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Tuesday, October 27, 2020

ASHG PacBio Workshop: Applications of third generation sequencing in unsolved disease

In this ASHG 2020 PacBio Workshop Emily Farrow of Children’s Mercy Kansas City shares how the incorporation of long-read sequencing into the Genomic Answers for Kids research study is increasing diagnostic yields through the identification of novel genetic variation. Emily highlights several cases in which PacBio HiFi sequencing was able to provide insights where short-read sequencing alone was inconclusive, due to limitations stemming from repetitive regions and large structural variants.

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Sunday, October 25, 2020

ASHI PacBio Workshop: KIR haplotypes – The long and short of it

KIR haplotypes can be determined by physical and computational and statistical methods. Martin Maiers from National Bone Marrow Donor Program (NMDP) presents a summary of their work to determine KIR genomic content for use in clinical transplantation, outcomes of HLA sequencing of KIR region across a variety of methods and shares their data from recent experiments using PacBio single-molecule sequencing of fosmid libraries.

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Sunday, October 25, 2020

Podcast: Long-read sequencing dramatically improves blood matching – Steven Marsh

One of the popular questions on the Mendelspod program is how those doing sequencing decide between the quality of PacBio’s long reads and the cheaper short read technology, such as that of Illumina or Thermo Fisher. Steve Marsh, the Director of Bioinformatics at the Anthony Nolan Research Institute in London, provides the most clear and dramatic answer yet: use the PacBio system exclusively. Established in 1974 by the mother of a boy with a rare blood disease, the Anthony Nolan Institute is a world leader in blood crossmatching and donor/patient registries. Steve and his team at the Institute have dramatically…

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Sunday, October 25, 2020

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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Sunday, October 25, 2020

AGBT PacBio Workshop: High-throughput HLA class I whole gene and HLA class II long range typing on PacBio RSII and Sequel Platforms

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…

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Sunday, October 25, 2020

ASHG PacBio Workshop: Characterization of a large, human-specific tandem repeat array associated with bipolar disorder and schizophrenia

In this ASHG workshop presentation, Janet Song of Stanford School of Medicine shared research on resolving a tandem repeat array implicated in bipolar disorder and schizophrenia. These psychiatric diseases share a number of genomic risk variants, she noted, but scientists continue to search for a specific causal variant in the CACNA1C gene suggested by previous genome-wide association studies. SMRT Sequencing of this region in 16 individuals identified a series of 30-mer repeats, containing a total of about 50 variants. Analysis showed that 10 variants were linked to protective or risk haplotypes. Song aims to study the function of these variants…

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Sunday, October 25, 2020

User Group Meeting: From long reads to transcript function: Bioinformatics tools for Iso-transcriptomics analysis

In this PacBio User Group Meeting presentation, Ana Conesa Cegarra from the University of Florida spoke about Iso-Seq analysis tools developed by her group, which created the popular SQANTI tools for Iso-Seq data QC. They’re also working on IsoAnnot to perform functional annotation at isoform resolution; validation has already been done on various species. Currently it’s a set of scripts, but her team is working to produce a more user-friendly version. Finally, tappAS is for functional diversity analysis and for prioritizing genes for validation.

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Tuesday, April 21, 2020

Tandem repeats lead to sequence assembly errors and impose multi-level challenges for genome and protein databases.

The widespread occurrence of repetitive stretches of DNA in genomes of organisms across the tree of life imposes fundamental challenges for sequencing, genome assembly, and automated annotation of genes and proteins. This multi-level problem can lead to errors in genome and protein databases that are often not recognized or acknowledged. As a consequence, end users working with sequences with repetitive regions are faced with ‘ready-to-use’ deposited data whose trustworthiness is difficult to determine, let alone to quantify. Here, we provide a review of the problems associated with tandem repeat sequences that originate from different stages during the sequencing-assembly-annotation-deposition workflow, and…

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Tuesday, April 21, 2020

The bracteatus pineapple genome and domestication of clonally propagated crops.

Domestication of clonally propagated crops such as pineapple from South America was hypothesized to be a ‘one-step operation’. We sequenced the genome of Ananas comosus var. bracteatus CB5 and assembled 513?Mb into 25 chromosomes with 29,412 genes. Comparison of the genomes of CB5, F153 and MD2 elucidated the genomic basis of fiber production, color formation, sugar accumulation and fruit maturation. We also resequenced 89 Ananas genomes. Cultivars ‘Smooth Cayenne’ and ‘Queen’ exhibited ancient and recent admixture, while ‘Singapore Spanish’ supported a one-step operation of domestication. We identified 25 selective sweeps, including a strong sweep containing a pair of tandemly duplicated…

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Tuesday, April 21, 2020

Cultured Epidermal Autografts from Clinically Revertant Skin as a Potential Wound Treatment for Recessive Dystrophic Epidermolysis Bullosa.

Inherited skin disorders have been reported recently to have sporadic normal-looking areas, where a portion of the keratinocytes have recovered from causative gene mutations (revertant mosaicism). We observed a case of recessive dystrophic epidermolysis bullosa treated with cultured epidermal autografts (CEAs), whose CEA-grafted site remained epithelized for 16 years. We proved that the CEA product and the grafted area included cells with revertant mosaicism. Based on these findings, we conducted an investigator-initiated clinical trial of CEAs from clinically revertant skin for recessive dystrophic epidermolysis bullosa. The donor sites were analyzed by genetic analysis, immunofluorescence, electron microscopy, and quantification of the…

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Tuesday, April 21, 2020

Evolution of a 72-kb cointegrant, conjugative multiresistance plasmid from early community-associated methicillin-resistant Staphylococcus aureus isolates.

Horizontal transfer of plasmids encoding antimicrobial-resistance and virulence determinants has been instrumental in Staphylococcus aureus evolution, including the emergence of community-associated methicillin-resistant S. aureus (CA-MRSA). In the early 1990s the first CA-MRSA isolated in Western Australia (WA), WA-5, encoded cadmium, tetracycline and penicillin-resistance genes on plasmid pWBG753 (~30 kb). WA-5 and pWBG753 appeared only briefly in WA, however, fusidic-acid-resistance plasmids related to pWBG753 were also present in the first European CA-MRSA at the time. Here we characterized a 72-kb conjugative plasmid pWBG731 present in multiresistant WA-5-like clones from the same period. pWBG731 was a cointegrant formed from pWBG753 and a…

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Tuesday, April 21, 2020

Transcriptional initiation of a small RNA, not R-loop stability, dictates the frequency of pilin antigenic variation in Neisseria gonorrhoeae.

Neisseria gonorrhoeae, the sole causative agent of gonorrhea, constitutively undergoes diversification of the Type IV pilus. Gene conversion occurs between one of the several donor silent copies located in distinct loci and the recipient pilE gene, encoding the major pilin subunit of the pilus. A guanine quadruplex (G4) DNA structure and a cis-acting sRNA (G4-sRNA) are located upstream of the pilE gene and both are required for pilin antigenic variation (Av). We show that the reduced sRNA transcription lowers pilin Av frequencies. Extended transcriptional elongation is not required for Av, since limiting the transcript to 32 nt allows for normal…

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Tuesday, April 21, 2020

Acquired N-Linked Glycosylation Motifs in B-Cell Receptors of Primary Cutaneous B-Cell Lymphoma and the Normal B-Cell Repertoire.

Primary cutaneous follicle center lymphoma (PCFCL) is a rare mature B-cell lymphoma with an unknown etiology. PCFCL resembles follicular lymphoma (FL) by cytomorphologic and microarchitectural criteria. FL B cells are selected for N-linked glycosylation motifs in their B-cell receptors (BCRs) that are acquired during continuous somatic hypermutation. The stimulation of mannosylated BCR by lectins on the tumor microenvironment is therefore a candidate driver in FL pathogenesis. We investigated whether the same mechanism could play a role in PCFCL pathogenesis. Full-length functional variable, diversity, and joining gene sequences of 18 PCFCL and 8 primary cutaneous diffuse large B-cell lymphoma, leg-type were…

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Tuesday, April 21, 2020

Plasmid-encoded tet(X) genes that confer high-level tigecycline resistance in Escherichia coli.

Tigecycline is one of the last-resort antibiotics to treat complicated infections caused by both multidrug-resistant Gram-negative and Gram-positive bacteria1. Tigecycline resistance has sporadically occurred in recent years, primarily due to chromosome-encoding mechanisms, such as overexpression of efflux pumps and ribosome protection2,3. Here, we report the emergence of the plasmid-mediated mobile tigecycline resistance mechanism Tet(X4) in Escherichia coli isolates from China, which is capable of degrading all tetracyclines, including tigecycline and the US FDA newly approved eravacycline. The tet(X4)-harbouring IncQ1 plasmid is highly transferable, and can be successfully mobilized and stabilized in recipient clinical and laboratory strains of Enterobacteriaceae bacteria. It…

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