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Tuesday, April 21, 2020

Characterization of Reference Materials for Genetic Testing of CYP2D6 Alleles: A GeT-RM Collaborative Project.

Pharmacogenetic testing increasingly is available from clinical and research laboratories. However, only a limited number of quality control and other reference materials currently are available for the complex rearrangements and rare variants that occur in the CYP2D6 gene. To address this need, the Division of Laboratory Systems, CDC-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Cell Repositories (Camden, NJ), has characterized 179 DNA samples derived from Coriell cell lines. Testing included the recharacterization of 137 genomic DNAs that were genotyped in previous Genetic Testing Reference Material Coordination…

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Tuesday, April 21, 2020

A comparison of immunoglobulin IGHV, IGHD and IGHJ genes in wild-derived and classical inbred mouse strains.

The genomes of classical inbred mouse strains include genes derived from all three major subspecies of the house mouse, Mus musculus. We recently posited that genetic diversity in the immunoglobulin heavy chain (IGH) gene loci of C57BL/6 and BALB/c mice reflect differences in subspecies origin. To investigate this hypothesis, we conducted high-throughput sequencing of IGH gene rearrangements to document IGH variable (IGHV), joining (IGHJ), and diversity (IGHD) genes in four inbred wild-derived mouse strains (CAST/EiJ, LEWES/EiJ, MSM/MsJ, and PWD/PhJ), and a single disease model strain (NOD/ShiLtJ), collectively representing genetic backgrounds of several major mouse subspecies. A total of 341 germline…

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Tuesday, April 21, 2020

Complete genome screening of clinical MRSA isolates identifies lineage diversity and provides full resolution of transmission and outbreak events

Whole-genome sequencing (WGS) of Staphylococcus aureus is increasingly used as part of infection prevention practices, but most applications are focused on conserved core genomic regions due to limitations of short-read technologies. In this study we established a long-read technology-based WGS screening program of all first-episode MRSA blood infections at a major urban hospital. A survey of 132 MRSA genomes assembled from long reads revealed widespread gain/loss of accessory mobile genetic elements among established hospital- and community-associated lineages impacting >10% of each genome, and frequent megabase-scale inversions between endogenous prophages. We also characterized an outbreak of a CC5/ST105/USA100 clone among 3…

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Tuesday, April 21, 2020

Dissemination of multiple carbapenem resistance genes in an in vitro gut model simulating the human colon.

Carbapenemase-producing Enterobacteriaceae (CPE) pose a major global health risk. Mobile genetic elements account for much of the increasing CPE burden.To investigate CPE colonization and the impact of antibiotic exposure on subsequent resistance gene dissemination within the gut microbiota using a model to simulate the human colon.Gut models seeded with CPE-negative human faeces [screened with BioMérieux chromID® CARBA-SMART (Carba-Smart), Cepheid Xpert® Carba-R assay (XCR)] were inoculated with distinct carbapenemase-producing Klebsiella pneumoniae strains (KPC, NDM) and challenged with imipenem or piperacillin/tazobactam then meropenem. Resistant populations were enumerated daily on selective agars (Carba-Smart); CPE genes were confirmed by PCR (XCR, Check-Direct CPE Screen…

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Tuesday, April 21, 2020

High-coverage, long-read sequencing of Han Chinese trio reference samples.

Single-molecule long-read sequencing datasets were generated for a son-father-mother trio of Han Chinese descent that is part of the Genome in a Bottle (GIAB) consortium portfolio. The dataset was generated using the Pacific Biosciences Sequel System. The son and each parent were sequenced to an average coverage of 60 and 30, respectively, with N50 subread lengths between 16 and 18?kb. Raw reads and reads aligned to both the GRCh37 and GRCh38 are available at the NCBI GIAB ftp site (ftp://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/data/ChineseTrio/). The GRCh38 aligned read data are archived in NCBI SRA (SRX4739017, SRX4739121, and SRX4739122). This dataset is available for anyone…

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Monday, March 30, 2020

Webinar: Complete genomes within reach – Closing bacterial genomes from the lakes of Minnesota to NYC hospitals

In this webinar, Ben Auch, Research Scientist, Innovation Lab, University of Minnesota Genomics Center, Cody Sheik, Assistant Professor of Biology, University of Minnesota Duluth, and Harm van Bakel, Assistant Professor of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai provide details of the newly updated microbial whole genome sequencing pipeline, which leverages the multiplexing capabilities of the Sequel System, share new insights into the ecophysiology of Minnesota microbes using long-read sequencing, and show of how whole genome sequencing is used in pathogen surveillance programs at hospitals.

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Monday, March 30, 2020

AGBT Roche and PacBio Workshop: Leveraging SMRT Sequencing technology for developing niche assays with diagnostic potential

Robert Sebra reports the use of SMRT Sequencing at the Icahn Institute and presents some early data from the new Sequel System. Topics include: Targeted sequencing applications for CYP2D6 metabolism and Gaucher disease, tandem repeat detection in FTD/ALS patients, structural variation detection for Goldenhar Syndrome, inverted PCR assays for detection of DNA damage in Glioblastome, whole gene BRCA sequencing, and sensitive somatic variant detection in heterogeneous tissues.

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Sunday, September 22, 2019

HIV-1 infection of primary CD4(+) T cells regulates the expression of specific HERV-K (HML-2) elements.

Endogenous retroviruses (ERVs) occupy extensive regions of the human genome. Although many of these retroviral elements have lost their ability to replicate, those whose insertion took place more recently, such as the HML-2 group of HERV-K elements, still retain intact open reading frames and the capacity to produce certain viral RNA and/or proteins. Transcription of these ERVs is, however, tightly regulated by dedicated epigenetic control mechanisms. Nonetheless, it has been reported that some pathologic states, such as viral infections and certain cancers, coincide with ERV expression suggesting transcriptional reawakening is possible. HML-2 elements are reportedly induced during HIV-1 infection, but…

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Sunday, September 22, 2019

Metagenomic binning and association of plasmids with bacterial host genomes using DNA methylation.

Shotgun metagenomics methods enable characterization of microbial communities in human microbiome and environmental samples. Assembly of metagenome sequences does not output whole genomes, so computational binning methods have been developed to cluster sequences into genome ‘bins’. These methods exploit sequence composition, species abundance, or chromosome organization but cannot fully distinguish closely related species and strains. We present a binning method that incorporates bacterial DNA methylation signatures, which are detected using single-molecule real-time sequencing. Our method takes advantage of these endogenous epigenetic barcodes to resolve individual reads and assembled contigs into species- and strain-level bins. We validate our method using synthetic…

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Sunday, September 22, 2019

Improved OTU-picking using long-read 16S rRNA gene amplicon sequencing and generic hierarchical clustering

BACKGROUND: High-throughput bacterial 16S rRNA gene sequencing followed by clustering of short sequences into operational taxonomic units (OTUs) is widely used for microbiome profiling. However, clustering of short 16S rRNA gene reads into biologically meaningful OTUs is challenging, in part because nucleotide variation along the 16S rRNA gene is only partially captured by short reads. The recent emergence of long-read platforms, such as single-molecule real-time (SMRT) sequencing from Pacific Biosciences, offers the potential for improved taxonomic and phylogenetic profiling. Here, we evaluate the performance of long- and short-read 16S rRNA gene sequencing using simulated and experimental data, followed by OTU…

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