Oligonucleotide-selective Sequencing Research Articles

High-resolution targeted bisulfite sequencing reveals blood cell type-specific DNA methylation patterns in IL13 and ORMDL3

BackgroundMethylation of DNA at CpG sites is an epigenetic modification and a potential modifier of disease risk, possibly mediating environmental effects. Currently, DNA methylation is commonly assessed using specific microarrays that sample methylation at a few % of all methylated sites.MethodsTo understand if significant information on methylation can be added by a more comprehensive analysis of methylation, we set up a quantitative method, bisulfite oligonucleotide-selective sequencing (Bs-OS-seq), and compared the data with microarray-derived methylation data. We assessed methylation at two asthma-associated genes, IL13 and ORMDL3, in blood samples collected from children with and without asthma and fractionated white blood cell types from healthy adult controls.ResultsOur results show that Bs-OS-seq can uncover vast amounts of methylation variation not detected by commonly used array methods. We found that high-density methylation information from even one gene can delineate the main white blood cell lineages.ConclusionsWe conclude that high-resolution methylation studies can yield clinically important information at selected specific loci missed by array-based methods, with potential implications for future studies of methylation-disease associations.

A robust targeted sequencing approach for low input and variable quality DNA from clinical samples

Next-generation deep sequencing of gene panels is being adopted as a diagnostic test to identify actionable mutations in cancer patient samples. However, clinical samples, such as formalin-fixed, paraffin-embedded specimens, frequently provide low quantities of degraded, poor quality DNA. To overcome these issues, many sequencing assays rely on extensive PCR amplification leading to an accumulation of bias and artifacts. Thus, there is a need for a targeted sequencing assay that performs well with DNA of low quality and quantity without relying on extensive PCR amplification. We evaluate the performance of a targeted sequencing assay based on Oligonucleotide Selective Sequencing, which permits the enrichment of genes and regions of interest and the identification of sequence variants from low amounts of damaged DNA. This assay utilizes a repair process adapted to clinical FFPE samples, followed by adaptor ligation to single stranded DNA and a primer-based capture technique. Our approach generates sequence libraries of high fidelity with reduced reliance on extensive PCR amplification—this facilitates the accurate assessment of copy number alterations in addition to delivering accurate single nucleotide variant and insertion/deletion detection. We apply this method to capture and sequence the exons of a panel of 130 cancer-related genes, from which we obtain high read coverage uniformity across the targeted regions at starting input DNA amounts as low as 10 ng per sample. We demonstrate the performance using a series of reference DNA samples, and by identifying sequence variants in DNA from matched clinical samples originating from different tissue types.

Abstract A1-41: Automated pipeline for high confidence variant calling and functional annotation, for matched tumor/normal samples sequenced by next-generation sequencing (NGS)

Abstract As the time required to sequence a genome using NGS sequencers is decreasing rapidly, the use of genomic data in the clinic to inform patient care, is becoming feasible. One area that needs to be addressed is the computational time required to align reads to a reference genome and call variants, which is now a larger proportion of the overall process. Standardization and repeatability of processing is critical in a clinical setting. To satisfy these criteria, we have developed a bioinformatics pipeline which starts with deep targeted resequencing from gene sets and related loci, and then automates the alignment, variant calling and functional annotation using an open source workflow tool. Given that tumor samples are often heterogenous mixtures - indicating either subclonal populations or fractions of normal tissue, deep sequencing is crucial in accurate detection of less prevalent minor allele somatic mutations. We used Oligonucleotide-Selective Sequencing (OS-Seq) as a targeted resequencing approach to provide deep coverage over the target region, with high specificity. With OS-Seq, the inner surface of an Illumina flow cell is modified with primer probe oligos, turning it into an enrichment platform for DNA sequencing libraries. The required steps from modification to a ready to sequence flow cell are fully automated and integrated into the standard Illumina sequencing preparation using cBot. The computational workflow for this project was developed using the open source tool bpipe. Once a library of bioinformatics steps has been defined in bpipe, these steps can be used as building blocks and put together in various ways to develop different pipelines. Bpipe also provides comprehensive audit trails and logs so that results are traceable and repeatable. For this particular workflow, steps for alignment to the genome using Burrows-Wheeler Aligner (BWA), steps for differentiation and filtering for each OS-Seq primer probe; variant calling using muTect; and variant filtering and annotation using custom scripts which access resources such as Single Nucleotide Polymorphisms Database (dbSNP), Combined Annotation Dependent Depletion (CADD), Catalogue of Somatic Mutations in Cancer (COSMIC), The Cancer Genome Atlas (TCGA), Drug Gene Interaction Database (DGIdb), are included. As a pilot for a larger study on the underlying genetics of Gastric cancer (GC [MIM 137215]) we analyzed matched tumor/normal samples from ten GC patients (6 intestinal, 4 diffuse) using this automated pipeline. We targeted the exons of 74 driver genes involved in several pathways commonly implicated in gastrointestinal cancers, such as RTK-RAS signaling. A median sequencing depth of 1300-2700 was achieved over these 20 samples, resulting in an average of four high-confidence somatic mutations in the target genes per patient, predicted to be deleterious by CADD. All variants were fully annotated with amino acid change, CADD score, and any match to mutations curated in COSMIC or reported in TCGA, plus possible drug targets as reported in DGIdb. By reducing the data processing turnaround time required to provide clinically actionable mutations, this study has significant implications in the development of precision cancer medicine. Our open source bioinformatics analysis approach provides mutations in a format readily accessible to the physician, and which can inform patient care. Citation Format: Susan M. Grimes, HoJoon Lee, Stephanie Greer, Jae-Ho Cheong, Hanlee P. Ji. Automated pipeline for high confidence variant calling and functional annotation, for matched tumor/normal samples sequenced by next-generation sequencing (NGS). [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-41.

Genetics and genotype-phenotype correlations in Finnish patients with dilated cardiomyopathy.

Despite our increased understanding of the genetic basis of dilated cardiomyopathy (DCM), the clinical utility and yield of clinically meaningful findings of comprehensive next-generation sequencing (NGS)-based genetic diagnostics in DCM has been poorly described. We utilized a high-quality oligonucleotide-selective sequencing (OS-Seq)-based targeted sequencing panel to investigate the genetic landscape of DCM in Finnish population and to evaluate the utility of OS-Seq technology as a novel comprehensive diagnostic tool. Using OS-Seq, we targeted and sequenced the coding regions and splice junctions of 101 genes associated with cardiomyopathies in 145 unrelated Finnish patients with DCM. We developed effective bioinformatic variant filtering strategy and implemented strict variant classification scheme to reveal diagnostic yield and genotype-phenotype correlations. Implemented OS-Seq technology provided high coverage of the target region (median coverage 410× and 99.42% of the nucleotides were sequenced at least 15× read depth). Diagnostic yield was 35.2% (familial 47.6% and sporadic 25.6%, P = 0.004) when both pathogenic and likely pathogenic variants are considered as disease causing. Of these, 20 (53%) were titin (TTN) truncations (non-sense and frameshift) affecting all TTN transcripts. TTN truncations accounted for 20.6% and 14.6% of the familial and sporadic DCM cases, respectively. Panel-based, high-quality NGS enables high diagnostic yield especially in the familial form of DCM, and bioinformatic variant filtering is a reliable step in the process of interpretation of genomic data in a clinical setting.

Accurate genetic diagnosis of Finnish pulmonary arterial hypertension patients using oligonucleotide-selective sequencing.

The genetic basis of pulmonary arterial hypertension (PAH) among Finnish PAH patients is poorly understood. We adopted a novel-targeted next-generation sequencing (NGS) approach called Oligonucleotide-Selective Sequencing (OS-Seq) and developed a custom data analysis and interpretation pipeline to identify pathogenic base substitutions, insertions, and deletions in seven genes associated with PAH (BMPR2, BMPR1B, ACVRL1, ENG, SMAD9, CAV1, and KCNK3) from Finnish PAH patients. This study represents the first clinical study with OS-Seq technology on patients suffering from a rare genetic disorder. We analyzed DNA samples from 21 Finnish PAH patients, whose BMPR2 and ACVRL1 mutation status had been previously studied using Sanger sequencing. Our sequencing panel covered 100% of the targeted base pairs with >15× sequencing depth. Pathogenic base substitutions were identified in the BMPR2 gene in 29% of the Finnish PAH cases. Two of the pathogenic variant-positive patients had been previously tested negative using Sanger sequencing. No clinically significant variants were identified in the six other PAH genes. Our study validates the use of targeted OS-Seq for genetic diagnostics of PAH and revealed pathogenic variants that had been previously missed using Sanger sequencing.

A programmable method for massively parallel targeted sequencing.

We have developed a targeted resequencing approach referred to as Oligonucleotide-Selective Sequencing. In this study, we report a series of significant improvements and novel applications of this method whereby the surface of a sequencing flow cell is modified in situ to capture specific genomic regions of interest from a sample and then sequenced. These improvements include a fully automated targeted sequencing platform through the use of a standard Illumina cBot fluidics station. Targeting optimization increased the yield of total on-target sequencing data 2-fold compared to the previous iteration, while simultaneously increasing the percentage of reads that could be mapped to the human genome. The described assays cover up to 1421 genes with a total coverage of 5.5 Megabases (Mb). We demonstrate a 10-fold abundance uniformity of greater than 90% in 1 log distance from the median and a targeting rate of up to 95%. We also sequenced continuous genomic loci up to 1.5 Mb while simultaneously genotyping SNPs and genes. Variants with low minor allele fraction were sensitively detected at levels of 5%. Finally, we determined the exact breakpoint sequence of cancer rearrangements. Overall, this approach has high performance for selective sequencing of genome targets, configuration flexibility and variant calling accuracy.

Oligonucleotide-selective sequencing for genetic diagnostics of pulmonary arterial hypertension

Oligonucleotide-selective sequencing for genetic diagnostics of pulmonary arterial hypertension

Targeted oligonucleotide-selective sequencing of 101 genes from 150 patients with idiopathic dilated cardiomyopathy in Finland

Targeted oligonucleotide-selective sequencing of 101 genes from 150 patients with idiopathic dilated cardiomyopathy in Finland

Illumina TruSight Cardiomyopathy Sequencing Panel: Application to hypertrophic cardiomyopathy

exon 8 mutations has been described, to date. In contrast to multiorgan disease caused by G406R either in exon 8A or 8, the G402S carrier manifested only an isolated cardiac phenotype with LQT and cardiac arrest. This isolated phenotype was suggested to be a cause of somatic mosaicism. We describe a teenage patient resuscitated from ventricular fibrillation and treated with ICD. She has no other organ manifestations, no syndactyly, her QTc is <480ms, and she has normal neurodevelopment. There is no family history of arrhythmias or sudden deaths. Targeted oligonucleotideselective sequencing of channelopathy genes revealed a de novo G402S in the exon 8 of CACNA1C. Direct sequencing of blood and oral mucosa derived DNA revealed an identical mutation peak suggesting the mutation is not expressed as mosaicism. The phenotype of our patient and the previously described patient show an isolated arrhythmia disease with no clinical manifestations of TS. Based on current evidence, we suggest the G402S mutation in CACNA1C should not be considered as causative for TS.

Abstract 3232: Oligonucleotide-selective sequencing (OS-Seq): a rapid and efficient targeting technology for ultra-deep detection of cancer mutations and rearrangements.

Abstract Somatic mutations, chromosomal rearrangements and other genomic aberrations in cancer genomes are critical events in the development of cancer and increasingly used in identifying oncogenic events that may be indicators for targeted therapies. For rapid and high accuracy analysis of targeted candidate genes and regions from cancer genomes, we developed OS-Seq that can be used to simultaneously discover cancer point mutations, copy number variants and rearrangements. For this new sequencing technology, we re-engineer the immobilized oligonucleotide primer lawn on a solid phase support (e.g. Illumina flow cell) with capture “primer-probes” such that it functions for both genomic target capture and sequencing (Myllykangas et al. 2011, Nature Biotech). As a result, in a matter of days we can rapidly sequence target regions in cancer genomes. We designed an in-silico exome capture probe set, enabling rapid design of disease-specific targeted capture primer probe sets. Subsequently, we designed assays to cover 313 and 1,400 cancer genes. Using the 313-gene assay targeting 5,000 exons, we can routinely sequence cancer genomes with on-target coverage greater than 500X, including for the entire length of exons as large as 1 Mb regions. Using a smaller gene set we have established coverage depths over 8,000 that enables the investigation of tumor evolution via mutations with low allelic fractions. In addition, we have designed OS-Seq assays for characterizing loci that are involved in rearrangements. We design capture probes positioned on opposing strands upstream and downstream of suspected rearrangement breakpoints and by localized assembly were able to confirm certain rearrangements, including a long deletion and inversions. For our current studies, we are using OS-Seq to identify cancer mutations in colorectal adenocarcinoma and determine the extent of genetic heterogeneity prevalent in these tumors. OS-Seq provides high-depth, high quality cancer genome sequence data, opening the door to potential diagnostic analysis of cancer genetic heterogeneity as seen in tumor evolution and cancer rearrangements. Citation Format: Erik S. Hopmans, Georges Natsoulis, Jason Buenrostro, Sue Grimes, John Bell, Hanlee P. Ji. Oligonucleotide-selective sequencing (OS-Seq): a rapid and efficient targeting technology for ultra-deep detection of cancer mutations and rearrangements. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3232. doi:10.1158/1538-7445.AM2013-3232

Oligonucleotide-selective sequencing

Oligonucleotide-selective sequencing

An efficient new targeted resequencing method

Resequencing of targeted genomic regions, such as exons, enables studies of genetic variation in large numbers of samples. This paper presents a new method called oligonucleotide-selective sequencing (OS-seq) in which oligonucleotides are immobilized on a substrate and used to capture sequences of interest before next-generation sequencing is performed on the same substrate. Additional preparation steps that are involved in other methods are avoided, thus increasing speed. To validate the method, OS-seq was successfully used to target the exons of ~350 cancer genes.

Efficient targeted resequencing of human germline and cancer genomes by oligonucleotide-selective sequencing

We describe an approach for targeted genome resequencing, called oligonucleotide-selective sequencing (OS-Seq), in which we modify the immobilized lawn of oligonucleotide primers of a next-generation DNA sequencer to function as both a capture and sequencing substrate. We apply OS-Seq to resequence the exons of either 10 or 344 cancer genes from human DNA samples. In our assessment of capture performance, >87% of the captured sequence originated from the intended target region with sequencing coverage falling within a tenfold range for a majority of all targets. Single nucleotide variants (SNVs) called from OS-Seq data agreed with >95% of variants obtained from whole-genome sequencing of the same individual. We also demonstrate mutation discovery from a colorectal cancer tumor sample matched with normal tissue. Overall, we show the robust performance and utility of OS-Seq for the resequencing analysis of human germline and cancer genomes.