yield near complete genomes from low viral load specimens.Methodology: A non-redundant biotin-labeled probe set (HIV-xGen; n = 652) was synthesized to tile all HIV-1 (groups M, N, O, and P) and HIV-2 (A and B) strains. Illumina Nextera barcoded libraries of either gene-specific or randomly primed cDNA derived from infected plasma were hybridized to probes in a single pool and unbound sequences were washed away. Captured viral cDNA was amplified by Illumina adaptor primers, sequenced on a MiSeq, and NGS reads were demultiplexed for alignment with CLC Bio software.Results: HIV-xGen probes selectively captured and amplified reads spanning the entirety of the HIV phylogenetic tree. HIV sequences clearly present in unenriched libraries of specimens but previously not observed due to high host background levels, insufficient sequencing depth or the extent of multiplexing, were now enriched by >1,000-fold. Thus, xGen selection not only substantially increased the depth of existing sequence, but also extended overall genome coverage by an average of 40%. We characterized 50 new, diverse HIV strains from clinical specimens and demonstrated a viral load cutoff of approximately log 3.5 copies/ml for full length coverage. Genome coverage was <20% for 5/10 samples with viral loads <log 3.5 copies/ml and >90% for 35/40 samples with higher viral loads.Conclusions: Characterization of >20 complete genomes at a time is now possible from a single probe hybridization and MiSeq run. With the versatility to capture all HIV strains and the sensitivity to detect low titer specimens, HIV-xGen will serve as an important tool for monitoring HIV sequence diversity.

Performance comparison of blood collection tubes as liquid biopsy storage system for minimizing cfDNA contamination from genomic DNA.

The ratio of target cfDNA in total plasma is low. The abundant gDNA background resulting from blood cell lysis caused by improper operation has become a major obstacle to accurately measure cfDNA. In this study, we investigated the storage capacity of three blood cell collection tubes (BCTs) in the prevention of genomic DNA contamination caused by white blood cell rupture and evaluated their performance when they were utilized combining with highly sensitive mutation detection technology. Blood samples were drawn from six healthy blood donors and stored in three types of BCTs (BD K2 -EDTA tube, Roche tube, and Streck tube). Plasma samples were isolated at specific time points (day 0, day 3, day 7, and day 14) for content analysis. Roche BCT was more capable for preventing cfDNA contamination caused by white blood cell disruption within 14days, comparing with BD K2 -EDTA tube and Streck tube. Severe white blood cell lysis and gDNA contamination were found in the BD tube. The impacts of Roche and Streck tubes on the quantity and complexity of next-generation sequencing (NGS) libraries did not differ significantly within 3days, satisfying most of our daily demands. In addition, the rupture of WBC was not synchronized with hemolysis in BCTs. This study showed that capacities of blood collection tubes differed considerably in preservation of blood samples. Therefore, suitable blood collection devices should be selected to minimize gDNA contamination and to standardize blood samples processing for achieving more accurate and reliable clinical analysis of cfDNA.

Development of High-Throughput Clinical Testing ofRPGRORF15 Using a Large Inherited Retinal Dystrophy Cohort

Mutations in the ORF15 region of RPGR account for approximately half of all X-linked retinitis pigmentosa cases. However, a robust high-throughput method for the detection of ORF15 mutations has yet to be validated. We set out to develop the first clinically validated next-generation sequencing (NGS) method for the detection of mutations in this difficult-to-sequence region, including test accuracy and coverage data. As part of a blind-test, 145 research samples, previously tested by Sanger sequencing, and 81 clinical samples were evaluated using NGS of long-range PCR products fragmented with Illumina's Nextera library preparation kit (method 1), or with Centrillion's OneTube technology, supplemented with duplication analysis using an ORF15-specific in-silico array (method 2). DNA fragments were analyzed using Agilent's DNA 1000 assay, and sequencing was done on Illumina's MiSeq 2×150 or HiSeq2500 2×100. NextGENe by SoftGenetics was used for data analysis and variant calling. The Nextera library preparation method produced 24 cases of discordance due to (in order of decreasing occurrence) false-negatives, incorrectly called variants, and a false-positive. Subsequent use of a new, OneTube NGS library preparation method, supplemented with duplication analyses, resolved discordance between Sanger and NGS data in all cases. This improvement in variant detection accuracy was largely attributed to improvement in random fragmentation offered by the enzymatic OneTube method, resulting in more complete coverage of the highly repetitive ORF15 region. Minimum coverage was roughly 320 reads for Nextera and 6800 reads for OneTube (normalized for total read counts). This paper documents the first clinically validated NGS method for reliable, high-throughput sequencing of RPGR ORF15. Sensitivity and specificity of the new method were 100%, with the caveat of unclear zygosity calling for one large duplication case. These findings demonstrate a reliable and practical implementation for NGS-based diagnosis of RPGR ORF15 mutations. They also provide the foundation for targeted, high-coverage sequencing of any other repetitive regions within the genome.

Biotinylated amplicon sequencing: A method for preserving DNA samples of limited quantity

BackgroundGenomic testing is often limited by the exhaustible nature of human tissue and blood samples. Here we describe biotinylated amplicon sequencing (BAmSeq), a method that allows for the creation of PCR amplicon based next-generation sequencing (NGS) libraries while retaining the original source DNA. Design and methodsBiotinylated primers for different loci were designed to create NGS libraries using human genomic DNA from cell lines, plasma, and formalin-fixed paraffin embedded (FFPE) tissues using the BAmSeq protocol. DNA from the original template used for each BAmSeq library was recovered after separation with streptavidin magnetic beads. The recovered DNA was then used for end-point, quantitative and droplet digital PCR (ddPCR) as well as NGS using a cancer gene panel. ResultsRecovered DNA was analyzed and compared to the original DNA after one or two rounds of BAmSeq. Recovered DNA revealed comparable genomic distributions and mutational allelic frequencies when compared to original source DNA. Sufficient quantities of recovered DNA after BAmSeq were obtained, allowing for additional downstream applications. ConclusionsWe demonstrate that BAmSeq allows original DNA template to be recovered with comparable quality and quantity to the source DNA. This recovered DNA is suitable for many downstream applications and may prevent sample exhaustion, especially when DNA quantity or source material is limiting.

Molecular cloning of novel transcripts of the adaptor-related protein complex 2 alpha 1 subunit (AP2A1) gene, using Next-Generation Sequencing

The adaptor-related protein (AP) complexes play important roles in cargo selection and vesicle formation, and hence in intracellular membrane trafficking. Five different AP complexes are currently known, each consisting of four subunits, known as adaptins. AP-2, the most thoroughly characterized of the five AP complexes, facilitates clathrin-mediated endocytosis. In this study, we describe the discovery and molecular cloning of seventy-seven novel alternatively spliced transcripts of the human AP2A1 gene, which encodes the αA adaptin of the AP-2 complex. For this purpose, we have used Next-Generation Sequencing (NGS), a powerful tool for studying alternative splicing. In brief, we subcultured fifty-five established human cell lines, originating from several distinct cancerous and normal tissues, extracted total RNA, and synthesized first-strand cDNA. Next, we used nested touchdown PCR to amplify the whole coding region of the AP2A1 transcripts of each cell line, mixed all PCR products, and proceeded to NGS library construction, template preparation, and semiconductor sequencing. Extensive bioinformatic analysis revealed thirteen novel splice junctions of previously annotated exons, as also verified via nested PCR with primers targeting these splice junctions. Moreover, consecutive nested PCRs led to the determination of the primary structure of seventy-seven novel AP2A1 transcripts, all of which were shown to comprise at least one premature translation termination codon, thus representing nonsense-mediated mRNA decay (NMD) candidates. NMD is a mechanism that cells use to control gene expression. Consequently, alterations in the levels of these potentially non-coding AP2A1 transcripts could lead to a decrease in the number of AP2A1 mRNA molecules, when needed. Undoubtedly, the exact role of these new APA1 splice variants merits elucidation.

Transcriptional analysis of viral mRNAs reveals common transcription patterns in cells infected by five different filoviruses.

Filoviruses are notorious viral pathogens responsible for high-consequence diseases in humans and non-human primates. Transcription of filovirus mRNA shares several common features with transcription in other non-segmented negative-strand viruses, including differential expression of genes located across the viral genome. Transcriptional patterns of Ebola virus (EBOV) and Marburg virus (MARV) have been previously described using traditional, laborious methods, such as northern blots and in vivo labeling of viral mRNAs. More recently, however, the availability of the next generation sequencing (NGS) technology has offered a more straightforward approach to assess transcriptional patterns. In this report, we analyzed the transcription patterns of four ebolaviruses—EBOV, Sudan (SUDV), Bundibugyo (BDBV), and Reston (RESTV) viruses—in two different cell lines using standard NGS library preparation and sequencing protocols. In agreement with previous reports mainly focused on EBOV and MARV, the remaining filoviruses used in this study also showed a consistent transcription pattern, with only minor variations between the different viruses. We have also analyzed the proportions of the three mRNAs transcribed from the GP gene, which are characteristic of the genus Ebolavirus and encode the glycoprotein (GP), the soluble GP (sGP), and the small soluble GP (ssGP). In addition, we used NGS methodology to analyze the transcription pattern of two previously described recombinant MARV. This analysis allowed us to correct our construction design, and to make an improved version of the original MARV expressing reporter genes.

ATAC2GRN: optimized ATAC-seq and DNase1-seq pipelines for rapid and accurate genome regulatory network inference

BackgroundChromatin accessibility profiling assays such as ATAC-seq and DNase1-seq offer the opportunity to rapidly characterize the regulatory state of the genome at a single nucleotide resolution. Optimization of molecular protocols has enabled the molecular biologist to produce next-generation sequencing libraries in several hours, leaving the analysis of sequencing data as the primary obstacle to wide-scale deployment of accessibility profiling assays. To address this obstacle we have developed an optimized and efficient pipeline for the analysis of ATAC-seq and DNase1-seq data.ResultsWe executed a multi-dimensional grid-search on the NIH Biowulf supercomputing cluster to assess the impact of parameter selection on biological reproducibility and ChIP-seq recovery by analyzing 4560 pipeline configurations. Our analysis improved ChIP-seq recovery by 15% for ATAC-seq and 3% for DNase1-seq and determined that PCR duplicate removal improves biological reproducibility by 36% without significant costs in footprinting transcription factors. Our analyses of down sampled reads identified a point of diminishing returns for increased library sequencing depth, with 95% of the ChIP-seq data of a 200 million read footprinting library recovered by 160 million reads.ConclusionsWe present optimized ATAC-seq and DNase-seq pipelines in both Snakemake and bash formats as well as optimal sequencing depths for ATAC-seq and DNase-seq projects. The optimized ATAC-seq and DNase1-seq analysis pipelines, parameters, and ground-truth ChIP-seq datasets have been made available for deployment and future algorithmic profiling.

PO-336 ThruPLEX® and PicoPLEX® technologies for rare alleles and copy number variation detection from cell-free DNA and single human cancer cells

IntroductionLiquid biopsies provide a non-invasive method to acquire the genetic information provided in cell-free DNA (cfDNA) as well as in single circulating tumour cells. Access to this genetic information through next-generation sequencing (NGS) identifies mutations and alterations such as Copy Number Variation (CNV) that play a role in cancer and other diseases.Material and methodsThe key to identifying rare mutations is improved sequencing accuracy and the ability to distinguish between biological and PCR duplicates. ThruPLEX Tag-seq was developed with the addition of unique molecular tags (UMTs) to improve sequencing accuracy by accounting for polymerase and sequencing errors and to increase confidence in rare allele identification. Whole Genome Amplification (WGA) for CNV detection was achieved by the thermal cycling quasi-random primed library chemistry of the PicoPLEX DNA-seq single-cell NGS library prep kit.Results and discussionsThruPLEX Tag-seq libraries were prepared using 10–30 ng of Horizon Discovery’s Multiplex I cfDNA Reference Standard Set containing six single nucleotide variants (SNV) for 4 different genes (EGFR, KRAS, NRAS, PIK3CA) present at 0.5%–5% allele frequency. The libraries were enriched with either a 110 kb or 240 kb custom panel or the Agilent ClearSeq Comprehensive Cancer Panel. Enriched libraries were sequenced witan average total read coverage of approximately 5,000X and analysed with and without the UMTs.For CNV analysis in single cells, a bar-coded PicoPLEX DNA-seq library was synthesised and amplified from 6 single cells from either PBMCs and clonally-expanded PC3 prostate cancer cells. Sequencing was performed on a MiSeq v2 and reads were mapped using BWA-MEM, processed in Picard_Mark_Duplicates, and further characterised in DNA nexus.classic. All PC3 cells showed reproducible CNV calling, however none of the lymphocyte samples showed any CNVs. Accurate CNV calls for PC3 cells were achieved even at when fastq files were randomly downsampled to 1 50 000 read pairs.ConclusionTherefore, use of UMTs in the preparation of NGS libraries from cfDNA enhances sequencing accuracy: by distinguishing between biological duplicates and PCR duplicates, increasing read coverage and decreasing background noise, reducing false positives, and in more confident mutation calls. PicoPLEX DNA-seq NGS libraries have a very simple and fast workflow that is suitable reproducible CNV detection in single cells even at low 0.002X average coverage.

Abstract 3414: Determining microsatellite instability (MSI) status of colorectal cancers through next-generation sequencing (NGS)

Abstract Introduction: The MSI status of a tumor is often a marker of deoxyribonucleic acid (DNA) mismatch repair deficiency. Recent clinical trials have shown MSI-High (MSI-H) tumors are more likely to respond to checkpoint inhibitor immunotherapy. The United States Food and Drug Administration recently granted approval for a checkpoint inhibitor in metastatic solid tumor that demonstrates high microsatellite instability. Here we show the accuracy of determining the MSI status of colorectal cancers (CRC) using either TruSight™ Tumor 170 (TST170), a NGS-based 170 gene panel for solid tumor profiling, or whole exome sequencing (WES). Further, we report the accuracy of identifying the MSI status through sequencing of tumor-only samples without the subject-matched normal DNA. Experimental Method: The MSI status of 52 CRC tumors (51 formalin-fixed, paraffin embedded and 1 fresh-frozen sample) were evaluated using a microsatellite instability (MSI) assay (Promega), which is a detection method that uses capillary electrophoresis to analyze PCR-products. For comparison to WES, the tumors and subject-matched normal DNA libraries were prepared and enriched using TruSight™ Oncology reagents (Illumina). Targeted NGS libraries were prepared and enriched for the TST170-gene panel using the TST170 library kit (Illumina). NGS libraries were sequenced with the HiSeq™ 2000, HiSeq™ 2500, or NextSeq™ 550 instruments (Illumina). MSI scores of the 52 tumors were calculated through a novel internally developed bioinformatics algorithm. Data Summary: From the 52 subjects assessed through Promega's MSI detection kit, 28 and 24 were found to be MSI-H and microsatellite stable (MSS), respectively. These data were used as the benchmark for the MSI status of these subjects. Both the NGS-based tumor-normal data from WES and TST170 achieved a 100% concordance with PCR-based MSI detection. In addition, when only the sequencing data of the tumor samples were used without the subject-matched normal DNA, the Illumina MSI algorithm could identify the MSI status of the 52 subjects with 98% concordance with Promega's MSI detection kit. Conclusion: Collectively, this data indicates that NGS can be utilized for determining tumor MSI status using WES or the TST170 panel. In addition, a bioinformatics algorithm was developed that successfully categorized tumors according to their MSI status without requiring subject-matched normal DNA sequence data. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Alex So, Shile Zhang, Shannon Kaplan, Joyee Yao, Phillip Le, Christine Glidewell-Kenney, Kristina Kruglyak, Jenny Jie Chen, Ali Kuraishy, Ina Deras, Karen Gutekunst. Determining microsatellite instability (MSI) status of colorectal cancers through next-generation sequencing (NGS) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3414.

PO-391 High-throughput single-cell T-cell receptor profiling by SMART technology

IntroductionSingle-cell T-cell receptor (scTCR) clonotype analysis permits the determination of the specific TCR alpha-beta (α/β) chain pairing expressed on each cell. This pairing information allows researchers to gain insight into Tcell heterogeneity and plasticity, determine the contribution of the pairing to antigen specificity of the individual TCR, and design therapeutic antibodies. Here we employ a 5’-RACE-like approach and SMART technology, in conjunction with two novel next-generation sequencing (NGS) library preparation kits, using the same primer pairs, to capture full-length variable regions of TCR-α and -β chains.Material and methodsMethod 1, using the SMARTer Human scTCRa/b Profiling Kit, permits NGS library preparation of FACS-sorted cells in 96-well plates. Method 2 is an adaptation of the process above that scales to ~1200 single cells using the SMARTer ICELL8 Single-Cell System, which enables single-cell isolation and nanoliter PCR in a nanowell chip.Results and discussionsWe present data showing α/β pairing from Jurkat, CCRF, PBMCs, and CD4 +T cells. In addition to the sensitivity of this method, the ability to pool the cDNA from 96wells into 12 sequencing libraries adds to the ease of use. Consistent with immunology reports, unstimulated CCRF-CEM cells examined with this kit expressed a TCR-β but not a TCR-α chain.scTCR pofiling with SMARTer ICELL8 Single-Cell System as a proof of principle of scTCRα/β was performed on Jurkat cells and CCRF-CEM cells.Jurkat cells and CCRF-CEM cells were processed using an ICELL8 chip preprinted with barcoded oligos. Paired TCRα/β Jurkat clonotypes were detected in 77% and 87% cells in mixed and single cell populations, respectively.ConclusionThe ability of the core biochemistry and PCR components of these kits to be used with either FACS-sorted cells in 96-well plates or >1,000 cells in novel ICELL8 chips (in development) points to the general utility and scalability of this approach in understanding paired scTCR clonotype diversity.

Abstract 3647: Prior DNA repair improves the read quality in next-generation sequencing of cell-free tumor DNA

Abstract The cell-free tumor DNA (ctDNA) in patient's plasma is a noninvasive tumor biomarker. The detection of ctDNA is helpful to evaluate tumor progression, tumor response to the therapy, and subtyping of tumor cells. Targeting next-generation sequencing (NGS) technology provides a sensitive and specific technique for ctDNA detection. It can detect multiple tumor-related gene mutations. However, collection of high quality of cell-free DNA (cfDNA) may not be available in some cases. The quality of cfDNA in plasma is impacted by multiple factors, such as plasma collection and storage conditions, DNase activity in the blood, and the apoptotic and necrotic status of the tumor. These factors affect the integrity of cfDNA, leading to full or partial degradation of cfDNA, including double-strand DNA breaks and single strand DNA nicking. Lesions on cfDNA decrease the usability of ctDNA by digital PCR and targeted NGS. Due to amount limitation of cfDNA in some cases, the repair of damaged cfDNA could be a necessary rescue step in producing viable libraries for NGS. CfDNA was extracted from the plasma with Zymo Research's Quick-cfDNA Serum and Plasma kit and was quantified by realtime PCR with Alu primer sets. The size of cfDNA was about 160 base pairs, as estimated by Agilent Tapestation 2200 with the High Sensitivity DNA kit. The DNA repair of cfDNA was conducted with PreCR Repair Mix of New England BioLabs. The sequencing library was prepared with Accel-Amplicon 56G Oncology Panel of Swift Biosciences and sequenced in Illumina's MiSeq. The quality of sequencing data was determined with FastQC. Compared to PreCR Repair Mix treated cfDNA samples, fastQ data from some of untreated cfDNA samples showed failed per base sequencing quality, about 50% of sequencing cycles with the mean quality score below 30. The per sequencing quality score of these cfDNA samples failed also to pass the quality filter. The read size distribution analysis indicated that about 60 to 70% of read sized between 30 to 45 bases and about 75 to 80% of read was smaller than 75 bases. However, sequencing data from same cfDNA samples treated with PreCR Repair Mix show a greatly improved sequencing quality. Both per base sequencing quality and per sequencing quality score passed quality filter. The above 97% of reads sized over 92 bases, complying with the requirement of 56G Oncology Panel. In conclusion, the DNA repair process before library preparation is necessary to rescue damaged cfDNA for amplicon-based NGS. Citation Format: Hong Yin, Adam H. Greer, Glenn Mills. Prior DNA repair improves the read quality in next-generation sequencing of cell-free tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3647.

Abstract 1623: Inter-laboratory harmonization of next generation sequencing somatic mutation assays for cancer response prediction

Abstract Background: The potential for next generation sequencing (NGS) analysis in personalized diagnosis and treatment of cancer is advancing rapidly. Measurement of mutation fraction less than 5% may be clinically relevant in analysis of circulating tumor DNA and/or small cytologic samples. However, due to uncertainty introduced by variation in sample quality, the mutation fraction lower limit of detection (LOD) reported by clinical laboratories does not typically extend beyond this 5% threshold. Even with a more conservative LOD criterion, (e.g., 20% mutation fraction), poor harmony may result from inter-laboratory variation in skills to assess sample quality as well as specimen characteristics that are not evident even to expert clinical pathologists. Thus, a critical unmet need is methods that harmonize reporting of mutation fraction measured by NGS by accurately estimating specimen-specific confidence limits for each measurement. Methods: We designed a mixture of spike-in competitive internal standards for tumor somatic mutations in 32 currently actionable genes that enable quality-controlled measurement by targeted NGS using either PCR-amplicon or hybrid-capture enrichment. We also developed a data analysis pipeline suitable to separate target and internal standard sequencing reads prior to analysis of target copy number and sequencing errors. Through participation in the FDA-led SEquencing Quality Control Project Phase 2 (SEQC2), where a set of highly characterized cell line mixtures was developed to assess cross-site reproducibility and mutation detection accuracy of multiple targeted sequencing platforms, we aim to comprehensively evaluate the utility of the spike-in mixture with various targeted NGS panels to measure 95% confidence limits around each somatic mutation fraction measurement. Results: In pilot studies, combining the mixture of internal standards with specimens prior to NGS library preparation enabled reliable measurement of 95% confidence limits around each somatic mutation measurement. Further, using the internal standards to control for specimen adequacy enabled reliable measurement of actionable mutation fraction at as low as 0.01% in suitable specimens. Conclusion: We expect that the full cross-site SEQC2 study will provide further support for the role of competitive spike-in mixtures to provide reliable 95% confidence limits around each measurement of somatic mutation fraction in a clinical specimen. Disclaimer: The views expressed here are those of the authors only and do not necessarily express the views/policies of the FDA. Citation Format: James C. Willey, Thomas M. Blomquist, Erin L. Crawford, Joshua Xu, Leihong Wu, Thomas Morrison. Inter-laboratory harmonization of next generation sequencing somatic mutation assays for cancer response prediction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1623.

Abstract 4651: Development of a targeted NGS panel for solid tumor actionable gene targets using multiplex PCR-based enrichment in an integrated fluidic circuit

Abstract Introduction: Next-generation sequencing (NGS) has been being rapidly adopted in clinical research to align actionable variants in tumors to targeted therapies or clinical trials. Comprehensive NGS-based tumor profiling assays are an efficient and effective method to characterize a variety of clinically relevant somatic mutations. We have developed a targeted NGS cancer gene panel that focuses on actionable gene mutations and employs a NGS library preparation method based on multiplex PCR enrichment in an integrated fluidic circuit (IFC) and the automated JunoTM System. Methods: 53 actionable cancer genes were selected based on both clinical and research knowledge that covers 15 solid tumor types. Assays for the selected regions of the 53 genes were designed by an internal assay design pipeline. Multiplex PCR using target-specific primers is conducted in an LP—48.48 IFC on the Fluidigm Juno™ system, where up to 48 DNA samples can be processed simultaneously. A unique barcode is incorporated in one of the PCR primers to distinguish individual samples. To evaluate the assay performance, three sets of cell line gDNA samples were identified from commercial sources: set 1: 22 commercial reference samples, set 2: 12 samples for single nucleotide variants (SNVs) and indels, and set 3: 12 samples for copy number variations (CNVs). Each sample was tested on the IFC in 4 replicates. PCR products harvested from the IFC were pooled and purified with Agencourt® AMPure® XP magnetic beads. A second PCR step using a universal primer pair was performed to add sequencing adapters. After a second purification, the DNA library was sequenced on a NextSeq™ 500 system. The data analysis was performed by a service provider partner (GenomOncology). Results: A total of 1,508 primer pairs with an average insert size of 155 bp were selected to cover SNVs, indels, and CNVs. The assays were separated into 44 pools to minimize the interaction between assay primers and improve performance. The percentage of reads mapped to the genome was 98.9%, and the percentage of reads that mapped to amplicons was 96.8%. Mutation detection sensitivity was 4% variant allele frequency (VAF.) In 47 selected samples, the total SNVs, CNVs and indels represented are 182, 154 and 28, respectively. For SNVs, the positive percent agreement (PPA) is 1.0 and positive predictive value (PPV) is 0.974. For CNVs, the PPA is 1.0 and PPV is 0.969 and for indels, the PPA and PPV are 1.0 and 0.966, respectively. Overall concordance is 0.99. Conclusions: A targeted NGS cancer panel employing PCR-based enrichment on an IFC has been developed and yielded high quality of libraries generated on the JunoTM system for detecting SNVs, indels and CNV in solid tumor samples. This panel covers actionable targets in 53 cancer genes and utilizes a microfluidic device to provide a simple streamlined workflow for library preparation of up to 48 DNA samples per IFC. Citation Format: Peilin Chen, Jaibiao Gong, David Wang, Devin Do, Lianne McLean, Tom Goralski. Development of a targeted NGS panel for solid tumor actionable gene targets using multiplex PCR-based enrichment in an integrated fluidic circuit [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4651.

Abstract 5356: Robust gene fusion detection in formalin-fixed tissue sample with QIAseq RNAscan Oncology Panel

Abstract Introduction: Gene fusions are frequently associated with tumorigenesismalignant tumors. Highly sensitive and reproducible detection of gene fusions in clinical samples, especially formalin-fixed paraffin-embedded (FFPE) specimens, is important for diagnosis, prognosis, and treatment guidance. Although many clinical tests utilize next-generation-sequencing (NGS) for detection of genetic alterations, reliable detection of gene fusions by NGS in FFPE samples remains a challenge. Methods: Five FFPE non-small cell lung cancer samples confirmed by FISH to contain EML4-ALK or KIF5B-RET fusions were selected for testing along with the Seraseq™ FFPE Tumor Fusion RNA Reference Material, v1, which evaluates forcontains 12 known fusions. Four operators prepared NGS libraries for each sample (89-250ng input) using the commercially available QIAseq RNAscan Oncology Panel, which targets 223 fusion gene pairs or 576 unique breakpoints. The resulting libraries were sequenced on an Illumina® NextSeq 500 sequencer. Demultiplexed FASTQ files were analyzed using cloud-based QIAseq RNAscan Analysis pipeline v1.7.20.2. Detected fusions that are on the panel's target list were reported as curated gene fusions and assigned a score based on unique molecules supporting the transcript. Results: For the Seraseq™ FFPE Tumor Fusion RNA Reference v1 sample, all 12 expectedknown fusions were detected in each of the libraries generated by four different operators. The analysis pipeline correctly called the expected fusion in all four replicates of the five clinical FFPE samples tested, which varied in both input quantityamount and quality. Reproducibility was evaluated by comparing the score values across replicate libraries for each sample; expected fusion detection was 100% consistent between users, and the average correlation coefficient for the fusion score was 0.995. Conclusions: The commercially available QIAseq RNAscan Oncology panel is a highly sensitive and robust product for detection of gene fusions in FFPE samples. QIAseq RNAscan methodology provides a useful tool for studying gene fusions in tumor samples and with optimization, customization, and validation can further fit the specific needs for of clinical laboratories. Citation Format: Jeffrey Falk, Parth Sitlani, Claire Orosco, Maya Panjikaran, Dana Weiner, Song Tian, Raed Samara, John DiCarlo, Yexun Wang, Eric Lader, Frank Reinecke. Robust gene fusion detection in formalin-fixed tissue sample with QIAseq RNAscan Oncology Panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5356.

Clinical Utility of the T1D Genetic Risk Score—Examples from the U.S. Monogenic Diabetes Registry

Monogenic forms of diabetes are often difficult to distinguish from type 1 (T1D) and type 2 diabetes due to overlapping clinical features. As a result, a growing majority of patients remain misdiagnosed and often inappropriately treated. We used a T1D genetic risk score (T1D-GRS), generated from 10 T1D-associated common genetic variants, to help identify patients with a low T1D-GRS, and thus a potential monogenic etiology. We genotyped 798 probands from the U.S. Monogenic Diabetes Registry: 37% (n=293) had a known monogenic cause and 63% (n=505) had an unknown cause or were not exhaustively sequenced. To improve the discriminatory power of the T1D-GRS, participants were categorized using a T1D-GRS equivalent to the 5th, 25th, 50th, 75th, and 95th centile of the UK T1D cohort. 45% (132) of the registry participants with a confirmed monogenic cause had a T1D-GRS below the 5th percentile, 35% (103) had a T1D-GRS between the 5-25th percentile, 12% (35) between the 25-50th percentile, 5% (15) between the 50-75th percentile, and 2% (7) between the 75-95th percentile. Of the participants with an unknown cause, 24% (n=108) had a T1D-GRS below the 5th percentile, 30% (n=137) had a T1D-GRS between the 5-25th percentile, 16% (72) between the 25-50th percentile, 15% (67) between the 50-75th percentile, and 15% (69) between the 75-95th percentile. We sequenced a total of 124 participants with a T1D-GRS below the 25th percentile using a targeted next-generation sequencing library that includes more than 140 genes known to be associated with diabetes. 21% (n=24) were found to have a pathogenic variant in a known diabetes gene. Further tests are ongoing. Our results show that the T1D-GRS is a useful discriminatory tool between diabetes types and a strong predictor of whether a monogenic cause is likely. More importantly, patients with a low T1D-GRS and negative for all known monogenic subtypes should be prioritized for exome or whole genome sequencing to maximize the chance of novel gene discoveries. Disclosure M. Sanyoura: None. L.R. Letourneau: Other Relationship; Self; Novo Nordisk Inc.. R.N. Naylor: None. L.H. Philipson: Research Support; Self; Novo Nordisk Inc.. Speaker's Bureau; Self; Merck &amp; Co., Inc.. Research Support; Self; JAEB Center For Health Research, JDRF, Janssen Pharmaceuticals, Inc., Medtronic MiniMed, Inc.. M.N. Weedon: None. R.A. Oram: Other Relationship; Self; Randox. S.W. Greeley: Research Support; Self; Novo Nordisk Inc..

Development and Verification of an RNA Sequencing (RNA-Seq) Assay for the Detection of Gene Fusions in Tumors

We assessed the performance characteristics of an RNA sequencing (RNA-Seq) assay designed to detect gene fusions in 571 genes to help manage patients with cancer. Polyadenylated RNA was converted to cDNA, which was then used to prepare next-generation sequencing libraries that were sequenced on an Illumina HiSeq 2500 instrument and analyzed with an in-house developed bioinformatic pipeline. The assay identified 38 of 41 gene fusions detected by another method, such as fluorescence in situ hybridization or RT-PCR, for a sensitivity of 93%. No false-positive gene fusions were identified in 15 normal tissue specimens and 10 tumor specimens that were negative for fusions by RNA sequencing or Mate Pair NGS (100% specificity). The assay also identified 22 fusions in 17 tumor specimens that had not been detected by other methods. Eighteen of the 22 fusions had not previously been described. Good intra-assay and interassay reproducibility was observed with complete concordance for the presence or absence of gene fusions in replicates. The analytical sensitivity of the assay was tested by diluting RNA isolated from gene fusion-positive cases with fusion-negative RNA. Gene fusions were generally detectable down to 12.5% dilutions for most fusions and as little as 3% for some fusions. This assay can help identify fusions in patients with cancer; these patients may in turn benefit from both US Food and Drug Administration-approved and investigational targeted therapies.

Next-generation sequencing library construction on a surface

BackgroundNext-generation sequencing (NGS) has revolutionized almost all fields of biology, agriculture and medicine, and is widely utilized to analyse genetic variation. Over the past decade, the NGS pipeline has been steadily improved, and the entire process is currently relatively straightforward. However, NGS instrumentation still requires upfront library preparation, which can be a laborious process, requiring significant hands-on time. Herein, we present a simple but robust approach to streamline library preparation by utilizing surface bound transposases to construct DNA libraries directly on a flowcell surface.ResultsThe surface bound transposases directly fragment genomic DNA while simultaneously attaching the library molecules to the flowcell. We sequenced and analysed a Drosophila genome library generated by this surface tagmentation approach, and we showed that our surface bound library quality was comparable to the quality of the library from a commercial kit. In addition to the time and cost savings, our approach does not require PCR amplification of the library, which eliminates potential problems associated with PCR duplicates.ConclusionsWe described the first study to construct libraries directly on a flowcell. We believe our technique could be incorporated into the existing Illumina sequencing pipeline to simplify the workflow, reduce costs, and improve data quality.

Triazole linking for preparation of a next-generation sequencing library from single-stranded DNA.

Next-generation sequencing of single-stranded DNA (ssDNA) is attracting increased attention from a wide variety of research fields. Accordingly, various methods are actively being tested for the efficient adaptor-tagging of ssDNA. We conceived a novel chemo-enzymatic method termed terminal deoxynucleotidyl transferase (TdT)-assisted, copper-catalyzed azide-alkyne cycloaddition (CuAAC)-mediated ssDNA ligation (TCS ligation). In this method, TdT is used to incorporate a single 3′-azide-modified dideoxyribonucleotide onto the 3′-end of target ssDNA, followed by CuAAC-mediated click ligation of the azide-incorporated 3′-end to a 5′-ethynylated synthetic adaptor. This report presents the first proof-of-principle application of TCS ligation with its use in the preparation of a next-generation sequencing library.