Generation Sequencing Workflow Research Articles

Abstract 4416: Rapid, low-input, targeted NGS workflow for DNA methylation

Abstract DNA methylation is a fundamental epigenetic process that regulates human gene expression. In cancer, methylation changes promote proliferation networks and metastasis. Development of biomarkers for methylation will be enabled by flexible, fast, low-input next generation sequencing workflows. We describe the first Ion AmpliSeq™ Methylation targeted panel and protocol on the turn-key Genexus™ Integrated Sequencer as part an ongoing collaboration with Ontario Institute for Cancer Research (OICR) to detect and predict response in early stage breast cancer and improve diagnostics for Black and Asian women. A 2-pool Ion AmpliSeq™ Methylation Panel for Breast Cancer Research was developed as a demonstration of design, workflow, and reporting for targeted, low-input methylation assessment in a multiplex setting across a variety of samples sources including FFPE. The panel contains 327 amplicons and was designed to target both strands and 10ng DNA input into bisulfite conversion was used for controls. A complete workflow begins at bisulfite conversion and progresses through the Genexus™ Integrated Sequencer which combines library construction, template preparation, and sequencing into a single run. The bioinformatics pipeline provides DNA methylation calls on both Watson and Crick strands at single base resolution and methylated:unmethylated ratios for each CpG. The entire end-to-end workflow was completed in 2 days, including a full analysis software solution. The panel was evaluated using 2 control gDNA samples. The first had an expected average methylation state across all CpGs of >98% and the second <5%. An equal mixture of these two samples was also tested. The Methylation Panel for Cancer Research performed well on control samples. The single lane Loading, Total Bases, Final Reads, and Raw Read Accuracy were >92%, >1G, >14 Million, and >90% respectively. The workflow was also demonstrated on research FFPE cancer samples. This Methylation Panel protocol offers a 2-day, end-to-end workflow with high resolution, targeted and quantitative methylation analysis from DNA input as low as 10ng into bisulfite conversion. The option to design custom methylation panels for interrogation of targets of interest without the need for whole genome methylation is now available. <For Research Use Only. Not for use in diagnostic procedures. Early access materials were provided by Thermo Fisher Scientific™ to OICR in support of this abstract, but no other financial support. This study was conducted with the support of the Ontario Institute for Cancer Research through funding provided by the Government of Ontario. © 2023 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.> Citation Format: Loni Pickle, Andrew Hatch, Gokhan Yavas, Melanie Spears, Louis Gasparini, Vida Talebian, Anna Ying-Wah Lee, Mathieu Lariviere, Jane Bayani, Seth Sadis, Jeffrey M. Smith. Rapid, low-input, targeted NGS workflow for DNA methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4416.

Abstract 263: Automating next generation sequencing workflows for custom hyb panels: high quality library preparation and target enrichment on the Biomek NGeniuS System

Abstract This study investigates a custom 2Mb xGen Hyb panel designed against mutated gene targets implicated in several cancers using an end-to-end assay workflow on the Biomek NGeniuS platform. Automation in laboratories is advantageous to generate reproducible next generation sequencing (NGS) sample libraries, however, assay automation differs from instrument vendor to vendor but also from lab to lab with different deck layouts of the same instrument. These differences require a significant amount of optimization to design the workflow and begin generating NGS libraries. In addition to this optimization, some research, e.g., cancer research utilizes low input and/or degraded samples such as cfDNA from plasma or FFPE biopsies. Integrated DNA Technologies (IDT) and Beckman Coulter - Life Sciences together provide a walk away NGS automation solution for these research samples directly upon instrument installation with no optimization required by combining Beckman Coulter’s Biomek NGeniuS with IDT’s xGen cfDNA & FFPE DNA Library Prep kit and xGen Hybridization Capture. This trio provides a solution for laboratories to minimize technician hands-on time, error reduction, and rework while improving flexibility. IDT’s xGen cfDNA & FFPE DNA Library Prep kit utilizes novel chemistry to maximize sample input conversion, suppress adapter-dimer formation, and facilitate consensus analysis. IDT’s xGen Hybridization Capture products maintain high library diversity, obtain high on-target, and provide consistent and uniform sequencing coverage regardless of panel size. We demonstrate performance of this complete workflow with a custom 2Mb xGen Hyb panel yielding high library complexity, high on-target coverage, and capture uniformity. The combination of IDT’s xGen library preparation solutions on the NGeniuS Biomek platform provides a reliable and consistent solution that can be instantly downloaded from the portal straight to analysis of precious low input and degraded samples. Citation Format: Nicole Roseman, Laura Tucker, Francesco Criscuolo, Lynette Lewis, Li Liu, Tyler Buit, Jasmeen Mandair, Zachary Smith, Calvin Cortes. Automating next generation sequencing workflows for custom hyb panels: high quality library preparation and target enrichment on the Biomek NGeniuS System [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 263.

Development of a long-read next generation sequencing workflow for improved characterization of fastidious respiratory mycoplasmas.

Mycoplasma cynos and Mycoplasma felis are often associated with canine and feline infectious respiratory disease in dogs and cats, respectively. Mycoplasmas have a reduced genome and dearth of many biosynthetic pathways, making them dependent on rich medium for growth. Due to this fastidious nature, mycoplasmas have been historically underdiagnosed. The aim of this study was to develop a cost-effective and accurate sequencing workflow for genotypic characterization of clinical isolates of M. cynos and M. felis using a rapid long-read sequencing platform. We explored the following critical aspects of bacterial whole genome sequencing, including: (i) five solid and liquid-based culture approaches based on a specialized media formulation for Mycoplasma culture, (ii) three DNA extraction methods modified for long-read sequencing purposes, and (iii) two de novo assembly platforms, Flye and Canu, as key components of a bioinformatics pipeline. DNA extraction method 1, a solid-phase and column-based kit with enzymatic lysis, provided the best DNA quality and concentration followed by high coverage and sequencing contiguity. This was obtained with a culture volume of 45 ml in modified Hayflick's broth incubated for 48 h. DNA extracted directly from colonies on agar or from small broth volumes (6 ml) did not meet the criteria required for long-read sequencing. Overall, Flye generated more contiguous assemblies than the Canu assembler and was more time efficient. This 4-5 day sample-to-sequence workflow provides the scientific and clinical communities with a more comprehensive tool than laborious conventional methods for complete genomic characterization of M. cynos and M. felis clinical isolates.

ABE8e adenine base editor precisely and efficiently corrects a recurrent COL7A1 nonsense mutation

Base editing introduces precise single-nucleotide edits in genomic DNA and has the potential to treat genetic diseases such as the blistering skin disease recessive dystrophic epidermolysis bullosa (RDEB), which is characterized by mutations in the COL7A1 gene and type VII collagen (C7) deficiency. Adenine base editors (ABEs) convert A-T base pairs to G-C base pairs without requiring double-stranded DNA breaks or donor DNA templates. Here, we use ABE8e, a recently evolved ABE, to correct primary RDEB patient fibroblasts harboring the recurrent RDEB nonsense mutation c.5047 C > T (p.Arg1683Ter) in exon 54 of COL7A1 and use a next generation sequencing workflow to interrogate post-treatment outcomes. Electroporation of ABE8e mRNA into a bulk population of RDEB patient fibroblasts resulted in remarkably efficient (94.6%) correction of the pathogenic allele, restoring COL7A1 mRNA and expression of C7 protein in western blots and in 3D skin constructs. Off-target DNA analysis did not detect off-target editing in treated patient-derived fibroblasts and there was no detectable increase in A-to-I changes in the RNA. Taken together, we have established a highly efficient pipeline for gene correction in primary fibroblasts with a favorable safety profile. This work lays a foundation for developing therapies for RDEB patients using ex vivo or in vivo base editing strategies.

Abstract 215: Oncomineprecision assay - an integrated and automatic next generation sequencing workflow for both FFPE tissue and plasma based cancer research

Abstract Introduction: Next generation sequencing technologies and platforms have been evolving rapidly over the last decade with improved throughput and cost-effectiveness. The application of various NGS assays using both genome wide or targeted sequencing has been widely employed in cancer research to characterize tumor biomarker profiles including SNV/indels, CNV, and fusion in various sample types. Here we will present the performance of OncomineTM Precision Assay developed at Thermo Fisher Scientific, which is compatible with both solid tumor and liquid biopsy sample types. It is the very first automated NGS assay developed with Ion Torrent Genexus system to provide a streamlined workflow directly from sample to results with minimal hands-on time. Methods: The assay employs a novel AmpliSeq™ HD library technology to provide optimal sensitivity and specificity for challenging sample types including degraded FFPE or cell-free nucleic acid from plasma. For library preparation, 10 ng DNA or RNA was used for FFPE and 20 ng cfTNA was used for plasma samples. Ion Torrent Genexus system provides an interface to guide the user to plan the run following a defined workflow according to sample type, and performs library preparation, templating and sequencing automatically. Results: The OncomineTM Precision Assay includes focused genetic content with clinical relevance including 45 hotpot genes (SNV/Indel), 14 CNVs, and 19 structural variants (fusion and exon variants). This study evaluated the assay performance using a set of controls for FFPE or cfDNA from positive cell line material or commercial sources such as Horizon discovery and SeraCare, which contain various SNV/Indels on several important gene loci (EGFR, KRAS, NRAS, BRAR, and PIK3CA), CNV (EGFR, MET amplification and pTEN deletion), and fusion variants across ALK, ROS1, RET, and NTRKs. The results showed that all the expected SNV variants were consistently detected with sensitivity at ≥95% for FFPE with 5% AF and ≥85% for cfDNA samples down to 0.1% LOD. For fusion variants and CNV targets including gain and loss, both sensitivity and PPV at ≥90% could be achieved for FFPE and cfTNA samples. Furthermore, a cohort of positive FFPE tissue was also analyzed and detected expected variants that had been previously confirmed through an orthogonal NGS test. Lastly, a set of matched FFPE and plasma samples was also evaluated using the assay and demonstrated high concordance between FFPE and plasma from each subject. Conclusion: Overall the study demonstrates that the OncomineTM Precision Assay provides an automated and sensitive NGS solution for efficient and effective mutation assessment using both FFPE and liquid biopsy samples. Citation Format: Ru Cao, Jian Gu, Jeff Schageman, Kris Lea, Priyanka Kshatriya, Amir Marcovitz, Paul Williams, Bo Ding, Khalid Hanif, Kelli Bramlett. Oncomineprecision assay - an integrated and automatic next generation sequencing workflow for both FFPE tissue and plasma based cancer research [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 215.

Integration of Liquid Biopsies into Clinical Laboratory Applications via NGS in Cancer Diagnostics.

Next Generation Sequencing is one of the latest advances in molecular testing and clinical laboratory applications. Next Generation Sequencing techniques involving liquid biopsies are emerging as important tools in cancer diagnostics and prognostics. Thus, integration of liquid biopsy studies into clinical laboratory applications has become a necessity. By virtue of liquid biopsies, determining potential treatment targets through metastasis and primary tumor sites in the right clinical context can result in a more comprehensive treatment. This also helps to overcome re-sampling difficulties which require an invasive procedure with the problem of tumor heterogeneity. As the literature involving liquid biopsies and next generation sequencing increases, the rate of laboratories with competencies and experience in this novel technology remains limited. Next generation sequencing was performed via a comprehensive multi-gene cancer panel (Actionable In-sight Solid Tumor Panel, Qiagen) consisting of 12 solid tumor related genes (EGFR, ALK, KRAS, PIK3CA, NRAS, PDGFRA, KIT, ERBB2, ERBB3, ESR1, BRAF and RAF1) from lung cancer patients who applied or were referred to CU AGENTEM (Cukurova University Adana Genetic Diseases Diagnosis and Treatment Center) for routine genetic testing. A modified next generation sequencing workflow was performed with a multi-gene solid tumor panel using liquid biopsies in comparison with formalin fixed paraffin embedded samples to integrate this technique into the routine clinical laboratory applications and bioinformatics. In this study, next generation sequencing of liquid biopsies in cancer patients was integrated into cancer diagnostics. Liquid biopsy studies provide numerous advantages when integrated with next generation sequencing through a well-optimized workflow.

Sequencing of full mitochondrial genomes for NIST population samples

The U.S. National Institute of Standards and Technology in collaboration with the Armed Forces Medical Examiner System’s Armed Forces DNA Identification Laboratory has undertaken a population sequencing study of 607 mitochondrial genomes (mtGenomes). The goal of the study is to increase the availability of high-quality whole mtGenome sequences for haplotype frequency estimations, allowing the increased discrimination power of the entire extranuclear genome to be used. Whole mtGenome sequencing is enabled by next generation sequencing workflows, which are vastly simplified compared to Sanger-type sequencing. Here we evaluated the relative performance of mtGenome versus sequencing the control region of the mtGenome alone.

PO-086 An efficient ion torrent™ next generation sequencing workflow for liquid biopsy research to assess cell-free total nucleic acid

IntroductionLiquid biopsy research contributes to precision medicine initiatives and enables minimally invasive and inexpensive sampling compared to traditional tissue biopsy. However, the low amount of circulating tumour nucleic acid fragments in the blood presents significant challenges for accurate variant detection using NGS technology. Utilisation of both cell free (cf) DNA and cf RNA requires methods capable of interrogating both types of analytes to maximise the utility of each blood sample. We described here a two days sample-to-report workflow using Oncomine Pan-Cancer Cell-Free Assay that surveys oncology variants across multiple tumour types and simultaneously detects single nucleotide variants (SNVs) and structural variants such as copy number variations (CNVs), gene fusions as well as exon skipping.Material and methodsCell Free Total Nucleic Acid (cfTNA) was extracted using MagMAX Cell-Free TNA Isolation Kit. Internal 0.1% or 0.5% cfDNA reference materials were used to evaluate SNV sensitivity and specificity. For CNV sensitivity and specificity evaluation, cfDNA from CNV positive cell lines were titrated into normal donor plasma cfTNA background. All controls were verified with orthogonal assays of dPCR. Libraries were manually prepared and sequenced with Ion Chef and S5 XL System. Data analysis was performed in Torrent SuiteTM 5.6 and Ion Reporter 5.6.Results and discussionsThe Pan-Cancer Cell Free Research Assay utilised a single-pool multiplex assay to query more than 900 tumour driver and resistance hotspots. The broad content panel encompasses SNVs, CNVs, fusions, exon skipping as well as expanded coverage of TP53 exon regions for TP53 mutation analysis. The entire work flow (sample-to-report) could be as less as 30 hours and is compatible with Oncomine knowledgebase that allows customers easy access to the variant reports. The limit of detection (LOD) of 0.5% and 0.1% allelic frequency can be achieved with sensitivity of >99% and 90% respectively, both specificity >99.9%. Detection as low as 1.3 fold gene amplification can be obtained for 12 CNV targets with sensitivity of >96% and specificity of >99%. For fusion detection, as low as 1% RNA fusions in cfTNA can be achieved.ConclusionThe Pan-Cancer Cell Free Research Assay provides an easy and quick NGS workflow that simultaneously analyses SNVs, CNVs, gene fusions and exon skipping across 52 genes associated multiple cancers. The 0.1% LOD enables accurate detection of low-abundance tumour variants for liquid biopsy research.

Evaluation of preimplantation genetic testing for chromosomal structural rearrangement by a commonly used next generation sequencing workflow

ObjectivesTo evaluate the applicability of a commonly used next generation sequencing workflow in detecting unbalanced meiotic segregation products for reciprocal translocation and inversion carriers. Study designAll preimplantation genetic testing treatment cycles performed for reciprocal translocation or inversion carriers from 2012 to April 2017 were included. Three hundreds and forty-two archived whole genome amplified DNA, which had previously analyzed by array comparative genomic hybridization (aCGH), were retrospectively analyzed by next generation sequencing (NGS). Concordance on overall diagnosis and segmental aneuploidies related to the translocation/inversion breakpoints between aCGH and NGS were determined. ResultsRetrospective analysis of 287 blastomere biopsies and 55 trophectoderm (TE) biopsies showed that the concordance rate on the overall diagnosis between aCGH and NGS on abnormal samples was 100% (266/266), irrespective to the type of biopsy. The concordance rates of normal biopsies were 98.4% (61/62) on blastomere and 78.6% (11/14) on TE biopsies. NGS detected a de novo segmental aneuploidy on one blastomere biopsy and three possible low level mosaic aneuploidies on 3 TE biopsies, which were previously concluded as euploid by aCGH. Using the karyotype of reciprocal translocation/inversion carriers, size of anticipated segmental aneuploidies could be calculated and be used to predict the applicability of NGS before proceeding to treatment. ConclusionThis is the first report to evaluate the applicability of a commercial NGS-based workflow for preimplantation testing for reciprocal translocations/inversions. Our study demonstrated that NGS can diagnose unbalanced translocation/inversion products with the same efficiency as aCGH. The applicability of NGS, with respect to individual karyotype, can be predicted before proceeding to treatment.

ANAQUIN: a software toolkit for the analysis of spike-in controls for next generation sequencing.

Spike-in controls are synthetic nucleic-acid sequences that are added to a user's sample and constitute internal standards for subsequent steps in the next generation sequencing workflow. : The software is implemented in C ++/R and is freely available under BSD license. The source code is available from github.com/student-t/Anaquin , binaries and user manual from www.sequin.xyz/software and R package from bioconductor.org/packages/Anaquin. anaquin@garvan.org.au or t.mercer@garvan.org.au. Supplementary data are available at Bioinformatics online.