Abstract 5272: Cloud-based informatics enables the design and analysis of massively multiplex custom gene fusion panels for next-generation sequencing on FFPE RNA samples
Fiona HylandRajesh GottimukkalaEfren BallesterosHeinz BreuYuandan LouScott P. MyrandMichael HoganKelli BramlettGuoying LiuSeth Sadis
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Abstract Gene fusions, a combination of two genes, comprising their coding and/or regulatory sequences, are caused by structural rearrangements in DNA or in RNA transcripts. Many gene fusions are strong driver mutations in neoplasia, and are important in understanding basic biology, interaction with targeted therapy, and research into risk stratification and outcomes. Next-generation sequencing enables sensitive, specific and precise detection of particular fusion isoforms for defined gene pairs. Massively multiplex Ampliseq gene fusion assays enable enrichment of fusion transcripts using as little as 10 ng of RNA extracted from FFPE samples. Sequencing on Ion Torrent instruments reveals the full sequence of the gene fusion, for precise definition of the breakpoint and the expressed exons or promoter regions of both genes. We developed cloud-based software to support the design of a custom Ampliseq gene fusion panel, comprising 1 to 1,000 fusion isoform assays and any gene expression assays for normalization. We extensively mined the scientific literature on fusions and the COSMIC database to identify over 1000 fusion isoforms. We rigorously curated this data using automated and manual methods, including mapping, confirmation and correction of reported sequence to obtain genomic coordinates, identification of breakpoints, annotation of exon junctions, and selected wet lab testing. We created a database containing over 1000 high quality curated and annotated fusion isoforms, including 70 ALK, 60 RET, 26 ROS1, and 21 NTRK1 fusions. We designed Ampliseq primer pairs for each of these fusions using advanced assay design and pooling algorithms, such that all fusion and gene expression assays can be multiplexed into 1 or 2 compatible pools. Assays can be selected by gene or gene pair; detailed information about each assay selected includes isoform, genes, exon numbers, and links to COSMIC and to relevant publications. We developed cloud-based analysis software to analyze the BAM file resulting from amplification and sequencing of custom Ampliseq fusion panels on an Ion Torrent sequencer. This analysis leverages the rich annotation information from the assay design. The reads are mapped to a custom reference sequence tailored to the custom Ampliseq fusion assay, and applying an optimized algorithm to select confidently mapped reads based on read length and overlap with each gene of the gene pair based on the reference and annotated breakpoint. Gene fusions are detected based on the total number of fusion reads and optionally frequency, and on the properties of those reads. Software QC steps for total number of mapped reads, number of reads for gene expression controls, and elimination of cross-talk artifacts result in a highly sensitive and specific detection of fusions, with LOD below 1%. Fusion results for any or all samples can be viewed, annotated, filtered, and visualized, and exported. Citation Format: Fiona Hyland, Rajesh Gottimukkala, Efren Ballesteros, Heinz Breu, Yuandan Lou, Scott Myrand, Michael Hogan, Kelli Bramlett, Guoying Liu, Seth Sadis. Cloud-based informatics enables the design and analysis of massively multiplex custom gene fusion panels for next-generation sequencing on FFPE RNA samples. [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 5272.Keywords:
Ion semiconductor sequencing
Massive parallel sequencing
Multiplex
Massive parallel sequencing
Personal genomics
Ion semiconductor sequencing
Hybrid genome assembly
Cancer genome sequencing
Illumina dye sequencing
Single cell sequencing
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Abstract The Early Access AmpliSeq™ Mitochondrial Panel amplifies whole mitochondrial genomes for phylogenetic and kinship identifications, using Ion Torrent™ technology. There is currently limited information on its performance with degraded DNA, a common occurrence in forensic samples. This study evaluated the performance of the Panel with DNA samples degraded in vitro, to mimic conditions commonly found in forensic investigations. Purified DNA from five individuals was heat‐treated at five time points each (125°C for 0, 30, 60, 120, and 240 min; total n = 25). The quality of DNA was assessed via a real‐time DNA assay of genomic DNA and prepared for massively parallel sequencing on the Ion Torrent™ platform. Mitochondrial sequences were obtained for all samples and had an amplicon coverage averaging between 66X to 2803X. Most amplicons (157/162) displayed high coverages (452 ± 333X), while reads with less than 100X coverage were recorded in five amplicons only (90 ± 5X). Amplicon coverage was decreased with prolonged heating. At 72% strand balance, reads were well balanced between forward and reverse strands. Using a coverage threshold of ten reads per SNP, complete sequences were recovered in all samples and resolved kinship and, haplogroup relations. Additionally, the HV1 and HV2 regions of the reference and 240‐min heat‐treated samples ( n = 10) were Sanger‐sequenced for concordance. Overall, this study demonstrates the efficacy of a novel forensic Panel that recovers high quality mitochondrial sequences from degraded DNA samples.
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Abstract Massively parallel DNA sequencing is a critical tool for genomics research and clinical diagnostics. Here, we describe the Association of Biomolecular Resource Facilities (ABRF) Next-Generation Sequencing Phase II Study to measure quality and reproducibility of DNA sequencing. Replicates of human and bacterial reference DNA samples were generated across multiple sequencing platforms, including well-established technologies such as Illumina, ThermoFisher Ion Torrent, and Pacific Biosciences, as well as emerging technologies such as BGI, Genapsys, and Oxford Nanopore. A total of 202 datasets were generated to investigate the performance of a total of 16 sequencing platforms, including mappability of reads, coverage and error rates in difficult genomic regions, and detection of small-scale polymorphisms and large-scale structural variants. This study provides a comprehensive baseline resource for continual benchmarking as chemistries, methods, and platforms evolve for DNA sequencing.
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The custom-designed single nucleotide polymorphism (SNP) panel amplified 231 autosomal SNPs in one PCR reaction and subsequently sequenced with massively parallel sequencing (MPS) technology and Ion Torrent personal genome machine (PGM). SNPs were chosen from SNPforID, IISNP, HapMap, dbSNP, and related published literatures. Full concordance was obtained between available MPS calling and Sanger sequencing with 9947A and 9948 controls. Ten SNPs (rs4606077, rs334355, rs430046, rs2920816, rs4530059, rs1478829, rs1498553, rs7141285, rs12714757 and rs2189011) with low coverage or heterozygote imbalance should be optimized or excluded from the panel. Sequence data had sufficiently high coverage and gave reliable SNP calling for the remaining 221 loci with the custom MPS-SNP panel. A default DNA input amount of 10 ng per reaction was recommended by Ampliseq technology but sensitivity testing revealed positive results from as little as 1 ng input DNA. Mixture testing with this panel is possible through analysis of the FMAR (frequency of major allele reads) values at most loci with enough high coverage depth and low level of sequencing noise. These results indicate the potential advantage of the custom MPS-SNP assays and Ion Torrent PGM platform for forensic study.
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The detection of gene fusion events is important for the diagnosis and management of malignancies. In this study, we describe the validation of a next-generation sequencing assay for multiplex detection of gene fusions.Based on previously described gene fusion events that occur in pediatric oncology, a custom anchored multiplex next-generation sequencing assay was designed to target 93 genes.A total of 24 previously characterized specimens were examined. Twenty specimens had 1 or more previously described fusion events, and 4 specimens were negative for fusion events. The accuracy across specimens was 100% (20 of 20 specimens). The analytical sensitivity and specificity were both 100%. Interday reproducibility for fusion events was 94%; in comparison, intraday reproducibility was 90%.This multiple-gene fusion assay demonstrated appropriate sensitivity, specificity, and accuracy for clinical use. We anticipate that this assay will improve the diagnosis and management of patients with pediatric solid tumors.
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Massive parallel sequencing
Ion semiconductor sequencing
Personal genomics
Hybrid genome assembly
Cancer genome sequencing
Single cell sequencing
Illumina dye sequencing
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Ion semiconductor sequencing
Illumina dye sequencing
Massive parallel sequencing
Hybrid genome assembly
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Massive parallel sequencing
Ion semiconductor sequencing
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