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June 1, 2021  |  

New discoveries from closing Salmonella genomes using Pacific Biosciences continuous long reads.

The newer hierarchical genome assembly process (HGAP) performs de novo assembly using data from a single PacBio long insert library. To assess the benefits of this method, DNA from several Salmonella enterica serovars was isolated from a pure culture. Genome sequencing was performed using Pacific Biosciences RS sequencing technology. The HGAP process enabled us to close sixteen Salmonella subsp. enterica genomes and their associated mobile elements: The ten serotypes include: Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) S. Bareilly, S. Heidelberg, S. Cubana, S. Javiana and S. Typhimurium, S. Newport, S. Montevideo, S. Agona, and S. Tennessee. In addition, we were able to detect novel methyltransferases (MTases) by using the Pacific Biosciences kinetic score distributions showing that each serovar appears to have a novel methylation pattern. For example while all Salmonella serovars examined so far have methylase specific activity for 5’-GATC-3’/3’-CTAG-5’ and 5’-CAGAG-3’/3’-GTCTC-5’ (underlined base indicates a modification), S. Heidelberg is uniquely specific for 5’-ACCANCC-3’/3’-TGGTNGG-5’, while S. Typhimurium has uniquely methylase specific for 5′-GATCAG-3’/3′- CTAGTC-5′ sites, for the samples examined so far. We believe that this may be due to the unique environments and phages that these serotypes have been exposed to. Furthermore, our analysis identified and closed a variety of plasmids such as mobilization plasmids, antimicrobial resistance plasmids and IncX plasmids carrying a Type IV secretion system (T4SS). The VirB/D4 T4SS apparatus is important in that it assists with rapid dissemination of antibiotic resistance and virulence determinants. Presently, only limited information exists regarding the genotypic characterization of drug resistance in S. Heidelberg isolates derived from various host species. Here, we characterize two S. Heidelberg outbreak isolates from two different outbreaks. Both isolates contain the IncX plasmid of approximately 35 kb, and carried the genes virB1, virB2, virB3/4, virB5, virB6, virB7, virB8, virB9, virB10, virB11, virD2, and virD4, that are associated with the T4SS. In addition, the outbreak isolate associated with ground turkey carries a 4,473 bp mobilization plasmid and an incompatibility group (Inc) I1 antimicrobial resistance plasmid encoding resistance to gentamicin (aacC2), beta-lactam (bl2b_tem), streptomycin (aadAI) and tetracycline (tetA, tetR) while the outbreak isolate associated with chicken breast carries the IncI1 plasmid encoding resistance to gentamicin (aacC2), streptomycin (aadAI) and sulfisoxazole (sul1). Using this new technology we explored the genetic elements present in resistant pathogens which will achieve a better understanding of the evolution of Salmonella.


June 1, 2021  |  

HLA variant identification techniques

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 use of statistical inference to suggest the most likely diploid alleles given the genotypes observed. The assumption of this approach is the availability of an extensive reference panel. Whilst there exists good population genetics data for imputing European populations, there remains a paucity of information about variation in African populations. Filling this gap is one of the aims of the Genome Diversity in Africa Project and as a first step we are performing a pilot study to identify the optimal method for determining HLA type information for large numbers of samples from African populations.To that end we have obtained samples from 125 consented African participants selected from 5 populations across Africa (Morrocan, Ashanti, Igbo, Kalenjin, and Zulu). The methods included in our pilot study are Sanger sequencing (ABI), NGS on HiSeqX Ten platform (Illumina); long-range PCR combined with single molecule real-time (SMRT) sequencing (PacBio); and for a subset of samples library preparation on GemCode Platform (10x Genomics), which delivers valuable long range contextual information, combined with Illumina NGS sequencing.Results from capillary sequencing suggests the presence of a minimum of two novel alleles. Long Range PCR have been performed initially on a subset of samples using both primers sourced from GenDX and designed as described in Shiina et al (2012). Initial results from both primer sets were promising on Promega DNA test samples but only the GenDX primers proved effective on the African samples, producing consistently PCR products of the expected size in the Igbo, Ashanti, Morrocan and Zulu samples. We will present early results from our evaluation of the different sequencing technologies


June 1, 2021  |  

Analysis of 37,000 Caucasian samples reveals tight linkage between SNP RS9277534 and high resolution typing of HLA-DPB1

HLA-DPB1 mismatching between patients and unrelated donors is known to increase the risk of acute graft-versus-host-disease (GvHD) after hematopoietic stem cell transplantation. If only HLA-DPB1 mismatched donors are available, the genotype defined by the Single Nucleotide Polymorphism (SNP) rs9277534 can be used to select mismatched donors that are well-tolerated. However, since rs9277534 resides within the 3’ untranslated region (UTR), it usually is not analyzed during DPB1 routine typing.


June 1, 2021  |  

SMRT-Cappable-seq reveals the complex operome of bacteria

SMRT-Cappable-seq combines the isolation of full-length prokaryotic primary transcripts with long read sequencing technology. It is the first experimental methodology to sequence entire prokaryotic transcripts. It identifies the transcription start site and termination site, thereby directly defines the operon structures genome-wide in prokaryotes. Applied to E.coli, SMRT-Cappable-seq identifies a total of ~2300 operons, among which ~900 are novel. Importantly, our result reveals a pervasive read-through of previous experimentally validated transcription termination sites. Termination read-through represents a powerful strategy to control gene expression. Taken together this data provides a first glance at the complexity of the ‘operome’ in bacteria and presents an invaluable resource for understanding gene regulation and function in bacteria.


June 1, 2021  |  

Full-length cDNA sequencing of prokaryotic transcriptome and metatranscriptome samples

Next-generation sequencing has become a useful tool for studying transcriptomes. However, these methods typically rely on sequencing short fragments of cDNA, then attempting to assemble the pieces into full-length transcripts. Here, we describe a method that uses PacBio long reads to sequence full-length cDNAs from individual transcriptomes and metatranscriptome samples. We have adapted the PacBio Iso-Seq protocol for use with prokaryotic samples by incorporating RNA polyadenylation and rRNA-depletion steps. In conjunction with SMRT Sequencing, which has average readlengths of 10-15 kb, we are able to sequence entire transcripts, including polycistronic RNAs, in a single read. Here, we show full-length bacterial transcriptomes with the ability to visualize transcription of operons. In the area of metatranscriptomics, long reads reveal unambiguous gene sequences without the need for post-sequencing transcript assembly. We also show full-length bacterial transcripts sequenced after being treated with NEB’s Cappable-Seq, which is an alternative method for depleting rRNA and enriching for full-length transcripts with intact 5’ ends. Combining Cappable-Seq with PacBio long reads allows for the detection of transcription start sites, with the additional benefit of sequencing entire transcripts.


June 1, 2021  |  

Comparative metagenome-assembled genome analysis of “Candidatus Lachnocurva vaginae”, formerly known as Bacterial Vaginosis Associated bacterium – 1 (BVAB1)

Bacterial Vaginosis Associated bacterium 1 (BVAB1) is an as-yet uncultured bacterial species found in the human vagina that belongs to the family Lachnospiraceae within the order Clostridiales. As its name suggests, this bacterium is often associated with bacterial vaginosis (BV), a common vaginal disorder that has been shown to increase a woman’s risk for HIV, Chlamydia trachomatis, and Neisseria gonorrhoeae infections as well as preterm birth. Further, BVAB1 is associated with the persistence of BV following metronidazole treatment, increased vaginal inflammation, and adverse obstetrics outcomes. There is no available complete genome sequence of BVAB1, which has made it di?cult to mechanistically understand its role in disease. We present here a circularized metagenome-assembled genome (cMAG) of B VAB1 as well as a comparative analysis including an additional six metagenome-assembled genomes (MAGs) of this species. These sequences were derived from cervicovaginal samples of seven separate women. The cMAG is 1.649 Mb in size and encodes 1,578 genes. We propose to rename BVAB1 to “Candidatus Lachnocurva vaginae” based on phylogenetic analyses, and provide genomic evidence that this candidate species may metabolize D-lactate, produce trimethylamine (one of the chemicals responsible for BV-associated odor), and be motile. The cMAG and the six MAGs are valuable resources that will further contribute to our understanding of the heterogeneous etiology of bacterial vaginosis.


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 Program studies and 42 additional samples that had not been characterized previously. DNA samples were distributed to volunteer testing laboratories for genotyping using a variety of commercially available and laboratory-developed tests. These publicly available samples will support the quality-assurance and quality-control programs of clinical laboratories performing CYP2D6 testing.Published by Elsevier Inc.


April 21, 2020  |  

Chromosome-level reference genome of X12, a highly virulent race of the soybean cyst nematode Heterodera glycines.

Soybean cyst nematode (SCN, Heterodera glycines) is a major pest of soybean that is spreading across major soybean production regions worldwide. Increased SCN virulence has recently been observed in both the United States and China. However, no study has reported a genome assembly for H. glycines at the chromosome scale. Herein, the first chromosome-level reference genome of X12, an unusual SCN race with high infection ability, is presented. Using whole-genome shotgun (WGS) sequencing, PacBio sequencing, Illumina paired-end sequencing, 10X Genomics linked reads and high-throughput chromatin conformation capture (Hi-C) genome scaffolding techniques, a 141.01-Mb assembled genome was obtained with scaffold and contig N50 sizes of 16.27 Mb and 330.54 kb, respectively. The assembly showed high integrity and quality, with over 90% of Illumina reads mapped to the genome. The assembly quality was evaluated using Core Eukaryotic Genes Mapping Approach (CEGMA) and Benchmarking Universal Single-Copy Orthologs (BUSCO). A total of 11,882 genes were predicted using De novo, Homolog and RNAseq data generated from eggs, second-stage juveniles (J2), third-stage juveniles (J3) and fourth-stage juveniles (J4) of X12, and 79.0% of homologous sequences were annotated in the genome. These high-quality X12 genome data will provide valuable resources for research in a broad range of areas, including fundamental nematode biology, SCN-plant interactions and coevolution, and also contribute to the development of technology for overall SCN management. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.


April 21, 2020  |  

Genome assembly provides insights into the genome evolution and flowering regulation of orchardgrass.

Orchardgrass (Dactylis glomerata L.) is an important forage grass for cultivating livestock worldwide. Here, we report an ~1.84-Gb chromosome-scale diploid genome assembly of orchardgrass, with a contig N50 of 0.93 Mb, a scaffold N50 of 6.08 Mb and a super-scaffold N50 of 252.52 Mb, which is the first chromosome-scale assembled genome of a cool-season forage grass. The genome includes 40 088 protein-coding genes, and 69% of the assembled sequences are transposable elements, with long terminal repeats (LTRs) being the most abundant. The LTRretrotransposons may have been activated and expanded in the grass genome in response to environmental changes during the Pleistocene between 0 and 1 million years ago. Phylogenetic analysis reveals that orchardgrass diverged after rice but before three Triticeae species, and evolutionarily conserved chromosomes were detected by analysing ancient chromosome rearrangements in these grass species. We also resequenced the whole genome of 76 orchardgrass accessions and found that germplasm from Northern Europe and East Asia clustered together, likely due to the exchange of plants along the ‘Silk Road’ or other ancient trade routes connecting the East and West. Last, a combined transcriptome, quantitative genetic and bulk segregant analysis provided insights into the genetic network regulating flowering time in orchardgrass and revealed four main candidate genes controlling this trait. This chromosome-scale genome and the online database of orchardgrass developed here will facilitate the discovery of genes controlling agronomically important traits, stimulate genetic improvement of and functional genetic research on orchardgrass and provide comparative genetic resources for other forage grasses. © 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.


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