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Friday, February 26, 2021

Highly sensitive and cost-effective detection of BRCA1 and BRCA2 cancer variants in FFPE samples using Multiplicom’s MASTR technology & Single Molecule, Real-Time (SMRT) Sequencing

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.

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Friday, February 26, 2021

WGS SMRT Sequencing of patient samples from a fecal microbiota transplant trial

Fecal samples were obtained from human subjects in the first blinded, placebo-controlled trial to evaluate the efficacy and safety of fecal microbiota transplant (FMT) for treatment of recurrent C. difficile infection. Samples included pre-and post-FMT transplant, post-placebo transplant, and the donor control; samples were taken at 2 and 8 week post-FMT. Sequencing was done on the PacBio Sequel System, with the goal of obtaining high quality sequences covering whole genes or gene clusters, which will be used to better understand the relationship between the composition and functional capabilities of intestinal microbiomes and patient health. Methods: Samples were randomly sheared to…

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Friday, February 26, 2021

Effect of coverage depth and haplotype phasing on structural variant detection with PacBio long reads

Each human genome has thousands of structural variants compared to the reference assembly, up to 85% of which are difficult or impossible to detect with Illumina short reads and are only visible with long, multi-kilobase reads. The PacBio RS II and Sequel single molecule, real-time (SMRT) sequencing platforms have made it practical to generate long reads at high throughput. These platforms enable the discovery of structural variants just as short-read platforms did for single nucleotide variants. Numerous software algorithms call structural variants effectively from PacBio long reads, but algorithm sensitivity is lower for insertion variants and all heterozygous variants. Furthermore,…

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Friday, February 26, 2021

Full-length cDNA sequencing on the PacBio Sequel platform

The protein coding potential of most plant and animal genomes is dramatically increased via alternative splicing. Identification and annotation of expressed mRNA isoforms is critical to the understanding of these complex organisms. While microarrays and other NGS-based methods have become useful for studying transcriptomes, these technologies yield short, fragmented transcripts that remain a challenge for accurate, complete reconstruction of splice variants. The Iso-Seq protocol developed at PacBio offers the only solution for direct sequencing of full-length, single-molecule cDNA sequences to survey transcriptome isoform diversity useful for gene discovery and annotation. Knowledge of the complete isoform repertoire is also key for…

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Friday, February 26, 2021

Profiling complex population genomes with highly accurate single molecule reads: cow rumen microbiomes

Determining compositions and functional capabilities of complex populations is often challenging, especially for sequencing technologies with short reads that do not uniquely identify organisms or genes. Long-read sequencing improves the resolution of these mixed communities, but adoption for this application has been limited due to concerns about throughput, cost and accuracy. The recently introduced PacBio Sequel System generates hundreds of thousands of long and highly accurate single-molecule reads per SMRT Cell. We investigated how the Sequel System might increase understanding of metagenomic communities. In the past, focus was largely on taxonomic classification with 16S rRNA sequencing. Recent expansion to WGS…

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Friday, February 26, 2021

Using the PacBio Sequel System to taxonomically and functionally classify metagenomic samples in a trial of patients undergoing fecal microbiota transplantation

Whole-sample shotgun sequencing can provide a more detailed view of a metagenomic community than 16S sequencing, but its use in multi-sample experiments is limited by throughput, cost and analysis complexity. While short-read sequencing technologies offer higher throughput, read lengthss less fewer than 500 bp will rarely cover a gene of interest, and necessitate assembly before further analysis. Assembling large fragments requires sampling each community member at a high depth, significantly increasing the amount of sequencing needed, and limiting the analysis of rare community members. Assembly methods also risk It is also possible to incorrectly combine combining sequences from different community…

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Friday, February 26, 2021

Profiling complex communities with highly accurate single molecule reads: cow rumen microbiomes

Determining compositions and functional capabilities of complex populations is often challenging, especially for sequencing technologies with short reads that do not uniquely identify organisms or genes. Long-read sequencing improves the resolution of these mixed communities, but adoption for this application has been limited due to concerns about throughput, cost and accuracy. The recently introduced PacBio Sequel System generates hundreds of thousands of long and highly accurate single-molecule reads per SMRT Cell. We investigated how the Sequel System might increase understanding of metagenomic communities. In the past, focus was largely on taxonomic classification with 16S rRNA sequencing. Recent expansion to WGS…

Read More »

Friday, February 26, 2021

Structural variant detection with low-coverage Pacbio sequencing

Despite amazing progress over the past quarter century in the technology to detect genetic variants, intermediate-sized structural variants (50 bp to 50 kb) have remained difficult to identify. Such variants are too small to detect with array comparative genomic hybridization, but too large to reliably discover with short-read DNA sequencing. Recent de novo assemblies of human genomes have demonstrated the power of PacBio Single Molecule, Real-Time (SMRT) Sequencing to fill this technology gap and sensitively identify structural variants in the human genome. While de novo assembly is the ideal method to identify variants in a genome, it requires high depth…

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Friday, February 26, 2021

T-cell receptor profiling using PacBio sequencing of SMARTer libraries

T-cells play a central part in the immune response in humans and related species. T-cell receptors (TCRs), heterodimers located on the T-cell surface, specifically bind foreign antigens displayed on the MHC complex of antigen-presenting cells. The wide spectrum of potential antigens is addressed by the diversity of TCRs created by V(D)J recombination. Profiling this repertoire of TCRs could be useful from, but not limited to, diagnosis, monitoring response to treatments, and examining T-cell development and diversification.

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Friday, February 26, 2021

Simplified sequencing of full-length isoforms in cancer on the PacBio Sequel platform

Tremendous flexibility is maintained in the human proteome via alternative splicing, and cancer genomes often subvert this flexibility to promote survival. Identification and annotation of cancer-specific mRNA isoforms is critical to understanding how mutations in the genome affect the biology of cancer cells. While microarrays and other NGS-based methods have become useful for studying transcriptomes, these technologies yield short, fragmented transcripts that remain a challenge for accurate, complete reconstruction of splice variants. In cancer proteomics studies, the identification of biomarkers from mass spectroscopy data is often limited by incomplete gene isoform expression information to support protein to transcript mapping. The…

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Friday, February 26, 2021

Detecting pathogenic structural variants with low-coverage PacBio sequencing.

Though a role for structural variants in human disease has long been recognized, it has remained difficult to identify intermediate-sized variants (50 bp to 5 kb), which are too small to detect with array comparative genomic hybridization, but too large to reliably discover with short-read DNA sequencing. Recent studies have demonstrated that PacBio Single Molecule, Real-Time (SMRT) sequencing fills this technology gap. SMRT sequencing detects tens of thousands of structural variants in the human genome, approximately five times the sensitivity of short-read DNA sequencing.

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Friday, February 26, 2021

Multiplexing strategies for microbial whole genome sequencing using the Sequel System

For microbial sequencing on the PacBio Sequel System, the current yield per SMRT Cell is in excess relative to project requirements. Multiplexing offers a viable solution; greatly increasing throughput, efficiency, and reducing costs per genome. This approach is achieved by incorporating a unique barcode for each microbial sample into the SMRTbell adapters and using a streamlined library preparation process. To demonstrate performance,12 unique barcodes assigned to B. subtilis and sequenced on a single SMRT Cell. To further demonstrate the applicability of this method, we multiplexed the genomes of 16 strains of H. pylori. Each DNA was sheared to 10 kb,…

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Friday, February 26, 2021

Detecting pathogenic structural variants with long-read PacBio SMRT Sequencing

Most of the base pairs that differ between two human genomes are in intermediate-sized structural variants (50 bp to 5 kb), which are too small to detect with array comparative genomic hybridization or optical mapping but too large to reliably discover with short-read DNA sequencing. Long-read sequencing with PacBio Single Molecule, Real-Time (SMRT) Sequencing platforms fills this technology gap. PacBio SMRT Sequencing detects tens of thousands of structural variants in a human genome with approximately five times the sensitivity of short-read DNA sequencing. Effective application of PacBio SMRT Sequencing to detect structural variants requires quality bioinformatics tools that account for…

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Friday, February 26, 2021

De novo assembly and preliminary annotation of the Schizocardium californicum genome

Animals in the phylum Hemichordata have provided key understanding of the origins and development of body patterning and nervous system organization. However, efforts to sequence and assemble the genomes of highly heterozygous non-model organisms have proven to be difficult with traditional short read approaches. Long repetitive DNA structures, extensive structural variation between haplotypes in polyploid species, and large genome sizes are limiting factors to achieving highly contiguous genome assemblies. Here we present the highly contiguous de novo assembly and preliminary annotation of an indirect developing hemichordate genome, Schizocardium californicum, using SMRT Sequening long reads.

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