Cost-effective Sequencing Research Articles

Abstract PD1-12: Identification of copy number alterations associated with the progression of high risk premalignant breast lesions to breast carcinoma

Abstract Background: Atypical ductal hyperplasia (ADH) is common finding in the mammographic era, but has wide variation in diagnosis and treatment. ADH is a high risk factor for invasive breast ductal carcinoma (IDC), and is also considered to be a non-obligate precursor to IDC. Although a few studies have revealed some of the common genomic characteristics of ADH, a clear understanding of the molecular changes associated with breast cancer progression has been limited by inadequately powered studies and low resolution methodology (Lopez-Garcia et al., 2010, Histopathology 57: 171-192). Copy number alterations (CNAs), one of the major genetic alterations in cancer, are present in IDC and ductal carcinoma in situ (DCIS) and given a suitable detection method could be used in research and potentially in the clinical setting to predict patient prognosis. Method: We optimised a robust, cost effective low-coverage whole genome sequencing (LCWGS) method for CNA detection, using as little as 5 ng of formalin-fixed paraffin-embedded tissue derived DNA. We applied this novel method to 21 cases of pure ADH (not associated with cancer) as well as 20 ADH with synchronous DCIS and/or IDC. Cases were reviewed independently by two pathologists, followed by micro-dissection and DNA extraction. CNA were analysed using the LCWGS method, using 5-10 ng total DNA input per sample. Results: Highly accurate copy number profiles produced by low input LCWGS are comparable to those obtained from an alternative method, Molecular Inversion Probe arrays, while requiring 10 fold less input DNA (Kader et al., 2016, Genome Medicine 8: 121). Genetic analysis of pure ADH found that 95% had at least one copy number event and the median fraction of the genome altered was 8% (0-19%). Surprisingly, while 29% of pure ADH showed 16q loss and 1q gain (CNAs common in low-grade (LG) breast cancer), 29% of pure ADH showed 8q gain, an event more frequently observed in high grade (HG) cancer. Analysis of ADH with synchronous DCIS and/or IDC showed that 67% of ADH was clonally related with both LG (6/8) and HG carcinoma (4/7). The final analysis of 20 synchronous cases (9 LG, 11 HG) will be presented. Conclusion: Here we report the most detailed molecular taxonomy of this high risk pre-malignant breast lesion with the largest cohort undertaken worldwide. Our observation that 67% of ADH is clonal to breast carcinoma suggests that both LG and HG carcinoma can evolve from a similar ancestor lesion. As ADH has the potential to develop both LG and HG carcinoma, a biomarker signifying breast cancer progression from pre-malignant lesions is highly desirable for the more precise treatment of individual patients diagnosed with ADH. Citation Format: Kader T, Hill P, Opeskin K, Goode DL, Elder K, Pang J-M, Fox SB, Mann GB, Campbell IG, Gorringe KL. Identification of copy number alterations associated with the progression of high risk premalignant breast lesions to breast carcinoma [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD1-12.

RNA-Seq as an Emerging Tool for Marine Dinoflagellate Transcriptome Analysis: Process and Challenges

Dinoflagellates are the large group of marine phytoplankton with primary studies interest regarding their symbiosis with coral reef and the abilities to form harmful algae blooms (HABs). Toxin produced by dinoflagellates during events of HABs cause severe negative impact both in the economy and health sector. However, attempts to understand the dinoflagellates genomic features are hindered by their complex genome organization. Transcriptomics have been employed to understand dinoflagellates genome structure, profile genes and gene expression. RNA-seq is one of the latest methods for transcriptomics study. This method is capable of profiling the dinoflagellates transcriptomes and has several advantages, including highly sensitive, cost effective and deeper sequence coverage. Thus, in this review paper, the current workflow of dinoflagellates RNA-seq starts with the extraction of high quality RNA and is followed by cDNA sequencing using the next-generation sequencing platform, dinoflagellates transcriptome assembly and computational analysis will be discussed. Certain consideration needs will be highlighted such as difficulty in dinoflagellates sequence annotation, post-transcriptional activity and the effect of RNA pooling when using RNA-seq.

Multiple single cell screening and DNA MDA amplification chip for oncogenic mutation profiling.

The oncogenic mutation heterogeneity of the cancer cell population has been proven to be essential for predicting both drug-response and drug-resistance of targeted therapies, such as tyrosine kinase inhibitors. It is necessary to accurately evaluate the mutation heterogeneity, oncogenic mutation and resistant mutation profiling at a single cell level. However, there are two major hurdles in the process. First, majority of the cells in tumor tissue are non-cancer cells, which cause background noise. Second, the work load and cost of next generation sequencing on dozens of single cells are prohibitive. To address both these issues, we developed a microfluidic chip for profiling of dozens of selected cells. With the help of a novel tri-states valve structure, which performs precise controlling of the cell/reagent movement, as well as active mixing of different reagents, trapping/identification/lysis and in situ MDA amplification was achieved at a single cell level on the same chip. Using a proof-of-concept assay mimicking EGFR targeting drug Gefitinib treatment of lung cancer cells, the new method was validated as capable of not only detecting the existence of multiple mutations, but also providing complete information of the mutation scenario at the single cell level by using cost-effective Sanger's sequencing.

Plant Genetics and Molecular Biology: An Introduction.

The rapidly evolving technologies can serve as a potential growth engine in agriculture as many of these technologies have revolutionized several industries in the recent past. The tremendous advancements in biotechnology methods, cost-effective sequencing technology, refinement of genomic tools, and standardization of modern genomics-assisted breeding methods hold great promise in taking the global agriculture to the next level through development of improved climate-smart seeds. These technologies can dramatically increase our capacity to understand the molecular basis of traits and utilize the available resources for accelerated development of stable high-yielding, nutritious, input-use efficient, and climate-smart crop varieties. This book aimed to document the monumental advances witnessed during the last decade in multiple fields of plant biotechnology such as genetics, structural and functional genomics, trait and gene discovery, transcriptomics, proteomics, metabolomics, epigenomics, nanotechnology, and analytical tools. This book will serve to update the scientific community, academicians, and other stakeholders in global agriculture on the rapid progress in various areas of agricultural biotechnology. This chapter provides a summary of the book, "Plant Genetics and Molecular Biology." Graphical Abstract.

Assessment of the cPAS-based BGISEQ-500 platform for metagenomic sequencing.

BackgroundMore extensive use of metagenomic shotgun sequencing in microbiome research relies on the development of high-throughput, cost-effective sequencing. Here we present a comprehensive evaluation of the performance of the new high-throughput sequencing platform BGISEQ-500 for metagenomic shotgun sequencing and compare its performance with that of 2 Illumina platforms.FindingsUsing fecal samples from 20 healthy individuals, we evaluated the intra-platform reproducibility for metagenomic sequencing on the BGISEQ-500 platform in a setup comprising 8 library replicates and 8 sequencing replicates. Cross-platform consistency was evaluated by comparing 20 pairwise replicates on the BGISEQ-500 platform vs the Illumina HiSeq 2000 platform and the Illumina HiSeq 4000 platform. In addition, we compared the performance of the 2 Illumina platforms against each other. By a newly developed overall accuracy quality control method, an average of 82.45 million high-quality reads (96.06% of raw reads) per sample, with 90.56% of bases scoring Q30 and above, was obtained using the BGISEQ-500 platform. Quantitative analyses revealed extremely high reproducibility between BGISEQ-500 intra-platform replicates. Cross-platform replicates differed slightly more than intra-platform replicates, yet a high consistency was observed. Only a low percentage (2.02%–3.25%) of genes exhibited significant differences in relative abundance comparing the BGISEQ-500 and HiSeq platforms, with a bias toward genes with higher GC content being enriched on the HiSeq platforms.ConclusionsOur study provides the first set of performance metrics for human gut metagenomic sequencing data using BGISEQ-500. The high accuracy and technical reproducibility confirm the applicability of the new platform for metagenomic studies, though caution is still warranted when combining metagenomic data from different platforms.

Interventions for hyperhidrosis in secondary care: a systematic review and value-of-information analysis.

Hyperhidrosis is uncontrollable excessive sweating that occurs at rest, regardless of temperature. The symptoms of hyperhidrosis can significantly affect quality of life. The management of hyperhidrosis is uncertain and variable. To establish the expected value of undertaking additional research to determine the most effective interventions for the management of refractory primary hyperhidrosis in secondary care. A systematic review and economic model, including a value-of-information (VOI) analysis. Treatments to be prescribed by dermatologists and minor surgical treatments for hyperhidrosis of the hands, feet and axillae were reviewed; as endoscopic thoracic sympathectomy (ETS) is incontestably an end-of-line treatment, it was not reviewed further. Fifteen databases (e.g. CENTRAL, PubMed and PsycINFO), conference proceedings and trial registers were searched from inception to July 2016. Systematic review methods were followed. Pairwise meta-analyses were conducted for comparisons between botulinum toxin (BTX) injections and placebo for axillary hyperhidrosis, but otherwise, owing to evidence limitations, data were synthesised narratively. A decision-analytic model assessed the cost-effectiveness and VOI of five treatments (iontophoresis, medication, BTX, curettage, ETS) in 64 different sequences for axillary hyperhidrosis only. Fifty studies were included in the effectiveness review: 32 randomised controlled trials (RCTs), 17 non-RCTs and one large prospective case series. Most studies were small, rated as having a high risk of bias and poorly reported. The interventions assessed in the review were iontophoresis, BTX, anticholinergic medications, curettage and newer energy-based technologies that damage the sweat gland (e.g. laser, microwave). There is moderate-quality evidence of a large statistically significant effect of BTX on axillary hyperhidrosis symptoms, compared with placebo. There was weak but consistent evidence for iontophoresis for palmar hyperhidrosis. Evidence for other interventions was of low or very low quality. For axillary hyperhidrosis cost-effectiveness results indicated that iontophoresis, BTX, medication, curettage and ETS was the most cost-effective sequence (probability 0.8), with an incremental cost-effectiveness ratio of £9304 per quality-adjusted life-year. Uncertainty associated with study bias was not reflected in the economic results. Patients and clinicians attending an end-of-project workshop were satisfied with the sequence of treatments for axillary hyperhidrosis identified as being cost-effective. All patient advisors considered that the Hyperhidrosis Quality of Life Index was superior to other tools commonly used in hyperhidrosis research for assessing quality of life. The evidence for the clinical effectiveness and safety of second-line treatments for primary hyperhidrosis is limited. This meant that there was insufficient evidence to draw conclusions for most interventions assessed and the cost-effectiveness analysis was restricted to hyperhidrosis of the axilla. Based on anecdotal evidence and inference from evidence for the axillae, participants agreed that a trial of BTX (with anaesthesia) compared with iontophoresis for palmar hyperhidrosis would be most useful. The VOI analysis indicates that further research into the effectiveness of existing medications might be worthwhile, but it is unclear that such trials are of clinical importance. Research that established a robust estimate of the annual incidence of axillary hyperhidrosis in the UK population would reduce the uncertainty in future VOI analyses. This study is registered as PROSPERO CRD42015027803. The National Institute for Health Research Health Technology Assessment programme.

Incorporation of unique molecular identifiers in TruSeq adapters improves the accuracy of quantitative sequencing.

Quantitative analysis of next-generation sequencing (NGS) data requires discriminating duplicate reads generated by PCR from identical molecules that are of unique origin. Typically, PCR duplicates are identified as sequence reads that align to the same genomic coordinates using reference-based alignment. However, identical molecules can be independently generated during library preparation. Misidentification of these molecules as PCR duplicates can introduce unforeseen biases during analyses. Here, we developed a cost-effective sequencing adapter design by modifying Illumina TruSeq adapters to incorporate a unique molecular identifier (UMI) while maintaining the capacity to undertake multiplexed, single-index sequencing. Incorporation of UMIs into TruSeq adapters (TrUMIseq adapters) enables identification of bona fide PCR duplicates as identically mapped reads with identical UMIs. Using TrUMIseq adapters, we show that accurate removal of PCR duplicates results in improved accuracy of both allele frequency (AF) estimation in heterogeneous populations using DNA sequencing and gene expression quantification using RNA-Seq.

Improving somatic variant identification through integration of genome and exome data

Cost-effective high-throughput sequencing technologies, together with efficient mapping and variant calling tools, have made it possible to identify somatic variants for cancer study. However, integrating somatic variants from whole exome and whole genome studies poses a challenge to researchers as the variants identified by whole genome analysis may not be identified by whole exome analysis and vice versa. Simply taking the union or intersection of the results may lead to too many false positives or too many false negatives. To tackle this problem, we use machine learning models to integrate whole exome and whole genome calling results from two representative tools, VCMM (with the highest sensitivity but very low precision) and MuTect (with the highest precision). The evaluation results, based on both simulated and real data, show that our framework improves somatic variant calling, and is more accurate in identifying somatic variants than either individual method used alone or using variants identified from only whole genome data or only whole exome data. Using machine learning approach to combine results from multiple calling methods on multiple data platforms (e.g., genome and exome) enables more accurate identification of somatic variants.

Dissecting differentiation landscapes: a linked selection's perspective.

Dissecting differentiation landscapes: a linked selection's perspective.

Efficiency of ddRAD target enriched sequencing across spiny rock lobster species (Palinuridae: Jasus)

Double digest restriction site-associated DNA sequencing (ddRADseq) and target capture sequencing methods are used to explore population and phylogenetic questions in non-model organisms. ddRADseq offers a simple and reliable protocol for population genomic studies, however it can result in a large amount of missing data due to allelic dropout. Target capture sequencing offers an opportunity to increase sequencing coverage with little missing data and consistent orthologous loci across samples, although this approach has generally been applied to conserved markers for deeper evolutionary questions. Here, we combine both methods to generate high quality sequencing data for population genomic studies of all marine lobster species from the genus Jasus. We designed probes based on ddRADseq libraries of two lobster species (Jasus edwardsii and Sagmariasus verreauxi) and evaluated the captured sequencing data in five other Jasus species. We validated 4,465 polymorphic loci amongst these species using a cost effective sequencing protocol, of which 1,730 were recovered from all species, and 4,026 were present in at least three species. The method was also successfully applied to DNA samples obtained from museum specimens. This data will be further used to assess spatial-temporal genetic variation in Jasus species found in the Southern Hemisphere.

Optimal sequencing strategies for identifying disease-associated singletons.

With the increasing focus of genetic association on the identification of trait-associated rare variants through sequencing, it is important to identify the most cost-effective sequencing strategies for these studies. Deep sequencing will accurately detect and genotype the most rare variants per individual, but may limit sample size. Low pass sequencing will miss some variants in each individual but has been shown to provide a cost-effective alternative for studies of common variants. Here, we investigate the impact of sequencing depth on studies of rare variants, focusing on singletons—the variants that are sampled in a single individual and are hardest to detect at low sequencing depths. We first estimate the sensitivity to detect singleton variants in both simulated data and in down-sampled deep genome and exome sequence data. We then explore the power of association studies comparing burden of singleton variants in cases and controls under a variety of conditions. We show that the power to detect singletons increases with coverage, typically plateauing for coverage > ~25x. Next, we show that, when total sequencing capacity is fixed, the power of association studies focused on singletons is typically maximized for coverage of 15-20x, independent of relative risk, disease prevalence, singleton burden, and case-control ratio. Our results suggest sequencing depth of 15-20x as an appropriate compromise of singleton detection power and sample size for studies of rare variants in complex disease.

Germline and somatic SNVS calling in NGS panel tumor samples: Approaches to optimize tumor only genomic analysis for cancer precision medicine.

e13011 Background: Clinical NGS is often limited by tumor only profiling. Discrimination between somatic and likely germline mutations when calling from tumor patient samples is a critical step for clinical genotyping. Many algorithms have been developed for somatic single nucleotide variant (SNV) detection in matched tumor-normal whole genome and whole exome sequencing. Here, we demonstrate approaches of how a cost-effective large gene panel sequencing can be used to call somatic and germline SNVs for tumor only samples. Methods: Tumor, adjacent normal, and matched normal samples are collected from five patients. The somatic mutations were called with GATK Mutect2 in tumor only and adjacent normal. The germline mutations were called individually for all 15 samples with GATK Haplotype caller. To remove germline mutations from tumor only somatic calls, the filters ExAc pop freq, 1000G pop freq, COSMIC were applied on the tumor only somatic calls. PPV (Positive predictive value) for each filter was calculated by dividing the number of somatic mutations in the post-filtering mutation data by the total number of unfiltered mutations. TPR (True Positive Rate, representing sensitivity) was calculated by dividing the number of true somatic mutations in the tumor-only post-filter. Results: Compared with germline mutations called from matched normal, 70% germline mutations were called in RAWE somatic calls. A PPV of 0.71 and a TPR of 0.95 were optimally provided when the filter ExAc pop freq > 0.01 and COSMIC ( > 5 occurrence) applied. For germline mutations called in tumor samples, when compared with those in blood samples and in adjacent normal samples, PPV is 0.99 and TPR is 0.97. For somatic mutations called with tumor-adjacent normal pair mode in Mutect2, PPV is 0.5 and TPR is 0.99. Conclusions: Optimization of tools and parameters in NGS large panels could detect somatic and germline variants with high specificity, sensitivity and accuracy, without matched or adjacent normal. For the germline variants, when adjacent normal is available, it could replace matched normal with high accuracy.

Functional soil metagenomics: elucidation of polycyclic aromatic hydrocarbon degradation potential following 12 years of in situ bioremediation.

A culture-independent function-based screening approach was used to assess the microbial aerobic catabolome for polycyclic aromatic hydrocarbons degradation of a soil subjected to 12 years of in situ bioremediation. A total of 422750 fosmid clones were screened for key aromatic ring-cleavage activities using 2,3-dihydroxybiphenyl as substrate. Most of the genes encoding ring-cleavage enzymes on the 768 retrieved positive fosmids could not be identified using primer-based approaches and, thus, 205 fosmid inserts were sequenced. Nearly two hundred extradiol dioxygenase encoding genes of three different superfamilies could be identified. Additional key genes of aromatic metabolic pathways were identified, including a high abundance of Rieske non-heme iron oxygenases that provided detailed information on enzymes activating aromatic compounds and enzymes involved in activation of the side chain of methylsubstituted aromatics. The gained insights indicated a complex microbial network acting at the site under study, which comprises organisms similar to recently identified Immundisolibacter cernigliae TR3.2 and Rugosibacter aromaticivorans Ca6 and underlined the great potential of an approach that combines an activity-screening, a cost-effective high-throughput sequencing of fosmid clones and a phylogenomic-routed and manually curated database to carefully identify key proteins dedicated to aerobic degradation of aromatic compounds.

Cost-utility of biological treatment sequences for luminal Crohn’s disease in Europe

ABSTRACTBackground: This study aims to compare the cost-effectiveness of treatment sequences with available biologics, including adalimumab (ADA), biosimilar infliximab (bsIFX), originator infliximab (IFX) and vedolizumab (VEDO) for luminal Crohn’s disease in nine European countries.Methods: A Markov-model was constructed to simulate five-year medical costs and quality-adjusted life years (QALYs). Data on clinical efficacy were obtained from randomised controlled trials. Country-specific unit costs, discount rates and a third-party payer perspective were applied.Results: The bsIFX versus conventional therapy resulted in the most favourable incremental cost-utility ratios (ICURs) ranging from €34,580 (Hungary) to €77,062/QALY (Sweden). Compared to bsIFX, the bsIFX-ADA sequence was more cost-effective than the bsIFX-VEDO sequence with ICURs varying between €70,277 (France) and €162,069/QALY (Germany). The ICURs of the bsIFX-ADA-VEDO sequence versus the bsIFX-ADA strategy were between €206,266 (The Netherlands) and €363,232/QALY (Spain).Conclusion: We are the first to compare cost-effectiveness of multiple biological sequences for luminal Crohn’s disease. Based on our findings, bsIFX can be recommended as a first-line treatment in patients unresponsive to conventional treatments. While biological sequences only slightly differ in their associated health gains, their costs vary greatly. The bsIFX-ADA-VEDO seems to be the most cost-effective sequence of the available biologics across Europe.

Rapid and cost-effective high-throughput sequencing for identification of germline mutations of BRCA1 and BRCA2.

Genetic testing for breast cancer predisposing genes, BRCA1 and BRCA2, can take advantage for early identification of carriers with pathogenic germline mutations. However, conventional approaches based on Sanger sequencing are laborious and expensive. Next-generation sequencing technology has a great impact on investigation of medical genomics and now applied clinical genetics. We provide a protocol based on a pool and capture method followed by high-throughput sequencing, which realizes a rapid, high-quality, high-accuracy and low-cost testing for mutations in BRCA1 and BRCA2 by using small amounts of input DNA. Custom capture probes were designed for 195 kb regions encompassing the entire BRCA1 and BRCA2. DNA libraries of 96 samples with distinct indices were pooled before hybridizing to the capture probes, which largely reduced labor and cost. The captured library was run on the Illumina MiSeq sequencer. We applied the method to 384 Japanese individuals including 11 patients with breast cancer whose mutation statuses had been determined by standard clinical testing and 373 individuals from a general population. 99.99% of coding exons and their 20 bp flanking regions were covered with a minimum of 20 reads and the average depth was 179.5, supporting confident variant detection. The sequencing method rendered concordant results for 11 patients with breast cancer compared with the standard clinical testing including nine mutations in eight patients. Among 373 individuals from the general population, novel stop gain and frameshift deletion in BRCA2 were identified, which led to truncated protein and were most likely to be pathogenic. The result suggests the importance of a large-scale population-wide screening for carriers of mutations in these genes.

High-Throughput Sequencing of the Major Histocompatibility Complex following Targeted Sequence Capture.

The Human Major Histocompatibility Complex (MHC) is a highly polymorphic region full of immunoregulatory genes. The MHC codes for the human leukocyte antigens (HLA), proteins that present on the cellular surface and that are involved in self-non-self recognition. For matching donors and recipients for organ and stem-cell transplants it is important to know an individual's HLA haplotype determinable in this region. Now, as next-generation sequencing (NGS) platforms mature and become more and more accepted as a standard method, NGS applications have spread from research laboratories to the clinic, where they provide valid genetic insights. Here, we describe a cost-effective microarray-based sequence capture, enrichment, and NGS sequencing approach to characterize MHC haplotypes. Using this approach, ~4 MB of MHC sequence for four DNA samples (donor, recipient and the parents of the recipient) were sequenced in parallel in one NGS instrument run. We complemented this approach using microarray-based genome-wide SNP analysis. Taken together, the use of recently developed tools and protocols for sequence capture and massively parallel sequencing allows for detailed MHC analysis and donor-recipient matching.

An Improved Barcoded Oligonucleotide Primers-based Next-generation Sequencing Approach for Direct Identification of Viral Pathogens in Clinical Specimens

ObjectiveTo provide a feasible and cost-effective next-generation sequencing (NGS) method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use of common NGS, such as high cost, complicated procedures, tremendous data analysis, and high background noise in clinical samples. MethodsViruses from cell culture materials or clinical specimens were identified following an improved NGS procedure: reduction of background noise by sample preprocessing, viral enrichment by barcoded oligonucleotide (random hexamer or non-ribosomal hexanucleotide) primer-based amplification, fragmentation-free library construction and sequencing of one-tube mixtures, as well as rapid data analysis using an in-house pipeline. ResultsNGS data demonstrated that both barcoded primer sets were useful to simultaneously capture multiple viral pathogens in cell culture materials or clinical specimens and verified that hexanucleotide primers captured as many viral sequences as hexamers did. Moreover, direct testing of clinical specimens using this improved hexanucleotide primer-based NGS approach provided further detailed genotypes of enteroviruses causing hand, foot, and mouth disease (HFMD) and identified other potential viruses or differentiated misdiagnosis events. ConclusionThe improved barcoded oligonucleotide primer-based NGS approach is simplified, time saving, cost effective, and appropriate for direct identification of viral pathogens in clinical practice.

Detection of false positive mutations in BRCA gene by next generation sequencing.

BRCA1 and BRCA2 genes are implicated in 20-25% of hereditary breast and ovarian cancers. New age sequencing platforms have revolutionized massively parallel sequencing in clinical practice by providing cost effective, rapid, and sensitive sequencing. This study critically evaluates the false positives in multiplex panels and suggests the need for careful analysis. We employed multiplex PCR based BRCA1 and BRCA2 community Panel with ion torrent PGM machine for evaluation of these mutations. Out of all 41samples analyzed for BRCA1 and BRCA2 five were found with 950_951 insA(Asn319fs) at Chr13:32906565 position and one sample with 1032_1033 insA(Asn346fs) at Chr13:32906647, both being frame-shift mutations in BRCA2 gene. 950_951 insA(Asn319fs) mutation is reported as pathogenic allele in NCBI dbSNP. On examination of IGV for all these samples, it was seen that both mutations had 'A' nucleotide insertion at 950, and 1032 position in exon 10 of BRCA2 gene. Sanger Sequencing did not confirm these insertions. Next-generation sequencing shows great promise by allowing rapid mutational analysis of multiple genes in human cancer but our results indicate the need for careful sequence analysis to avoid false positive results.

Copy number analysis by low coverage whole genome sequencing using ultra low-input DNA from formalin-fixed paraffin embedded tumor tissue.

Unlocking clinically translatable genomic information, including copy number alterations (CNA), from formalin-fixed paraffin-embedded (FFPE) tissue is challenging due to low yields and degraded DNA. We describe a robust, cost-effective low-coverage whole genome sequencing (LC WGS) method for CNA detection using 5 ng of FFPE-derived DNA. CN profiles using 100 ng or 5 ng input DNA were highly concordant and comparable with molecular inversion probe (MIP) array profiles. LC WGS improved CN profiles of samples that performed poorly using MIP arrays. Our technique enables identification of driver and prognostic CNAs in archival patient samples previously deemed unsuitable for genomic analysis due to DNA limitations.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-016-0375-z) contains supplementary material, which is available to authorized users.

Full-Length Mitochondrial-DNA Sequencing on the PacBio RSII.

Conventional mitochondrial-DNA (MT DNA) sequencing approaches use Sanger sequencing of 20-40 partially overlapping PCR fragments per individual, which is a time- and resource-consuming process. We have developed a high-throughput, accurate, fast, and cost-effective human MT DNA sequencing approach. In this setup we first generate long-range PCR products for two partially overlapping 7.7 and 9.2 kb MT DNA-specific amplicons, add sample-specific barcodes, and sequence these on the PacBio RSII system to obtain full-length MT DNA sequences for genotyping/haplotyping purposes.