Next-generation Sequencing Library Preparation Method Research Articles

A novel NGS library preparation method to characterize native termini of fragmented DNA

Biological and chemical DNA fragmentation generates DNA molecules with a variety of termini, including blunt ends and single-stranded overhangs. We have developed a Next Generation Sequencing (NGS) assay, XACTLY, to interrogate the termini of fragmented DNA, information traditionally lost in standard NGS library preparation methods. Here we describe the XACTLY method, showcase its sensitivity and specificity, and demonstrate its utility in in vitro experiments. The XACTLY assay is able to report relative abundances of all lengths and types (5′ and 3′) of single-stranded overhangs, if present, on each DNA fragment with an overall accuracy between 80–90%. In addition, XACTLY retains the sequence of each native DNA molecule after fragmentation and can capture the genomic landscape of cleavage events at single nucleotide resolution. The XACTLY assay can be applied as a novel research and discovery tool for fragmentation analyses and in cell-free DNA.

P.295Linked-read whole genome sequencing in patients with congenital myopathy

Over the past decade, Next Generation Sequencing (NGS) methods have revolutionised the field of genetics. Currently, the usage of Whole Genome Sequencing (WGS) in addition to Whole Exome Sequencing (WES) and panel sequencing is increasing in research and in clinical diagnostics. In this study, we wanted to test whether a new NGS application — the Linked-Read (LR) library preparation — can improve the identification of disease-causing variants in patients with congenital myopathy. For LR library preparation, gDNA molecules are encapsulated inside emulsion droplets. Every microreaction provides a unique barcode to each of the DNA fragments inside the droplet. In the data analysis step, the barcoded short-read sequences are linked up to each other, providing long-range information. In addition to regular WGS information, this enables the identification of structural variants such as insertions or translocations. It also resolves the haplotypes of the patient. Moreover, LRs have the potential to improve the short-read alignment of repetitive regions in genes such as nebulin and titin. We selected three patients with a congenital myopathy for this pilot study. The prior analysis using targeted Sanger sequencing of candidate genes, a custom neuromuscular gene panel or WES had failed to identify the genetic cause of their disorder. Furthermore, all patient samples had previously been analysed for Copy Number Variation (CNV) using a Comparative Genomic Hybridisation array (CGH-array) for neuromuscular disorders designed by our group, but the molecular diagnosis had remained unresolved. Our preliminary results indicate that LR NGS library preparation method adds valuable information, especially of structural variation, and is likely to improve the diagnostics of neuromuscular disorders.

SMARTcleaner: identify and clean off-target signals in SMART ChIP-seq analysis

BackgroundNoises and artifacts may arise in several steps of the next-generation sequencing (NGS) process. Recently, an NGS library preparation method called SMART, or Switching Mechanism At the 5′ end of the RNA Transcript, is introduced to prepare ChIP-seq (chromatin immunoprecipitation and deep sequencing) libraries from small amount of DNA material, using the DNA SMART ChIP-seq Kit. The protocol adds Ts to the 3′ end of DNA templates, which is subsequently recognized and used by SMART poly(dA) primers for reverse transcription and then addition of PCR primers and sequencing adapters. The poly(dA) primers, however, can anneal to poly(T) sequences in a genome and amplify DNA fragments that are not enriched in the immunoprecipitated DNA templates. This off-target amplification results in false signals in the ChIP-seq data.ResultsHere, we show that the off-target ChIP-seq reads derived from false amplification of poly(T/A) genomic sequences have unique and strand-specific features. Accordingly, we develop a tool (called “SMARTcleaner”) that can exploit these features to remove SMART ChIP-seq artifacts. Application of SMARTcleaner to several SMART ChIP-seq datasets demonstrates that it can remove reads from off-target amplification effectively, leading to significantly improved ChIP-seq peaks and results.ConclusionsSMARTcleaner could identify and clean the false signals in SMART-based ChIP-seq libraries, leading to improvement in peak calling, and downstream data analysis and interpretation.

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.

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.

Next-generation sequencing library preparation method for identification of RNA viruses on the Ion Torrent Sequencing Platform.

Next generation sequencing (NGS) is a powerful tool for the characterization, discovery, and molecular identification of RNA viruses. There were multiple NGS library preparation methods published for strand-specific RNA-seq, but some methods are not suitable for identifying and characterizing RNA viruses. In this study, we report a NGS library preparation method to identify RNA viruses using the Ion Torrent PGM platform. The NGS sequencing adapters were directly inserted into the sequencing library through reverse transcription and polymerase chain reaction, without fragmentation and ligation of nucleic acids. The results show that this method is simple to perform, able to identify multiple species of RNA viruses in clinical samples.

Tagmentation on Microbeads: Restore Long-Range DNA Sequence Information Using Next Generation Sequencing with Library Prepared by Surface-Immobilized Transposomes.

The next generation sequencing (NGS) technologies have been rapidly evolved and applied to various research fields, but they often suffer from losing long-range information due to short library size and read length. Here, we develop a simple, cost-efficient, and versatile NGS library preparation method, called tagmentation on microbeads (TOM). This method is capable of recovering long-range information through tagmentation mediated by microbead-immobilized transposomes. Using transposomes with DNA barcodes to identically label adjacent sequences during tagmentation, we can restore inter-read connection of each fragment from original DNA molecule by fragment-barcode linkage after sequencing. In our proof-of-principle experiment, more than 4.5% of the reads are linked with their adjacent reads, and the longest linkage is over 1112 bp. We demonstrate TOM with eight barcodes, but the number of barcodes can be scaled up by an ultrahigh complexity construction. We also show this method has low amplification bias and effectively fits the applications to identify copy number variations.

Abstract 3624: Development of a comprehensive and highly sensitive next-generation sequencing assay for detection of copy number variations

Abstract Background: As our knowledge of how DNA alterations can drive cancer progression increases, assays that can simultaneously detect multiple types of variants in a simple and cost-effective manner are becoming increasingly crucial. This holds true of copy number variations (CNVs), where evaluation of this type of variant is an important and necessary feature of any solid tumor profiling assay. Conventional methods for detecting CNVs such as immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and quantitative PCR (qPCR) are limited to detecting only one gene amplification at a time. This can be a significant drawback with FFPE samples, where the DNA is of low abundance and often heavily degraded, while the number of gene amplifications known to be important in cancer continues to grow. Additionally, different tumor types may express amplifications at different rates and expression may be heterogeneous within the tumor, potentially with irregular staining patterns - all of which illustrate the need for new approaches to CNV detection. Methods: Next-generation sequencing (NGS) offers the ability to assess variants in multiple genes using one sample. To that end, Illumina is developing a comprehensive, hybrid capture-based NGS assay targeting 170 key cancer genes for sequencing on the NextSeq1 platform. The assay consists of a DNA workflow for the identification of single-nucleotide variants (SNVs), small insertions and deletions (indels), as well as an RNA workflow for the identification of splice variants and gene fusions. In addition, using the DNA workflow, a novel analysis pipeline, and CNV caller, CNVs from 57 different genes can be simultaneously assessed all by sequencing of a single sample. Results: Here we present data on both cell lines and FFPE samples of SNVs and indels down to 5% allele fraction and CNVs down to ∼2-fold amplification, all from 40 ng of DNA. To demonstrate the accuracy and precision of our CNV detection method, we tested 7 samples for CNVs using orthogonal CNV detection methods. The Illumina NGS assay detected ERBB2 amplifications in 4 out of 7 samples. Of the 4 Illumina NGS positive samples, 3 samples were positive by FISH and all 4 were positive by droplet digital PCR (ddPCR) and the Illumina TruSight Tumor 15 panel. The 3 samples that were negative for ERBB2 amplifications by the Illumina NGS assay were also negative by both FISH and ddPCR. Within these samples we also found a previously unknown FGFR1 amplification. Conclusions: The novel Illumina NGS library preparation method is an innovative and useful tool to find multiple CNVs, along with other variant types, within a single sample. The assay can detect multiple CNVs within a single FFPE sample and identify previously uncharacterized CNVs that could be important in finding the correct treatment for a cancer patient. Citation Format: Chia-Ling Hsieh, Clare Zlatkov, Byron Luo, Chen Zhao, Kathryn Stephens, Han-Yu Chuang, Lisa Kelly, Katherine Chang, Rachel Liang, Jianli Cao, Scott Lang, Ashley Adams, Naseem Ajili, Laurel Ball, Glorianna Caves, Danny Chou, Katie Clark, Brian Crain, Anthony Daulo, Sarah Dumm, Ridwana Ekram, Yonmee Han, Anne Jager, Suzanne Johansen, Li Teng, Jenn Lococo, Jaime McLean, Juli Parks, Jason Rostron, Jennifer Sayne, Jennifer Silhavy, June Snedecor, Mckenzi Toh, Stephanie Tong, Elizabeth Upsall, Paulina Walichiewicz, Xiao Chen, Amanda Young, Ali Kuraishy, Karen Gutekunst, Matt Friedenberg, Charles Lin. Development of a comprehensive and highly sensitive next-generation sequencing assay for detection of copy number variations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3624.

Abstract 3953: Highly accurate genetic profiling of circulating tumor cells using a label-free inertial microfluidic approach coupled with droplet PCR-based next-generation sequencing

Abstract Background: Circulating tumor cells (CTCs) could provide significant insights into cancer metastatic events and potentially even inform clinical decisions. However, the scarcity of CTCs, compounded by the abundance of circulating white blood cells, poses a major technical challenge in the isolation, yield and downstream molecular analysis. Moreover, next-generation sequencing (NGS) of CTCs has been challenged by low CTC purity and nucleic acid yields commonly derived from existing technologies. Here, we present a label-free microfluidic approach that utilizes curvilinear microchannel geometry for high purity CTC enrichment from 7.5 ml whole blood. Methods: We showed high purity isolation of CTCs using a label-free inertial focusing microfluidics platform, ClearCell® FX. The lung cancer cell line, NCI-H1975, with known EGFR, CDKN2A and TP53 mutations, were spiked into 7.5ml healthy donor peripheral blood samples at varying concentrations (50, 100, 200 cells). DNA was extracted using the QIAamp Micro kit. The Thunderbolts Cancer Panel droplet digital PCR NGS library preparation method was used to enrich 230 loci in 50 known cancer genes from the isolated CTC gDNA. The libraries were sequenced on a MiSeq system. Results: The CTCs were sufficiently enriched with an average recovery of 45.8% and purities ranging from 1% - 70%. All libraries were successfully generated and sequenced with ≥1400× mean coverage, even with as little as 2 ng of starting material. Known EGFR, CDKN2A and TP53 mutations in H1975 were successfully detected in all the samples (n = 9), with a significant variant frequency that correlates with CTC purity levels. Conclusion: We have demonstrated a highly accurate and sensitive workflow utilizing a combination of inertial focusing and digital droplet microfluidics to detect low numbers of rare CTCs and interrogate their molecular profiles from as little as 10 cells/mL frequency. This workflow has the potential for further development and adoption as a liquid biopsy approach for non-invasive genomic profiling of metastatic cancers. Citation Format: Yi Fang Lee, Mark Consugar, Kimberly Helzer, Mei Hui Tan, Lori Emrick, Ali Asgar Bhagat. Highly accurate genetic profiling of circulating tumor cells using a label-free inertial microfluidic approach coupled with droplet PCR-based next-generation sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3953.

Control for stochastic sampling variation and qualitative sequencing error in next generation sequencing

BackgroundClinical implementation of Next-Generation Sequencing (NGS) is challenged by poor control for stochastic sampling, library preparation biases and qualitative sequencing error. To address these challenges we developed and tested two hypotheses. MethodsHypothesis 1: Analytical variation in quantification is predicted by stochastic sampling effects at input of (a) amplifiable nucleic acid target molecules into the library preparation, (b) amplicons from library into sequencer, or (c) both. We derived equations using Monte Carlo simulation to predict assay coefficient of variation (CV) based on these three working models and tested them against NGS data from specimens with well characterized molecule inputs and sequence counts prepared using competitive multiplex-PCR amplicon-based NGS library preparation method comprising synthetic internal standards (IS). Hypothesis 2: Frequencies of technically-derived qualitative sequencing errors (i.e., base substitution, insertion and deletion) observed at each base position in each target native template (NT) are concordant with those observed in respective competitive synthetic IS present in the same reaction. We measured error frequencies at each base position within amplicons from each of 30 target NT, then tested whether they correspond to those within the 30 respective IS. ResultsFor hypothesis 1, the Monte Carlo model derived from both sampling events best predicted CV and explained 74% of observed assay variance. For hypothesis 2, observed frequency and type of sequence variation at each base position within each IS was concordant with that observed in respective NTs (R2=0.93). ConclusionIn targeted NGS, synthetic competitive IS control for stochastic sampling at input of both target into library preparation and of target library product into sequencer, and control for qualitative errors generated during library preparation and sequencing. These controls enable accurate clinical diagnostic reporting of confidence limits and limit of detection for copy number measurement, and of frequency for each actionable mutation.

Next-generation sequencing of custom amplicons to improve coverage of HaloPlex multigene panels.

Next-generation sequencing (NGS) of multigene panels performed for genetic clinical diagnostics requires 100% coverage of all targeted genes. In the genetic diagnostics laboratory, coverage gaps are typically filled with Sanger sequencing after NGS data are collected and analyzed. Libraries prepared using the hybridization-based custom capture HaloPlex method are covered at ~98% and include gaps in coverage because of the location of the restriction enzyme sites used for fragmentation and differences in the designed and actual library insert size. We describe a method for improving the coverage of HaloPlex libraries by generating a set of amplicons spanning known low-coverage regions that are pooled, indexed by sample, and sequenced together with the HaloPlex libraries. This approach reduces the number of post-NGS Sanger sequencing reactions required and complements any NGS library preparation method when complete gene coverage is necessary.