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Sunday, October 25, 2020

AGBT Virtual Poster: Direct-Seq – towards library-prep free PacBio sequencing

Paul Coupland and his team at the Wellcome Trust Sanger Institute have developed a sequencing method on the PacBio System for small DNA molecules that avoids the need for a standard library preparation. To date this approach has been applied toward sequencing single-stranded and double-stranded viral genomes, bacterial plasmids, plasmid vector models for DNA-modification analysis, and linear DNA fragments covering an entire bacterial genome. Using direct sequencing it is possible to generate sequence data from as little as 1 ng of DNA, offering a significant advantage over current protocols which typically require 400–500 ng of sheared DNA for the library…

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Sunday, October 25, 2020

User Group Meeting: New genotype to phenotype associations in viral metagenomes enabled by SMRT Sequencing

In this PacBio User Group Meeting lightning talk, Shawn Polson of the University of Delaware speaks about viral metagenomes, which are more challenging to distinguish than their bacterial counterparts because viruses have no 16S equivalent. By using SMRT Sequencing, his team generated higher-resolution data about viral genomes and aims to use this information as a guide to how these genomes function.

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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

The replication-competent HIV-1 latent reservoir is primarily established near the time of therapy initiation.

Although antiretroviral therapy (ART) is highly effective at suppressing HIV-1 replication, the virus persists as a latent reservoir in resting CD4+ T cells during therapy. This reservoir forms even when ART is initiated early after infection, but the dynamics of its formation are largely unknown. The viral reservoirs of individuals who initiate ART during chronic infection are generally larger and genetically more diverse than those of individuals who initiate therapy during acute infection, consistent with the hypothesis that the reservoir is formed continuously throughout untreated infection. To determine when viruses enter the latent reservoir, we compared sequences of replication-competent viruses…

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Tuesday, April 21, 2020

MSC: a metagenomic sequence classification algorithm.

Metagenomics is the study of genetic materials directly sampled from natural habitats. It has the potential to reveal previously hidden diversity of microscopic life largely due to the existence of highly parallel and low-cost next-generation sequencing technology. Conventional approaches align metagenomic reads onto known reference genomes to identify microbes in the sample. Since such a collection of reference genomes is very large, the approach often needs high-end computing machines with large memory which is not often available to researchers. Alternative approaches follow an alignment-free methodology where the presence of a microbe is predicted using the information about the unique k-mers…

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Tuesday, April 21, 2020

Analysis of the Complete Genome Sequence of a Novel, Pseudorabies Virus Strain Isolated in Southeast Europe.

Pseudorabies virus (PRV) is the causative agent of Aujeszky’s disease giving rise to significant economic losses worldwide. Many countries have implemented national programs for the eradication of this virus. In this study, long-read sequencing was used to determine the nucleotide sequence of the genome of a novel PRV strain (PRV-MdBio) isolated in Serbia.In this study, a novel PRV strain was isolated and characterized. PRV-MdBio was found to exhibit similar growth properties to those of another wild-type PRV, the strain Kaplan. Single-molecule real-time (SMRT) sequencing has revealed that the new strain differs significantly in base composition even from strain Kaplan, to…

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Tuesday, April 21, 2020

RNA-seq of HaHV-1-infected abalones reveals a common transcriptional signature of Malacoherpesviruses.

Haliotid herpesvirus-1 (HaHV-1) is the viral agent causative of abalone viral ganglioneuritis, a disease that has severely affected gastropod aquaculture. Although limited, the sequence similarity between HaHV-1 and Ostreid herpesvirus-1 supported the assignment of both viruses to Malacoherpesviridae, a Herpesvirales family distantly related with other viruses. In this study, we reported the first transcriptional data of HaHV-1, obtained from an experimental infection of Haliotis diversicolor supertexta. We also sequenced the genome draft of the Chinese HaHV-1 variant isolated in 2003 (HaHV-1-CN2003) by PacBio technology. Analysis of 13 million reads obtained from 3 RNA samples at 60?hours post injection (hpi) allowed…

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Tuesday, April 21, 2020

Metaepigenomic analysis reveals the unexplored diversity of DNA methylation in an environmental prokaryotic community.

DNA methylation plays important roles in prokaryotes, and their genomic landscapes-prokaryotic epigenomes-have recently begun to be disclosed. However, our knowledge of prokaryotic methylation systems is focused on those of culturable microbes, which are rare in nature. Here, we used single-molecule real-time and circular consensus sequencing techniques to reveal the ‘metaepigenomes’ of a microbial community in the largest lake in Japan, Lake Biwa. We reconstructed 19 draft genomes from diverse bacterial and archaeal groups, most of which are yet to be cultured. The analysis of DNA chemical modifications in those genomes revealed 22 methylated motifs, nine of which were novel. We…

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Tuesday, April 21, 2020

Linking CRISPR-Cas9 interference in cassava to the evolution of editing-resistant geminiviruses.

Geminiviruses cause damaging diseases in several important crop species. However, limited progress has been made in developing crop varieties resistant to these highly diverse DNA viruses. Recently, the bacterial CRISPR/Cas9 system has been transferred to plants to target and confer immunity to geminiviruses. In this study, we use CRISPR-Cas9 interference in the staple food crop cassava with the aim of engineering resistance to African cassava mosaic virus, a member of a widespread and important family (Geminiviridae) of plant-pathogenic DNA viruses.Our results show that the CRISPR system fails to confer effective resistance to the virus during glasshouse inoculations. Further, we find…

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Tuesday, April 21, 2020

Origin and recent expansion of an endogenous gammaretroviral lineage in domestic and wild canids.

Vertebrate genomes contain a record of retroviruses that invaded the germlines of ancestral hosts and are passed to offspring as endogenous retroviruses (ERVs). ERVs can impact host function since they contain the necessary sequences for expression within the host. Dogs are an important system for the study of disease and evolution, yet no substantiated reports of infectious retroviruses in dogs exist. Here, we utilized Illumina whole genome sequence data to assess the origin and evolution of a recently active gammaretroviral lineage in domestic and wild canids.We identified numerous recently integrated loci of a canid-specific ERV-Fc sublineage within Canis, including 58…

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Wednesday, February 26, 2020

Rapid sequencing of HIV-1 genomes as single molecules from simple and complex samples.

Background: To better understand the relationships among HIV-1 viruses in linked transmission pairs, we sequenced several samples representing HIV transmission pairs from the Zambia Emory HIV Research Project (Lusaka, Zambia) using Single Molecule, Real-Time (SMRT) Sequencing. Methods: Single molecules were sequenced as full-length (9.6 kb) amplicons directly from PCR products without shearing. This resulted in multiple, fully-phased, complete HIV-1 genomes for each patient. We examined Single Genome Amplification (SGA) prepped samples, as well as samples containing complex mixtures of genomes. We detail mathematical techniques used in viral variant subspecies identification, including clustering distance metrics and mutual information, which were used…

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Wednesday, February 26, 2020

Sequencing complex mixtures of HIV-1 genomes with single-base resolution.

A large number of distinct HIV-1 genomes can be present in a single clinical sample from a patient chronically infected with HIV-1. We examined samples containing complex mixtures of near-full-length HIV-1 genomes. Single molecules were sequenced as near-full-length (9.6 kb) amplicons directly from PCR products without shearing. Mathematical analysis techniques deconvolved the complex mixture of reads into estimates of distinct near-full-length viral genomes with their relative abundances. We correctly estimated the originating genomes to single-base resolution along with their relative abundances for mixtures where the truth was known exactly by independent sequencing methods. Correct estimates were made even when genomes…

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Wednesday, February 26, 2020

High-accuracy, single-base resolution of near-full-length HIV genomes.

Background: The HIV-1 proviral reservoir is incredibly stable, even while undergoing antiretroviral therapy, and is seen as the major barrier to HIV-1 eradication. Identifying and comprehensively characterizing this reservoir will be critical to achieving an HIV cure. Historically, this has been a tedious and labor intensive process, requiring high-replicate single-genome amplification reactions, or overlapping amplicons that are then reconstructed into full-length genomes by algorithmic imputation. Here, we present a deep sequencing and analysis method able to determine the exact identity and relative abundances of near-full-length HIV genomes from samples containing mixtures of genomes without shearing or complex bioinformatic reconstruction. Methods:…

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Wednesday, February 26, 2020

High-throughput SMRT Sequencing of clinically relevant targets

Targeted sequencing with Sanger as well as short read based high throughput sequencing methods is standard practice in clinical genetic testing. However, many applications beyond SNP detection have remained somewhat obstructed due to technological challenges. With the advent of long reads and high consensus accuracy, SMRT Sequencing overcomes many of the technical hurdles faced by Sanger and NGS approaches, opening a broad range of untapped clinical sequencing opportunities. Flexible multiplexing options, highly adaptable sample preparation method and newly improved two well-developed analysis methods that generate highly-accurate sequencing results, make SMRT Sequencing an adept method for clinical grade targeted sequencing. The…

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