As a cost-effective alternative to whole genome human sequencing, targeted sequencing of specific regions, such as exomes or panels of relevant genes, has become increasingly common. These methods typically include direct PCR amplification of the genomic DNA of interest, or the capture of these targets via probe-based hybridization. Commonly, these approaches are designed to amplify or capture exonic regions and thereby result in amplicons or fragments that are a few hundred base pairs in length, a length that is well-addressed with short-read sequencing technologies. These approaches typically provide very good coverage and can identify SNPs in the targeted region, but…
Recent advances in next-generation sequencing have led to an increased use of formalin-fixed and paraffin-embedded (FFPE) tissues for medical samples in disease and scientific research. Single Molecule, Real-Time (SMRT) Sequencing offers a unique advantage for direct analysis of FFPE samples without amplification. However, obtaining ample long-read information from FFPE samples has been a challenge due to the quality and quantity of the extracted DNA. FFPE samples often contain damaged sites, including breaks in the backbone and missing or altered nucleotide bases, which directly impact sequencing and target enrichment. Additionally, the quality and quantity of the recovered DNA vary depending on…
Specific mutations in BRCA1 and BRCA2 have been shown to be associated with several types of cancers. Molecular profiling of cancer samples requires assays capable of accurately detecting the entire spectrum of variants, including those at relatively low frequency. Next-Generation Sequencing (NGS) has been a powerful tool for researchers to better understand cancer genetics. Here we describe a targeted re-sequencing workflow that combines barcoded amplification of BRCA1 and BRCA2 exons from 12 FFPE tumor samples using Multiplicom’s MASTR technology with PacBio SMRT Sequencing. This combination allows for the accurate detection of variants in a cost-effective and timely manner.
With the increasing availability of whole-genome sequencing, haplotype reconstruction of individual genomes, or haplotype assembly, remains unsolved. Like the de novo genome assembly problem, haplotype assembly is greatly simplified by having more long-range information. The Targeted Locus Amplification (TLA) technology from Cergentis has the unique capability of targeting a specific region of the genome using a single primer pair and yielding ~2 kb DNA circles that are comprised of ~500 bp fragments. Fragments from the same circle come from the same haplotype and follow an exponential decay in distance from the target region, with a span that reaches the multi-megabase…
Targeted sequencing employing PCR amplification is a fundamental approach to studying human genetic disease. PacBio’s Sequel System and supporting products provide an end-to-end solution for amplicon sequencing, offering better performance to Sanger technology in accuracy, read length, throughput, and breadth of informative data. Sample multiplexing is supported with three barcoding options providing the flexibility to incorporate unique sample identifiers during target amplification or library preparation. Multiplexing is key to realizing the full capacity of the 1 million individual reactions per Sequel SMRT Cell. Two analysis workflows that can generate high-accuracy results support a wide range of amplicon sizes in two…
Melissa Laird Smith discussed how the Icahn School of Medicine at Mount Sinai uses long-read sequencing for translational research. She gave several examples of targeted sequencing projects run on the Sequel System including CYP2D6, phased mutations of GLA in Fabry’s disease, structural variation breakpoint validation in glioblastoma, and full-length immune profiling of TCR sequences.
Targeted sequencing experiments commonly rely on either PCR or hybrid capture to enrich for targets of interest. When using short read sequencing platforms, these amplicons or fragments are frequently targeted to a few hundred base pairs to accommodate the read lengths of the platform. Given PacBio’s long readlength, it is straightforward to sequence amplicons or captured fragments that are multiple kilobases in length. These long sequences are useful for easily visualizing variants that include SNPs, CNVs and other structural variants, often without assembly. We will review methods for the sequencing of long amplicons and provide examples using amplicons that range…
This webinar, presented by Nisha Pillai, provides an overview of amplicon sequencing to target specific regions of a genome using PacBio Single Molecule, Real-Time (SMRT) Sequencing. This session provides an overview of bioinformatics approaches for PacBio amplicon analysis including circular consensus sequencing and long amplicon analysis.
In this webinar, Lori Aro and Cheryl Heiner of PacBio describe how high-throughput amplicon sequencing using Single Molecule, Real-Time (SMRT) Sequencing and the Sequel System allows for the easy and cost-effective generation of high-fidelity, long reads from amplicons ranging in size from several hundred base pairs to 20 kb. Topics covered include the latest advances in SMRT Sequencing performance for detection of all variant types even in difficult to sequence regions of the genome, multiplexing options to increase throughput and improve efficiency, and examples of amplicon sequencing of clinically relevant targets.
We analyzed transcriptomes (n = 211), whole exomes (n = 99) and targeted exomes (n = 103) from 216 malignant pleural mesothelioma (MPM) tumors. Using RNA-seq data, we identified four distinct molecular subtypes: sarcomatoid, epithelioid, biphasic-epithelioid (biphasic-E) and biphasic-sarcomatoid (biphasic-S). Through exome analysis, we found BAP1, NF2, TP53, SETD2, DDX3X, ULK2, RYR2, CFAP45, SETDB1 and DDX51 to be significantly mutated (q-score = 0.8) in MPMs. We identified recurrent mutations in several genes, including SF3B1 (~2%; 4/216) and TRAF7 (~2%; 5/216). SF3B1-mutant samples showed a splicing profile distinct from that of wild-type tumors. TRAF7 alterations occurred primarily in the WD40 domain…
Epstein-Barr virus (EBV) was the first human tumor virus discovered more than 50 years ago. EBV-associated lymphomagenesis is still a significant viral-associated disease as it involves a diverse range of pathologies, especially B-cell lymphomas. Recent development of high-throughput next-generation sequencing technologies and in vivo mouse models have significantly promoted our understanding of the fundamental molecular mechanisms which drive these cancers and allowed for the development of therapeutic intervention strategies. This review will highlight the current advances in EBV-associated B-cell lymphomas, focusing on transcriptional regulation, chromosome aberrations, in vivo studies of EBV-mediated lymphomagenesis, as well as the treatment strategies to target viral-associated…
In recent years long-read technologies have moved from being a niche and specialist field to a point of relative maturity likely to feature frequently in the genomic landscape. Analogous to next generation sequencing, the cost of sequencing using long-read technologies has materially dropped whilst the instrument throughput continues to increase. Together these changes present the prospect of sequencing large numbers of individuals with the aim of fully characterizing genomes at high resolution. In this article, we will endeavour to present an introduction to long-read technologies showing: what long reads are; how they are distinct from short reads; why long reads…
Structural variants (SVs), including small insertion and deletion variants (indels), are challenging to detect through standard alignment-based variant calling methods. Sequence assembly offers a powerful approach to identifying SVs, but is difficult to apply at scale genome-wide for SV detection due to its computational complexity and the difficulty of extracting SVs from assembly contigs. We describe SvABA, an efficient and accurate method for detecting SVs from short-read sequencing data using genome-wide local assembly with low memory and computing requirements. We evaluated SvABA’s performance on the NA12878 human genome and in simulated and real cancer genomes. SvABA demonstrates superior sensitivity and…
Loss-of-function pathogenic variants in BRCA1 confer a predisposition to breast and ovarian cancer. Genetic testing for sequence changes in BRCA1 frequently reveals a missense variant for which the impact on cancer risk and on the molecular function of BRCA1 is unknown. Functional BRCA1 is required for the homology-directed repair (HDR) of double-strand DNA breaks, a critical activity for maintaining genome integrity and tumor suppression. Here, we describe a multiplex HDR reporter assay for concurrently measuring the effects of hundreds of variants of BRCA1 for their role in DNA repair. Using this assay, we characterized the effects of 1,056 amino acid…