Targeted resequencing of candidate genes using selector probes

Targeted genome enrichment is a powerful tool for making use of the massive throughput of novel DNA-sequencing instruments. We herein present a simple and scalable protocol for multiplex amplification of target regions based on the Selector technique. The updated version exhibits improved coverage and compatibility with next-generation-sequencing (NGS) library-construction procedures for shotgun sequencing with NGS platforms. To demonstrate the performance of the technique, all 501 exons from 28 genes frequently involved in cancer were enriched for and sequenced in specimens derived from cell lines and tumor biopsies. DNA from both fresh frozen and formalin-fixed paraffin-embedded biopsies were analyzed and 94% specificity and 98% coverage of the targeted region was achieved. Reproducibility between replicates was high (R2 = 0, 98) and readily enabled detection of copy-number variations. The procedure can be carried out in <24 h and does not require any dedicated instrumentation

Target enrichment using parallel nanoliter quantitative PCR amplification

Background: Next generation targeted resequencing is replacing Sanger sequencing at high pace in routine genetic diagnosis. The need for well validated, high quality enrichment platforms to complement the bench-top next generation sequencing devices is high. Results: We used the WaferGen Smartchip platform to perform highly parallelized PCR based target enrichment for a set of known cancer genes in a well characterized set of cancer cell lines from the NCI60 panel. Optimization of PCR assay design and cycling conditions resulted in a high enrichment efficiency. We provide proof of a high mutation rediscovery rate and have included technical replicates to enable SNP calling validation demonstrating the high reproducibility of our enrichment platform. Conclusions: Here we present our custom developed quantitative PCR based target enrichment platform. Using highly parallel nanoliter singleplex PCR reactions makes this a flexible and efficient platform. The high mutation validation rate shows this platform’s promise as a targeted resequencing method for multi-gene routine sequencing diagnostics

Diagnostic applications of next generation sequencing: working towards quality standards

Over the past 6 years, next generation sequencing (NGS) has been established as a valuable high-throughput method for research in molecular genetics and has successfully been employed in the identification of rare and common genetic variations. All major NGS technology companies providing commercially available instruments (Roche 454, Illumina, Life Technologies) have recently marketed bench top sequencing instruments with lower throughput and shorter run times, thereby broadening the applications of NGS and opening the technology to the potential use for clinical diagnostics. Although the high expectations regarding the discovery of new diagnostic targets and an overall reduction of cost have been achieved, technological challenges in instrument handling, robustness of the chemistry and data analysis need to be overcome. To facilitate the implementation of NGS as a routine method in molecular diagnostics, consistent quality standards need to be developed. Here the authors give an overview of the current standards in protocols and workflows and discuss possible approaches to define quality criteria for NGS in molecular genetic diagnostics

Performance of Microarray and Liquid Based Capture Methods for Target Enrichment for Massively Parallel Sequencing and SNP Discovery

Targeted sequencing is a cost-efficient way to obtain answers to biological questions in many projects, but the choice of the enrichment method to use can be difficult. In this study we compared two hybridization methods for target enrichment for massively parallel sequencing and single nucleotide polymorphism (SNP) discovery, namely Nimblegen sequence capture arrays and the SureSelect liquid-based hybrid capture system. We prepared sequencing libraries from three HapMap samples using both methods, sequenced the libraries on the Illumina Genome Analyzer, mapped the sequencing reads back to the genome, and called variants in the sequences. 74–75% of the sequence reads originated from the targeted region in the SureSelect libraries and 41–67% in the Nimblegen libraries. We could sequence up to 99.9% and 99.5% of the regions targeted by capture probes from the SureSelect libraries and from the Nimblegen libraries, respectively. The Nimblegen probes covered 0.6 Mb more of the original 3.1 Mb target region than the SureSelect probes. In each sample, we called more SNPs and detected more novel SNPs from the libraries that were prepared using the Nimblegen method. Thus the Nimblegen method gave better results when judged by the number of SNPs called, but this came at the cost of more over-sampling

Next Generation Molecular Diagnosis of Hereditary Spastic Paraplegias: An Italian Cross-Sectional Study

Hereditary spastic paraplegia (HSP) refers to a group of genetically heterogeneous neurodegenerative motor neuron disorders characterized by progressive age-dependent loss of corticospinal motor tract function, lower limb spasticity, and weakness. Recent clinical use of next generation sequencing (NGS) methodologies suggests that they facilitate the diagnostic approach to HSP, but the power of NGS as a first-tier diagnostic procedure is unclear. The larger-than-expected genetic heterogeneity-there are over 80 potential disease-associated genes-and frequent overlap with other clinical conditions affecting the motor system make a molecular diagnosis in HSP cumbersome and time consuming. In a single-center, cross-sectional study, spanning 4 years, 239 subjects with a clinical diagnosis of HSP underwent molecular screening of a large set of genes, using two different customized NGS panels. The latest version of our targeted sequencing panel (SpastiSure3.0) comprises 118 genes known to be associated with HSP. Using an in-house validated bioinformatics pipeline and several in silico tools to predict mutation pathogenicity, we obtained a positive diagnostic yield of 29% (70/239), whereas variants of unknown significance (VUS) were found in 86 patients (36%), and 83 cases remained unsolved. This study is among the largest screenings of consecutive HSP index cases enrolled in real-life clinical-diagnostic settings. Its results corroborate NGS as a modern, first-step procedure for molecular diagnosis of HSP. It also disclosed a significant number of new mutations in ultra-rare genes, expanding the clinical spectrum, and genetic landscape of HSP, at least in Italy

Nimbus: A design-driven analyses suite for amplicon-based NGS data

Motivation: PCR-based DNA enrichment followed by massively parallel sequencing is a straightforward and cost effective method to sequence genes up to high depth. The full potential of ampliconbased sequencing assays is currently not achieved as analysis methods do not take into account the source amplicons of the detected variants. Tracking the source amplicons has the potential to identify systematic biases, enhance variant calling and improve the designs of future assays. Results: We present Nimbus, a software suite for the analysis of amplicon-based sequencing data. Nimbus includes tools for data pre-processing, alignment, single nucleotide polymorphism (SNP), insertion and deletion calling, quality control and visualization. Nimbus can detect SNPs in its alignment seeds and reduces alignment issues by the usage of decoy amplicons. Tracking the amplicons throughout analysis allows easy and fast design optimization by amplicon performance comparison. It enables detection of probable false positive variants present in a single amplicon from real variants present in multiple amplicons and provides multiple sample visualization. Nimbus was tested using HaloPlex Exome datasets and outperforms other callers for low-frequency variants. The variants called by Nimbus were highly concordant between twin samples and SNP-arrays. The Nimbus suite provides an end-to-end solution for variant calling, design optimization and visualization of amplicon-derived next-generation sequencing datasets

Next-generation sequencing technologies and applications for human genetic history and forensics

Rapid advances in the development of sequencing technologies in recent years have enabled an increasing number of applications in biology and medicine. Here, we review key technical aspects of the preparation of DNA templates for sequencing, the biochemical reaction principles and assay formats underlying next-generation sequencing systems, methods for imaging and base calling, quality control, and bioinformatic approaches for sequence alignment, variant calling and assembly. We also discuss some of the most important advances that the new sequencing technologies have brought to the fields of human population genetics, human genetic history and forensic genetics

DNA repair genes are selectively mutated in diffuse large B cell lymphomas

DNA repair mechanisms are fundamental for B cell development, which relies on the somatic diversification of the immunoglobulin genes by V(D)J recombination, somatic hypermutation, and class switch recombination. Their failure is postulated to promote genomic instability and malignant transformation in B cells. By performing targeted sequencing of 73 key DNA repair genes in 29 B cell lymphoma samples, somatic and germline mutations were identified in various DNA repair pathways, mainly in diffuse large B cell lymphomas (DLBCLs). Mutations in mismatch repair genes (EXO1, MSH2, and MSH6) were associated with microsatellite instability, increased number of somatic insertions/deletions, and altered mutation signatures in tumors. Somatic mutations in nonhomologous end-joining (NHEJ) genes (DCLRE1C/ARTEMIS, PRKDC/DNA-PKcs, XRCC5/KU80, and XRCC6/KU70) were identified in four DLBCL tumors and cytogenetic analyses revealed that translocations involving the immunoglobulin-heavy chain locus occurred exclusively in NHEJ-mutated samples. The novel mutation targets, CHEK2 and PARP1, were further screened in expanded DLBCL cohorts, and somatic as well as novel and rare germline mutations were identified in 8 and 5% of analyzed tumors, respectively. By correlating defects in a subset of DNA damage response and repair genes with genomic instability events in tumors, we propose that these genes play a role in DLBCL lymphomagenesis

Molecular epidemiologic studies on helicobacter pylori infection and stomach cancer risk

Helicobacter pylori (H. pylori) infection increases stomach cancer risk. The aim of this thesis was to study genetic susceptibility from the host and to develop molecular methods for future characterization of bacterial virulence factors in longitudinal cohorts. In Study I, we investigated the association between genetic variation in an O-glycan transferase encoding gene (a4GnT) and H. pylori infection and gastric cancer risk in a Polish population-based case–control study (273 gastric cancer patients and 377 controls). A haplotype at rs2622694–rs397266 was associated with H. pylori infection, with the A-A haplotype associated with a higher risk compared with the most frequent G-G haplotype (odds ratio 2.30; 95% confidence intervals 1.35– 3.92). Neither this haplotype nor the tagSNPs were associated with overall gastric cancer risk. In Study II, we characterized genomic evolution of H. pylori over 20 years in the stomach. Whole genome of 21 sequential isolates 20 years apart, from 7 patients, were sequenced using 454 sequencing platform. There were on average 260 point mutations (range 70 to 488) per isolate over 20 years, and 45 recombinations (range 18 to 92). Genes in the cell motility category were overrepresented in point mutations and recombinations. Specifically, mutations often affected genes involved in chemotaxis, vacuolating cytotoxin-like protein, restriction and type IV secretory pathway; and recombinations affected glycosyltransferase involved in lipopolysaccharide biosynthesis. The major form of single nucleotide substitutions was transition (85%) and the minor form was transversion (15%). Mutation was sequence contextdependent. Clinical samples are often precious and of trace amounts. In Study III, we developed novel methods for DNA shotgun library construction and quantification. As compared with the standard procedure, our double-stranded and Y library construction methods are simpler and more efficient. A highly sensitive Taqman MGB-probe-based quantitative polymerase chain reaction (qPCR) was developed to quantify the amount of effective library. We also demonstrated that the distribution of library molecules on capture beads follows a Poisson distribution. Combining the qPCR and Poisson statistics, the labor intensive and costly titration can be eliminated and trace amounts of starting material is applicable. Archived formalin-fixed and paraffin-embedded (FFPE) biopsies, coupled with long term follow-up, are valuable resources for molecular epidemiologic studies. Study IV presented a method based on laser capture micro-dissection and modified whole genome sequencing methods to obtain metagenomic profiles of H. pylori from 15-year old FFPE biopsy sections