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Asset Tag: Third generation sequencing

July 19, 2019

Long-read Single-Molecule Real-Time (SMRT) full gene sequencing of cytochrome P450-2D6 (CYP2D6).

The CYP2D6 enzyme metabolizes ~25% of common medications, yet homologous pseudogenes and copy-number variants (CNVs) make interrogating the polymorphic CYP2D6 gene with short-read sequencing challenging. Therefore, we developed a novel long-read, full gene CYP2D6 single-molecule real-time (SMRT) sequencing method using the Pacific Biosciences platform. Long-range PCR and CYP2D6 SMRT sequencing of 10 previously genotyped controls identified expected star (*) alleles, but also enabled suballele resolution, diplotype refinement, and discovery of novel alleles. Coupled with an optimized variant calling pipeline, CYP2D6 SMRT sequencing was highly reproducible as triplicate intra- and inter-run non-reference genotype results were completely concordant. Importantly, targeted SMRT sequencing of upstream and downstream CYP2D6 gene copies characterized the duplicated allele in 15 control samples with CYP2D6 CNVs. The utility of CYP2D6 SMRT sequencing was further underscored by identifying the diplotypes of 14 samples with discordant or unclear CYP2D6 configurations from previous targeted genotyping, which again included suballele resolution, duplicated allele characterization, and discovery of a novel allele and tandem arrangement (CYP2D6*36+*41). Taken together, long-read CYP2D6 SMRT sequencing is an innovative, reproducible, and validated method for full-gene characterization, duplication allele-specific analysis and novel allele discovery, which will likely improve CYP2D6 metabolizer phenotype prediction for both research and clinical testing applications. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.

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July 19, 2019

Detection and screening of chromosomal rearrangements in uterine leiomyomas by long-distance inverse PCR.

Genome instability is a hallmark of many tumors and recently, next-generation sequencing methods have enabled analyses of tumor genomes at an unprecedented level. Studying rearrangement-prone chromosomal regions (putative “breakpoint hotspots”) in detail, however, necessitates molecular assays that can detect de novo DNA fusions arising from these hotspots. Here we demonstrate the utility of a long-distance inverse PCR-based method for the detection and screening of de novo DNA rearrangements in uterine leiomyomas, one of the most common types of human neoplasm. This assay allows in principle any genomic region suspected of instability to be queried for DNA rearrangements originating there. No prior knowledge of the identity of the fusion partner chromosome is needed. We used this method to screen uterine leiomyomas for rearrangements at genomic locations known to be rearrangement-prone in this tumor type: upstream HMGA2 and within RAD51B. We identified a novel DNA rearrangement upstream of HMGA2 that had gone undetected in an earlier whole-genome sequencing study. In more than 30 additional uterine leiomyoma samples, not analyzed by whole-genome sequencing previously, no rearrangements were observed within the 1,107 bp and 1,996 bp assayed in the RAD51B and HMGA2 rearrangement hotspots. Our findings show that long-distance inverse PCR is a robust, sensitive, and cost-effective method for the detection and screening of DNA rearrangements from solid tumors that should be useful for many diagnostic applications. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

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July 19, 2019

The power of Single Molecule Real-Time sequencing technology in the de novo assembly of a eukaryotic genome.

Second-generation sequencers (SGS) have been game-changing, achieving cost-effective whole genome sequencing in many non-model organisms. However, a large portion of the genomes still remains unassembled. We reconstructed azuki bean (Vigna angularis) genome using single molecule real-time (SMRT) sequencing technology and achieved the best contiguity and coverage among currently assembled legume crops. The SMRT-based assembly produced 100 times longer contigs with 100 times smaller amount of gaps compared to the SGS-based assemblies. A detailed comparison between the assemblies revealed that the SMRT-based assembly enabled a more comprehensive gene annotation than the SGS-based assemblies where thousands of genes were missing or fragmented. A chromosome-scale assembly was generated based on the high-density genetic map, covering 86% of the azuki bean genome. We demonstrated that SMRT technology, though still needed support of SGS data, achieved a near-complete assembly of a eukaryotic genome.

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July 19, 2019

DNA methylation assessed by SMRT Sequencing is linked to mutations in Neisseria meningitidis isolates.

The Gram-negative bacterium Neisseria meningitidis features extensive genetic variability. To present, proposed virulence genotypes are also detected in isolates from asymptomatic carriers, indicating more complex mechanisms underlying variable colonization modes of N. meningitidis. We applied the Single Molecule, Real-Time (SMRT) sequencing method from Pacific Biosciences to assess the genome-wide DNA modification profiles of two genetically related N. meningitidis strains, both of serogroup A. The resulting DNA methylomes revealed clear divergences, represented by the detection of shared and of strain-specific DNA methylation target motifs. The positional distribution of these methylated target sites within the genomic sequences displayed clear biases, which suggest a functional role of DNA methylation related to the regulation of genes. DNA methylation in N. meningitidis has a likely underestimated potential for variability, as evidenced by a careful analysis of the ORF status of a panel of confirmed and predicted DNA methyltransferase genes in an extended collection of N. meningitidis strains of serogroup A. Based on high coverage short sequence reads, we find phase variability as a major contributor to the variability in DNA methylation. Taking into account the phase variable loci, the inferred functional status of DNA methyltransferase genes matched the observed methylation profiles. Towards an elucidation of presently incompletely characterized functional consequences of DNA methylation in N. meningitidis, we reveal a prominent colocalization of methylated bases with Single Nucleotide Polymorphisms (SNPs) detected within our genomic sequence collection. As a novel observation we report increased mutability also at 6mA methylated nucleotides, complementing mutational hotspots previously described at 5mC methylated nucleotides. These findings suggest a more diverse role of DNA methylation and Restriction-Modification (RM) systems in the evolution of prokaryotic genomes.

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July 19, 2019

Quantifying influenza virus diversity and transmission in humans.

Influenza A virus is characterized by high genetic diversity. However, most of what is known about influenza evolution has come from consensus sequences sampled at the epidemiological scale that only represent the dominant virus lineage within each infected host. Less is known about the extent of within-host virus diversity and what proportion of this diversity is transmitted between individuals. To characterize virus variants that achieve sustainable transmission in new hosts, we examined within-host virus genetic diversity in household donor-recipient pairs from the first wave of the 2009 H1N1 pandemic when seasonal H3N2 was co-circulating. Although the same variants were found in multiple members of the community, the relative frequencies of variants fluctuated, with patterns of genetic variation more similar within than between households. We estimated the effective population size of influenza A virus across donor-recipient pairs to be approximately 100-200 contributing members, which enabled the transmission of multiple lineages, including antigenic variants.

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July 19, 2019

A diamond in the ruff.

Reference genomes are only as valuable as the scientific questions they can address. The ruff genome sequence papers exemplify three of the most important aspects of a useful genome: new biological insights, a high-quality resource and population variation data.

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July 19, 2019

Precision methylome characterization of Mycobacterium tuberculosis complex (MTBC) using PacBio single-molecule real-time (SMRT) technology.

Tuberculosis (TB) remains one of the most common infectious diseases caused by Mycobacterium tuberculosis complex (MTBC). To panoramically analyze MTBC’s genomic methylation, we completed the genomes of 12 MTBC strains (Mycobacterium bovis; M. bovis BCG; M. microti; M. africanum; M. tuberculosis H37Rv; H37Ra; and 6 M. tuberculosis clinical isolates) belonging to different lineages and characterized their methylomes using single-molecule real-time (SMRT) technology. We identified three (m6)A sequence motifs and their corresponding methyltransferase (MTase) genes, including the reported mamA, hsdM and a newly discovered mamB. We also experimentally verified the methylated motifs and functions of HsdM and MamB. Our analysis indicated the MTase activities varied between 12 strains due to mutations/deletions. Furthermore, through measuring ‘the methylated-motif-site ratio’ and ‘the methylated-read ratio’, we explored the methylation status of each modified site and sequence-read to obtain the ‘precision methylome’ of the MTBC strains, which enabled intricate analysis of MTase activity at whole-genome scale. Most unmodified sites overlapped with transcription-factor binding-regions, which might protect these sites from methylation. Overall, our findings show enormous potential for the SMRT platform to investigate the precise character of methylome, and significantly enhance our understanding of the function of DNA MTase.© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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July 19, 2019

Lifespan of restriction-modification systems critically affects avoidance of their recognition sites in host genomes.

Avoidance of palindromic recognition sites of Type II restriction-modification (R-M) systems was shown for many R-M systems in dozens of prokaryotic genomes. However the phenomenon has not been investigated systematically for all presently available genomes and annotated R-M systems. We have studied all known recognition sites in thousands of prokaryotic genomes and found factors that influence their avoidance.Only Type II R-M systems consisting of independently acting endonuclease and methyltransferase (called ‘orthodox’ here) cause avoidance of their sites, both palindromic and asymmetric, in corresponding prokaryotic genomes; the avoidance takes place for?~?50 % of 1774 studied cases. It is known that prokaryotes can acquire and lose R-M systems. Thus it is possible to talk about the lifespan of an R-M system in a genome. We have shown that the recognition site avoidance correlates with the lifespan of R-M systems. The sites of orthodox R-M systems that are encoded in host genomes for a long time are avoided more often (up to 100 % in certain cohorts) than the sites of recently acquired ones. We also found cases of site avoidance in absence of the corresponding R-M systems in the genome. An analysis of closely related bacteria shows that such avoidance can be a trace of lost R-M systems. Sites of Type I, II?/G, IIM, III, and IV R-M systems are not avoided in vast majority of cases.The avoidance of orthodox Type II R-M system recognition sites in prokaryotic genomes is a widespread phenomenon. Presence of an R-M system without an underrepresentation of its site may indicate that the R-M system was acquired recently. At the same time, a significant underrepresentation of a site may be a sign of presence of the corresponding R-M system in this organism or in its ancestors for a long time. The drastic difference between site avoidance for orthodox Type II R-M systems and R-M systems of other types can be explained by a higher rate of specificity changes or a less self-toxicity of the latter.

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July 19, 2019

AnnoTALE: bioinformatics tools for identification, annotation, and nomenclature of TALEs from Xanthomonas genomic sequences.

Transcription activator-like effectors (TALEs) are virulence factors, produced by the bacterial plant-pathogen Xanthomonas, that function as gene activators inside plant cells. Although the contribution of individual TALEs to infectivity has been shown, the specific roles of most TALEs, and the overall TALE diversity in Xanthomonas spp. is not known. TALEs possess a highly repetitive DNA-binding domain, which is notoriously difficult to sequence. Here, we describe an improved method for characterizing TALE genes by the use of PacBio sequencing. We present ‘AnnoTALE’, a suite of applications for the analysis and annotation of TALE genes from Xanthomonas genomes, and for grouping similar TALEs into classes. Based on these classes, we propose a unified nomenclature for Xanthomonas TALEs that reveals similarities pointing to related functionalities. This new classification enables us to compare related TALEs and to identify base substitutions responsible for the evolution of TALE specificities.

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July 19, 2019

TCR sequencing of single cells reactive to DQ2.5-glia-a2 and DQ2.5-glia-?2 reveals clonal expansion and epitope-specific V-gene usage.

CD4+ T cells recognizing dietary gluten epitopes in the context of disease-associated human leukocyte antigen (HLA)-DQ2 or HLA-DQ8 molecules are the key players in celiac disease pathogenesis. Here, we conducted a large-scale single-cell paired T-cell receptor (TCR) sequencing study to characterize the TCR repertoire for two homologous immunodominant gluten epitopes, DQ2.5-glia-a2 and DQ2.5-glia-?2, in blood of celiac disease patients after oral gluten challenge. Despite sequence similarity of the epitopes, the TCR repertoires are unique but shared several overall features. We demonstrate that clonally expanded T cells dominate the T-cell responses to both epitopes. Moreover, we find V-gene bias of TRAV26, TRAV4, and TRBV7 in DQ2.5-glia-a2 reactive TCRs, while DQ2.5-glia-?2 TCRs displayed significant bias toward TRAV4 and TRBV4. The knowledge that antigen-specific TCR repertoire in chronic inflammatory diseases tends to be dominated by a few expanded clones that use the same TCR V-gene segments across patients is important information for HLA-associated diseases where the antigen is unknown.

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July 19, 2019

The epigenomic landscape of prokaryotes.

DNA methylation acts in concert with restriction enzymes to protect the integrity of prokaryotic genomes. Studies in a limited number of organisms suggest that methylation also contributes to prokaryotic genome regulation, but the prevalence and properties of such non-restriction-associated methylation systems remain poorly understood. Here, we used single molecule, real-time sequencing to map DNA modifications including m6A, m4C, and m5C across the genomes of 230 diverse bacterial and archaeal species. We observed DNA methylation in nearly all (93%) organisms examined, and identified a total of 834 distinct reproducibly methylated motifs. This data enabled annotation of the DNA binding specificities of 620 DNA Methyltransferases (MTases), doubling known specificities for previously hard to study Type I, IIG and III MTases, and revealing their extraordinary diversity. Strikingly, 48% of organisms harbor active Type II MTases with no apparent cognate restriction enzyme. These active ‘orphan’ MTases are present in diverse bacterial and archaeal phyla and show motif specificities and methylation patterns consistent with functions in gene regulation and DNA replication. Our results reveal the pervasive presence of DNA methylation throughout the prokaryotic kingdoms, as well as the diversity of sequence specificities and potential functions of DNA methylation systems.

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July 19, 2019

Major improvements to the Heliconius melpomene genome assembly used to confirm 10 chromosome fusion events in 6 million years of butterfly evolution.

The Heliconius butterflies are a widely studied adaptive radiation of 46 species spread across Central and South America, several of which are known to hybridize in the wild. Here, we present a substantially improved assembly of the Heliconius melpomene genome, developed using novel methods that should be applicable to improving other genome assemblies produced using short read sequencing. First, we whole-genome-sequenced a pedigree to produce a linkage map incorporating 99% of the genome. Second, we incorporated haplotype scaffolds extensively to produce a more complete haploid version of the draft genome. Third, we incorporated ~20x coverage of Pacific Biosciences sequencing, and scaffolded the haploid genome using an assembly of this long-read sequence. These improvements result in a genome of 795 scaffolds, 275 Mb in length, with an N50 length of 2.1 Mb, an N50 number of 34, and with 99% of the genome placed, and 84% anchored on chromosomes. We use the new genome assembly to confirm that the Heliconius genome underwent 10 chromosome fusions since the split with its sister genus Eueides, over a period of about 6 million yr. Copyright © 2016 Davey et al.

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July 19, 2019

Phase variation of a Type IIG restriction-modification enzyme alters site-specific methylation patterns and gene expression in Campylobacter jejuni strain NCTC11168.

Phase-variable restriction-modification systems are a feature of a diverse range of bacterial species. Stochastic, reversible switches in expression of the methyltransferase produces variation in methylation of specific sequences. Phase-variable methylation by both Type I and Type III methyltransferases is associated with altered gene expression and phenotypic variation. One phase-variable gene of Campylobacter jejuni encodes a homologue of an unusual Type IIG restriction-modification system in which the endonuclease and methyltransferase are encoded by a single gene. Using both inhibition of restriction and PacBio-derived methylome analyses of mutants and phase-variants, the cj0031c allele in C. jejuni strain NCTC11168 was demonstrated to specifically methylate adenine in 5’CCCGA and 5’CCTGA sequences. Alterations in the levels of specific transcripts were detected using RNA-Seq in phase-variants and mutants of cj0031c but these changes did not correlate with observed differences in phenotypic behaviour. Alterations in restriction of phage growth were also associated with phase variation (PV) of cj0031c and correlated with presence of sites in the genomes of these phages. We conclude that PV of a Type IIG restriction-modification system causes changes in site-specific methylation patterns and gene expression patterns that may indirectly change adaptive traits.© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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July 19, 2019

Large genomic differences between Moraxella bovoculi isolates acquired from the eyes of cattle with infectious bovine keratoconjunctivitis versus the deep nasopharynx of asymptomatic cattle.

Moraxella bovoculi is a recently described bacterium that is associated with infectious bovine keratoconjunctivitis (IBK) or “pinkeye” in cattle. In this study, closed circularized genomes were generated for seven M. bovoculi isolates: three that originated from the eyes of clinical IBK bovine cases and four from the deep nasopharynx of asymptomatic cattle. Isolates that originated from the eyes of IBK cases profoundly differed from those that originated from the nasopharynx of asymptomatic cattle in genome structure, gene content and polymorphism diversity and consequently placed into two distinct phylogenetic groups. These results suggest that there are genetically distinct strains of M. bovoculi that may not associate with IBK.

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