Mark Andersen

Variation in the promoter region of the myeloperoxidase gene is not directly related to lung cancer risk among male smokers in Finland

In order to examine whether a polymorphism in the promoter region of the myeloperoxidase (MPO) gene is associated with lung cancer among male smokers, we conducted a case-control study nested within a Finnish clinical trial cohort. Although we found no evidence of an overall association between lung cancer risk and MPO genotype, the variant MPO genotype was associated with an increased risk of lung cancer among a subset of older men. These findings contrast with those from previous studies that report decreased lung cancer risk among MPO variant individuals.

Assessing copy number alterations in targeted, amplicon-based next-generation sequencing data.

Changes in gene copy number are important in the setting of precision medicine. Recent studies have established that copy number alterations (CNAs) can be detected in sequencing libraries prepared by hybridization-capture, but there has been comparatively little attention given to CNA assessment in amplicon-based libraries prepared by PCR. In this study, we developed an algorithm for detecting CNAs in amplicon-based sequencing data. CNAs determined from the algorithm mirrored those from a hybridization-capture library. In addition, analysis of 14 pairs of matched normal and breast carcinoma tissues revealed that sequence data pooled from normal samples could be substituted for a matched normal tissue without affecting the detection of clinically relevant CNAs (>|2| copies). Comparison of CNAs identified by array comparative genomic hybridization and amplicon-based libraries across 10 breast carcinoma samples showed an excellent correlation. The CNA algorithm also compared favorably with fluorescence in situ hybridization, with agreement in 33 of 38 assessments across four different genes. Factors that influenced the detection of CNAs included the number of amplicons per gene, the average read depth, and, most important, the proportion of tumor within the sample. Our results show that CNAs can be identified in amplicon-based targeted sequencing data, and that their detection can be optimized by ensuring adequate tumor content and read coverage.

Deep sequencing of HPV16 genomes: A new high-throughput tool for exploring the carcinogenicity and natural history of HPV16 infection

For unknown reasons, there is huge variability in risk conferred by different HPV types and, remarkably, strong differences even between closely related variant lineages within each type. HPV16 is a uniquely powerful carcinogenic type, causing approximately half of cervical cancer and most other HPV-related cancers. To permit the large-scale study of HPV genome variability and precancer/cancer, starting with HPV16 and cervical cancer, we developed a high-throughput next-generation sequencing (NGS) whole-genome method. We designed a custom HPV16 AmpliSeq™ panel that generated 47 overlapping amplicons covering 99% of the genome sequenced on the Ion Torrent Proton platform. After validating with Sanger, the current “gold standard” of sequencing, in 89 specimens with concordance of 99.9%, we used our NGS method and custom annotation pipeline to sequence 796 HPV16-positive exfoliated cervical cell specimens. The median completion rate per sample was 98.0%. Our method enabled us to discover novel SNPs, large contiguous deletions suggestive of viral integration (OR of 27.3, 95% CI 3.3–222, P=0.002), and the sensitive detection of variant lineage coinfections. This method represents an innovative high-throughput, ultra-deep coverage technique for HPV genomic sequencing, which, in turn, enables the investigation of the role of genetic variation in HPV epidemiology and carcinogenesis.

Abstract 1631: Sequencing the circulating and infiltrating T-cell repertoire on the Ion S5TM

T-cell repertoire (TCR) sequencing by next-generation sequencing (NGS) is a valuable tool for building a deeper understanding of the adaptive immune system. As immunotherapies, particularly T-cell dependent therapies, show increasing potential in treating cancer, the ability to gain a detailed, unbiased view of the TCR becomes imperative for biomarker discovery, immune response to treatment, and study of tumor microenvironments. A key question the field seeks to understand is the relationship between circulating T-cells and infiltrating T-cells at the tumor site. Here, we present a novel approach for TCR sequencing using the Ion S5 ™ sequencer which leverages simplified library construction workflows and offers a more complete characterization of the entire V(D)J region of TCRB. This method can leverage mRNA as input, minimizing requirements in starting materials and focusing sequencing to productive TCRB arrangements. This approach targets the constant (C) and the FR1 regions, minimizing the potential for primer bias and greatly increasing the phylogenetic information content compared to techniques that exclusively characterize the CDR3 domain. Our results show that the observed circulating T-cell repertoire size is approximately 2 orders of magnitude higher than the infiltrating T-cell repertoire. Accordingly, while it is difficult to fully capture the complete repertoire of circulating T-cells due to its vast diversity, we show that it is possible to reliably capture the complete infiltrating T-cell repertoire with as high as 10 samples on the Ion 530 ™ chip. Replicate sequencing runs of infiltrating T-cells offers correlation of ~0.9, indicating that the results were reproducible, and the samples were sequenced to appropriate depth. In summary, we believe that this workflow will allow researchers to more routinely characterize the infiltrating T-cell repertoire and offers the field a better understanding of the impact of repertoire diversity on tumor elimination. Citation Format: Geoffrey Lowman, Elizabeth Linch, Lauren Miller, Denise Topacio-Hall, Timothy Looney, Alex Pankov, Yongming Sun, Xinzhan Peng, Mark Andersen, Fiona Hyland, Ann Mongan. Sequencing the circulating and infiltrating T-cell repertoire on the Ion S5TM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1631. doi:10.1158/1538-7445.AM2017-1631

Abstract 3567: Sequencing the human TCRβ repertoire on the Ion S5™ System

Next-generation Sequencing (NGS) is proving an important tool in increasing understanding of the human immune system, and thereby cancer immunology. αβ-T cells are the primary constituents of human cell-mediated adaptive immunity. The antigen specificity of each αβ-T cell is encoded in the 500-600 bp transcript encompassing the variable portion of the rearranged TCRα and TCRβ subunits, which can be read via NGS in a process termed repertoire sequencing. Until now, the main challenge the field faces is the lack of a technology that can provide a contiguous read of 600 bp to minimize the complexity of designing bias-prone primers and informatics challenges of stitching short reads. Here we leverage the long read capability of Ion 530™ chip to comprehensively sequence all three CDR domains of the TCRβ chain. The Ion 530™ chip offers greater than 15 M productive reads, allowing a multiplex of 2-4 samples with sufficient coverage for most repertoire profiling studies. Initial testing with Leukocyte total RNA demonstrates that this multiplex PCR assay produced repertoires that were much more similar to data derived from 5’RACE protocol than the commonly used BIOMED2 primer set. This result suggested that the use of long reads minimizes bias by allowing targeting of less variable regions. To further assess the performance of the assay, we designed a model system of 30 plasmid controls containing common human T-cell CDR3 sequences. Each plasmid was amplified individually and sequenced to confirm the detection of a single clonal population. Analytical sensitivity of the assay and accuracy of the accompanied analysis solution were further evaluated by spiking in plasmid concentrations from 10 pg to 0.0001 pg (5 million to 50 copies) in a background of 100 ng cDNA reverse transcribed from leukocyte total RNA. Results showed the assay offers linearity over 5 orders of magnitude of decreasing input concentration. In summary, we have demonstrated a NGS workflow for TCRβ sequencing that offers multiplex flexibility on Ion S5 with sample to answer in less than 48 hours. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Denise S. Topacio-Hall, Tim Looney, Yongming Sun, Lauren Miller, Elizabeth Linch, Geoffrey Lowman, Lifeng Lin, Mark Andersen, Fiona Hyland, Ann Mongan. Sequencing the human TCRβ repertoire on the Ion S5™ System [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3567. doi:10.1158/1538-7445.AM2017-3567

Abstract 3627: Automated, purification-free, Ion AmpliSeq™ library preparation from FFPE samples using the Ion Torrent™ Ion Chef™ System

The ability to produce high quality libraries from FFPE samples is ever more important for next-generation sequencing. Quick turnaround time from sample to answer and simple protocols are critical components for this process. The Ion Chef and Ion AmpliSeq Kit for Chef DL8 is an automated system that has proven to reliably generate quality libraries from purified high molecular weight genomic DNA, in addition to formalin-fixed paraffin embedded (FFPE) DNA samples. We have further developed a simple and robust protocol for preparing DNA libraries from FFPE samples with no nucleic acid purification required. Our protocol is compatible with all Ion AmpliSeq panels and generates eight balanced libraries on the Ion Chef Instrument in under 8 hours, with less than 1 hour hands-on time. We observed equivalent or better sequencing performance using this method compared to a 16-hour manual FFPE DNA extraction protocol. This method has shown to work with low, medium, and high input FFPE samples from different tissue types on a single run. We evaluated a variety of FFPE samples using 1 or 2 pool Ion AmpliSeq DNA research panels containing assays designed to known oncogenes. We were able to generate and successfully sequence DNA libraries from a single FFPE slice as small as 2 mm X 2 mm. Minimal sample handling is an advantage for the NGS platform with this robust protocol. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Tanya Biorac, Michael Allen, Francis B. Peters, Cristina Van Loy, Kate Rhodes, Mark Andersen. Automated, purification-free, Ion AmpliSeq™ library preparation from FFPE samples using the Ion Torrent™ Ion Chef™ System. [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 3627.

Abstract 2042: A next-generation sequencing-based sample-to-result pharmacogenomics research solution enables both SNV and CNV detection at once

Pharmacogenomics (PGx) is the study of genetic variations in terms of their response to drugs. Variations in gene sequence or copy number may result in complete loss of function, partial decrease or increase in enzyme activity, or an altered affinity for substrates, which may in turn significantly impact a drug9s efficacy. PGx studies are increasing in significance as precision medicine is becoming a reality in standard practice. Different technologies have been developed to measure the sequence variation and copy number variation (CNV) in the PGx genes. Among them, a complete sample-to-result PGx workflow solution using the QuantStudio™ 12k Flex Real-Time PCR System is the most notable high throughput solution and has broad adoption by advanced PGx laboratories. Both PGx SNP/INDEL genotyping assays on OpenArray™ plates and copy number analysis on 384-well plates can be performed on the QuantStudio™ 12k Flex System. Integrated analysis software translates genotyping and copy number assay results into star allele genotypes for ease of interpretation. Recently we have developed a next generation sequencing (NGS) based PGx research solution with increased flexibility on the assay targets and combined detection of SNP/INDEL genotyping and CNV using Ion AmpliSeq™ technology for low to medium throughput laboratories. With a highly multiplexed PGx research panel, we can profile a set of 136 genetic markers in 40 known PGx related genes and CYP2D6 copy number variation in a single reaction using Ion Torrent™ semiconductor sequencing. The number of genetic markers can be customized easily based on the user need. To systematically compare these two end-to-end PGx workflows, we collected buccal swab samples from 20 individuals and performed both QuantStudio™ based assays and PGM™ based Ion AmpliSeq™ PGx research assay on them. Both systems generated high quality results. Compared with OpenArray™ plate genotyping results and 384-plate CYP2D6 copy number assay results from the QuantStudio™ system, the Ion AmpliSeq™ PGx research solution demonstrated >99.9% genotyping concordance, 100% CYP2D6 gene CNV concordance, >99.7% reproducibility, Citation Format: Melvin S. Wei, Zhoutao Chen, Shann-Ching Chen, Manimozhi Manivannan, Emily Zeringer, Sunali Patel, Toinette Hartshorne, Guoying Liu, Fiona Hyland, Mark Andersen. A next-generation sequencing-based sample-to-result pharmacogenomics research solution enables both SNV and CNV detection at once. [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 2042.

Abstract 4933: Sanger sequencing of low amount of genomic DNA and FFPE DNA with PCR primers derived from the Ion AmpliSeq cancer hotspot panel

The introduction of defined Ion AmpliSeq™ panels for detection and characterization of actionable mutations occurring in tumor tissue has the potential to revolutionize translational oncology research. The Ion Ampliseq™ cancer hotspot panel version 2 (CHP v2) by Ion Torrent includes 207 actionable mutation targets present in 50 genes and the more comprehensive Ion Oncomine™ cancer panel (OCP) contains over 2000 mutations. A hallmark of these Ion Torrent Ampliseq cancer panels is the low amount of input DNA needed which is critical when the clinical specimen material is limited such as with fine needle biopsy or FFPE samples. Typically, 10 ng of DNA obtained from these sources is sufficient to produce informative sequencing data. Often, cancer-causing or promoting mutations are detected at relatively low allele frequencies like 10-20% compared to the major normal allele. Many researchers wish to verify these findings of low frequency mutations by an orthologous method such as traditional dye-fluorescent Sanger sequencing on a capillary electrophoresis (CE) instrument such as the Applied Biosystems 3500 genetic analyzer. To that end, we have developed a workflow that enables the amplification and traditional Sanger sequencing of individual Ion AmpliSeq targets directly from the AmpliSeq library starting material. The method requires a retainer of 1 μl (∼ 5%) of the original AmpliSeq preamplification material. A dilution of this aliquot is used as template source for individualized PCR/sequencing reactions. We show that a random selection of 48 targets from the CHPv2 panel could be successfully amplified and Sanger-sequenced from an Ion Torrent Ampliseq library originally prepared from 10 ng of FFPE DNA. Furthermore, we show the successful Sanger-re-sequencing of all individual 24 targets covering the TP53 exons from the same sample processed and pre-amplified with the OncoMine AmpliSeq panel. Taken together, this method will enable researchers to reflex-test potential mutations of interest from very material-limited specimen using Sanger CE sequencing. Citation Format: Edgar H. Schreiber, Adam Broomer, Mark Andersen, Kamini Varma. Sanger sequencing of low amount of genomic DNA and FFPE DNA with PCR primers derived from the Ion AmpliSeq cancer hotspot panel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4933. doi:10.1158/1538-7445.AM2015-4933