Highly accurate long reads – HiFi reads – with single-molecule resolution make Single Molecule, Real-Time (SMRT) Sequencing ideal for full-length 16S rRNA sequencing, shotgun metagenomic profiling, and metagenome assembly.
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…
At AGBT 2017, Lars Paulin from the University of Helsinki presented this poster on whole genome sequencing of the virus responsible for progressive multifocal leukoencephalopathy, a rare and dangerous brain infection. His team used long amplicon analysis to resolve the whole virus genome from three patient samples, pooled them for SMRT Sequencing, and identified variants and rearrangements. This work represents the first time the viral genome was sequenced from patients.
In a talk at AGBT 2017, Histogenetics CEO Nezih Cereb reported on how SMRT Sequencing is allowing his team to produce full-length, phased sequences for HLA alleles, which are important for matching organ transplants to recipients. The company is typing thousands of samples per day on their PacBio RS II systems and their new Sequel System. Cereb noted that SMRT Sequencing is unique in its ability to reliably phase mutations in the HLA alleles without imputation. Cereb concluded with his plans to use this approach for other complex regions, such as KIR, and announced their continued increasing HLA typing capacity…
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.…
In this PacBio User Group Meeting lightning talk, Masako Nakanishi presents a study of how the gut microbiome alters an organism’s susceptibility to colonic ulceration; next, she plans to examine cause and effect by evaluating results of fecal transplants in mice.
In this Labroots webinar, Meredith Ashby, Director of Microbial Genomics at PacBio, describes the utility of highly accurate long-read sequencing, known as HiFi sequencing, to understand the SARs-CoV-2 viral genome. HiFi sequencing enables mutation phasing and rare variant detection to understand viral stability and mutation rates, as well as providing insights into viral population structure for monitoring viral evolution. Ashby also shares how HiFi sequencing can be used to explore the host immune response to COVID-19, specifically by providing full-length sequencing of the B cell repertoire, IGH locus and HLA genes. Access additional COVID-19 Sequencing Tools and Resources.
Allelic-level resolution HLA typing is known to improve survival prognoses post Unrelated Donor (UD) Haematopoietic Stem Cell Transplantation (HSCT). Currently, many commonly used HLA typing methodologies are limited either due to the fact that ambiguity cannot be resolved or that they are not amenable to high-throughput laboratories. Pacific Biosciences’ Single Molecule Real-Time (SMRT) DNA sequencing technology enables sequencing of single molecules in isolation and has read-length capabilities to enable whole gene sequencing for HLA. DNA barcode technology labels samples with unique identifiers that can be traced throughout the sequencing process. The use of DNA barcodes means that multiple samples can…
There are many sequencing-based approaches to understanding complex metagenomic communities spanning targeted amplification to whole-sample shotgun sequencing. While targeted approaches provide valuable data at low sequencing depth, they are limited by primer design and PCR amplification. Whole-sample shotgun experiments generally use short-read, second-generation sequencing, which results in data processing difficulties. For example, reads less than 1 kb in length will likely not cover a complete gene or region of interest, and will require assembly. This not only introduces the possibility of incorrectly combining sequence from different community members, it requires a high depth of coverage. As such, rare community members…
The Human Leukocyte Antigen (HLA) genes located on chromosome 6 are responsible for regulating immune function via antigen presentation and are one of the determining factors for stem cell and organ transplantation compatibility. Additionally various alleles within this region have been implicated in autoimmune disorders, cancer, vaccine response and both non-infectious and infectious disease risk. The HLA region is highly variable; containing repetitive regions; and co-dominantly expressed genes. This complicates short read mapping and means that assessing the effect of variation within a gene requires full phase information to resolve haplotypes.One solution to the problem of HLA identification is the…
There are many sequencing-based approaches to understanding complex metagenomic communities, spanning targeted amplification to whole-sample shotgun sequencing. While targeted approaches provide valuable data at low sequencing depth, they are limited by primer design and PCR amplification. Whole-sample shotgun experiments require a high depth of coverage. As such, rare community members may not be represented in the resulting assembly. Circular-consensus, Single Molecule, Real-Time (SMRT) Sequencing reads in the 1-2 kb range, with >99% consensus accuracy, can be efficiently generated for low amounts of input DNA, e.g. as little as 10 ng of input DNA sequenced in 4 SMRT Cells can generate…
There are many sequencing-based approaches to understanding complex metagenomic communities spanning targeted amplification to whole-sample shotgun sequencing. While targeted approaches provide valuable data at low sequencing depth, they are limited by primer design and PCR. Whole-sample shotgun experiments generally use short-read sequencing, which results in data processing difficulties. For example, reads less than 500bp in length will rarely cover a complete gene or region of interest, and will require assembly. This not only introduces the possibility of incorrectly combining sequence from different community members, it requires a high depth of coverage. As such, rare community members may not be represented…
There are many sequencing-based approaches to understanding complex metagenomic communities spanning targeted amplification to whole-sample shotgun sequencing. While targeted approaches provide valuable data at low sequencing depth, they are limited by primer design and PCR. Whole-sample shotgun experiments generally use short-read sequencing, which results in data processing difficulties. For example, reads less than 500 bp in length will rarely cover a complete gene or region of interest, and will require assembly. This not only introduces the possibility of incorrectly combining sequence from different community members, it requires a high depth of coverage. As such, rare community members may not be…
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…
The killer-cell immunoglobulin-like receptor (KIR) gene family are involved in immune modulation during viral infection, autoimmune disease and in allogeneic stem cell transplantation. Most KIR gene diversity studies and their impact on the transplant outcome is performed by gene absence/presence assays. However, it is well known that KIR gene allelic variations have biological significance. Allele level typing of KIR genes has been very challenging until recently due to the homologous nature of those genes and very long intronic sequences. SMRT (Single Molecule Real-Time) Sequencing generates average long reads of 10 to 15 kb and allows us to obtain in-phase long…