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Tuesday, June 1, 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…

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Tuesday, June 1, 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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Tuesday, June 1, 2021

Joint calling and PacBio SMRT Sequencing for indel and structural variant detection in populations

Fast and effective variant calling algorithms have been crucial to the successful application of DNA sequencing in human genetics. In particular, joint calling – in which reads from multiple individuals are pooled to increase power for shared variants – is an important tool for population surveys of variation. Joint calling was applied by the 1000 Genomes Project to identify variants across many individuals each sequenced to low coverage (about 5-fold). This approach successfully found common small variants, but broadly missed structural variants and large indels for which short-read sequencing has limited sensitivity. To support use of large variants in rare…

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Tuesday, June 1, 2021

Microbiome profiling at the strain level using rRNA amplicons

Strain level microbiome profiling is needed for a full understanding of how microbial communities influence human health. Microbiome profiling of rRNA gene amplicons is a well-understood method that is rapid and inexpensive, but standard 16S rRNA gene methods generally cannot differentiate closely related strains. Whole genome/shotgun microbiome profiling is considered a higher-resolution alternative, but with decreased throughput and significantly increased sequencing costs and analysis burden. With both methods there are also challenges with microbial lysis, DNA preparation, and taxonomic analysis. Specialized microbiome-focused protocols were developed to achieve strain-level taxonomic differentiation using a rapid, high throughput rRNA gene assay. The protocol…

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Tuesday, June 1, 2021

High-quality human genomes achieved through HiFi sequence data and FALCON-Unzip assembly

De novo assemblies of human genomes from accurate (85-90%), continuous long reads (CLR) now approach the human reference genome in contiguity, but the assembly base pair accuracy is typically below QV40 (99.99%), an order-of-magnitude lower than the standard for finished references. The base pair errors complicate downstream interpretation, particularly false positive indels that lead to false gene loss through frameshifts. PacBio HiFi sequence data, which are both long (>10 kb) and very accurate (>99.9%) at the individual sequence read level, enable a new paradigm in human genome assembly. Haploid human assemblies using HiFi data achieve similar contiguity to those using…

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Tuesday, June 1, 2021

Improving long-read assembly of microbial genomes and plasmids

Complete, high-quality microbial genomes are very valuable across a broad array of fields, from environmental studies, to human microbiome health, food pathogen surveillance, etc. Long-read sequencing enables accurate resolution of complex microbial genomes and is becoming the new standard. Here we report our novel Microbial Assembly pipeline to facilitate rapid, large-scale analysis of microbial genomes. We sequenced a 48-plex library with one SMRT Cell 8M on the Sequel II System, demultiplexed, then analyzed the data with Microbial Assembly.

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Tuesday, June 1, 2021

Metagenomic analysis of type II diabetes gut microbiota using PacBio HiFi reads reveals taxonomic and functional differences

In the past decade, the human microbiome has been increasingly shown to play a major role in health. For example, imbalances in gut microbiota appear to be associated with Type II diabetes mellitus (T2DM) and cardiovascular disease. Coronary artery disease (CAD) is a major determinant of the long-term prognosis among T2DM patients, with a 2- to 4-fold increased mortality risk when present. However, the exact microbial strains or functions implicated in disease need further investigation. From a large study with 523 participants (185 healthy controls, 186 T2DM patients without CAD, and 106 T2DM patients with CAD), 3 samples from each…

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Friday, February 5, 2021

ASMS Conference: Approaching the ‘perfect’ database – single-molecule, full-length transcript sequencing to create sample-specific, full-length protein databases

Recent advances in DNA sequencing technologies based on single-molecule detection now enable determination of full-length transcript sequences and, thus, all protein sequences in a sample. Utilizing data from this exciting technology, we have constructed customized, full-length protein databases that offer unprecedented advantages in proteomics database searching. Protein inference from bottom-up proteomics data can now be conducted using the set of correct protein sequences actually expressed in the sample, meaning that peptide identifications can be understood in the context of their corresponding full-length protein sequences. And most importantly, novel peptides or proteins originating from variations in the genome or transcriptome can…

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Friday, February 5, 2021

AGBT Virtual Poster: Genome variation in chronic viral infection – SMRT Sequencing for HCV

Ellen Paxinos, a scientist at PacBio, shares her AGBT poster on work done in collaboration with reference lab Monogram Biosciences using Single Molecule, Real-Time (SMRT) sequencing to detect minor species and variants in HCV. Using two genotypes mixed together, the team was able to detect variants down to 1% and to identify both viral haplotypes from the data. Paxinos says the study is a model for looking at genomic variation in chronic viral infection.

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Friday, February 5, 2021

ASHG Virtual Poster: The MHC Diversity in Africa Project (MDAP) pilot – 125 African high resolution HLA types from 5 populations

In this ASHG 2016 poster video, Martin Pollard from the Wellcome Trust Sanger Institute and the University of Cambridge describes an ambitious project to better represent natural variation in the complex MHC region by sequencing the locus in thousands of people from various populations in Africa. A pilot project in five populations has already revealed a lot of diversity in the region, which is important for human disease, vaccine response, and organ transplantation. Pollard says SMRT Sequencing is the only technology that can deliver the full-length haplotypes necessary to identify complete variation in this highly polymorphic complex. Plus: plans to…

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Friday, February 5, 2021

AGBT Virtual Poster: Using the PacBio Iso-Seq method to search for novel colorectal cancer biomarkers

Early detection of colorectal cancer (CRC) and its precursor lesions (adenomas) is crucial to reduce mortality rates. The fecal immunochemical test (FIT) is a non-invasive CRC screening test that detects the blood-derived protein hemoglobin. However, FIT sensitivity is suboptimal especially in detection of CRC precursor lesions. As adenoma-to-carcinoma progression is accompanied by alternative splicing, tumor-specific proteins derived from alternatively spliced RNA transcripts might serve as candidate biomarkers for CRC detection.

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Friday, February 5, 2021

AGBT Virtual Poster: Generation of local reference genomes using PacBio and BioNano data, and analysis of the “dark matter” of structural variants in 1000 Swedish genomes

In this AGBT 2017 poster, Ulf Gyllensten from Uppsala University presents two local reference genomes generated with PacBio and Bionano Genomics data. These assemblies include structural variation and repetitive regions that have been missed with previous short-read efforts, including some new genes not annotated in the human reference genome.

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Friday, February 5, 2021

Webinar: A paradigm shift in HLA sequencing: from exons to high-resolution allele-level HLA yyping

Human MHC class I genes HLA-A, -B, -C, and class II genes HLA -DR, -DQ, and -DP play a critical role in the immune system as primary factors responsible for organ transplant rejection. Additionally, the HLA genes are important targets for clinical and drug sensitivity research because of their direct or linkage-based association with several diseases, including cancer, and autoimmune diseases. HLA genes are highly polymorphic, and their diversity originates from exonic combinations as well as recombination events. With full-length gene sequencing, a significant increase of new alleles in the HLA database is expected, stressing the need for high-resolution sequencing.…

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Friday, February 5, 2021

Webinar: Beginner’s guide to PacBio SMRT Sequencing data analysis

PacBio SMRT Sequencing is fast changing the genomics space with its long reads and high consensus sequence accuracy, providing the most comprehensive view of the genome and transcriptome. In this webinar, I will talk about the various data analysis tools available in PacBio’s data analysis suite – SMRT Link – as well as 3rd party tools available. Key applications addressed in this talk are: Genome Assemblies, Structural Variant Analysis, Long Amplicon and Targeted Sequencing, Barcoding Strategies, Iso-Seq Analysis for Full-length Transcript Sequencing

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Friday, February 5, 2021

Webinar: PacBio targeted sequencing of long amplicons using PCR or hybrid capture

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…

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