scholarly journals SMRT sequencing reveals differential patterns of methylation in two O111:H- Shiga toxigenicEscherichia coliisolates from a historic hemolytic uremic syndrome outbreak in Australia

2017 ◽  
Author(s):  
Brian M. Forde ◽  
Lauren J. McAllister ◽  
James C. Paton ◽  
Adrienne W. Paton ◽  
Scott A. Beatson
Keyword(s):  
Single Molecule ◽  
Genomic Analysis ◽  
Smrt Sequencing ◽  
Promoter Regions ◽  
Food Borne ◽  
Long Reads ◽  
Long Read ◽  
Shiga Toxin 2 ◽  
Uremic Syndrome ◽  
Potential Promoter

AbstractShiga toxigenicEscherichia coli(STEC) are important food-borne pathogens and a major cause of haemorrhagic colitis and haemolytic-uremic syndrome (HUS) worldwide. In 1995 a severe HUS outbreak in Adelaide occurred. A recent genomic analysis of STEC O111:H-strains 95JB1 and 95NR1 from this outbreak found that the more virulent isolate, 95NR1, harboured two additional copies of the Shiga toxin 2 (Stx2) genes although the structure of the Stx2-converting prophages could not be fully resolved due to the fragmented assembly. In this study we have used Pacific Biosciences (PacBio) single molecule real-time (SMRT) long read sequencing to characterise the complete epigenome (genome and methylome) of 95JB1 and 95NR1. Using long reads we completely resolved the structure of two, tandemly inserted, stx2-converting phage in 95NR1. Our analysis of the methylome of 95NR1 and 95JB1 identified hemi-methylation of a novel motif (5’-CTGCm6AG-3’) in more than 4000 sites in the 95NR1 genome. These sites were entirely unmethalyted in the 95JB1, including at least 180 potential promoter regions that could explain regulatory differences between the strains. We identified a Type IIG methyltransferase encoded in both genomes in association with three additional genes in an operon-like arrangement. IS1203mediated disruption of this operon in 95JB1 is the likely cause of the observed differential patterns of methylation between 95NR1 and 95JB1. This study demonstrates the enormous potential of PacBio SMRT sequencing to resolve complex prophage regions and reveal the genetic and epigenetic heterogeneity within a clonal population of bacteria.

scholarly journals Assembling Large Genomes with Single-Molecule Sequencing and Locality Sensitive Hashing

2014 ◽  
Author(s):  
Konstantin Berlin ◽  
Sergey Koren ◽  
Chen-Shan Chin ◽  
James Drake ◽  
Jane M Landolin ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Locality Sensitive Hashing ◽  
Model Organisms ◽  
Smrt Sequencing ◽  
High Coverage ◽  
Celera Assembler ◽  
Single Molecule Sequencing ◽  
Long Reads ◽  
Long Read

We report reference-grade de novo assemblies of four model organisms and the human genome from single-molecule, real-time (SMRT) sequencing. Long-read SMRT sequencing is routinely used to finish microbial genomes, but the available assembly methods have not scaled well to larger genomes. Here we introduce the MinHash Alignment Process (MHAP) for efficient overlapping of noisy, long reads using probabilistic, locality-sensitive hashing. Together with Celera Assembler, MHAP was used to reconstruct the genomes of Escherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster, and human from high-coverage SMRT sequencing. The resulting assemblies include fully resolved chromosome arms and close persistent gaps in these important reference genomes, including heterochromatic and telomeric transition sequences. For D. melanogaster, MHAP achieved a 600-fold speedup relative to prior methods and a cloud computing cost of a few hundred dollars. These results demonstrate that single-molecule sequencing alone can produce near-complete eukaryotic genomes at modest cost.

scholarly journals Hapo-G, haplotype-aware polishing of genome assemblies with accurate reads

2021 ◽  
Vol 3 (2) ◽  
Author(s):  
Jean-Marc Aury ◽  
Benjamin Istace
Keyword(s):  
Single Molecule ◽  
Direct Consequence ◽  
High Quality ◽  
Sequencing Errors ◽  
Coding Regions ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read ◽  
Genome Assemblies

Abstract Single-molecule sequencing technologies have recently been commercialized by Pacific Biosciences and Oxford Nanopore with the promise of sequencing long DNA fragments (kilobases to megabases order) and then, using efficient algorithms, provide high quality assemblies in terms of contiguity and completeness of repetitive regions. However, the error rate of long-read technologies is higher than that of short-read technologies. This has a direct consequence on the base quality of genome assemblies, particularly in coding regions where sequencing errors can disrupt the coding frame of genes. In the case of diploid genomes, the consensus of a given gene can be a mixture between the two haplotypes and can lead to premature stop codons. Several methods have been developed to polish genome assemblies using short reads and generally, they inspect the nucleotide one by one, and provide a correction for each nucleotide of the input assembly. As a result, these algorithms are not able to properly process diploid genomes and they typically switch from one haplotype to another. Herein we proposed Hapo-G (Haplotype-Aware Polishing Of Genomes), a new algorithm capable of incorporating phasing information from high-quality reads (short or long-reads) to polish genome assemblies and in particular assemblies of diploid and heterozygous genomes.

Fatal Respiratory Diphtheria Caused by ß-Lactam–Resistant Corynebacterium diphtheriae

2020 ◽  
Author(s):  
Brian M Forde ◽  
Andrew Henderson ◽  
Elliott G Playford ◽  
David Looke ◽  
Belinda C Henderson ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genomic Analysis ◽  
Carbapenem Resistance ◽  
Corynebacterium Diphtheriae ◽  
Penicillin Binding Protein ◽  
Long Read ◽  
Amoxicillin Clavulanic Acid ◽  
Resistance To Penicillin ◽  
High Level

Abstract Background Diphtheria is a potentially fatal respiratory disease caused by toxigenic Corynebacterium diphtheriae. Although resistance to erythromycin has been recognized, β-lactam resistance in toxigenic diphtheria has not been described. Here, we report a case of fatal respiratory diphtheria caused by toxigenic C. diphtheriae resistant to penicillin and all other β-lactam antibiotics, and describe a novel mechanism of inducible carbapenem resistance associated with the acquisition of a mobile resistance element. Methods Long-read whole-genome sequencing was performed using Pacific Biosciences Single Molecule Real-Time sequencing to determine the genome sequence of C. diphtheriae BQ11 and the mechanism of β-lactam resistance. To investigate the phenotypic inducibility of meropenem resistance, short-read sequencing was performed using an Illumina NextSeq500 sequencer on the strain both with and without exposure to meropenem. Results BQ11 demonstrated high-level resistance to penicillin (benzylpenicillin minimum inhibitory concentration [MIC] ≥ 256 μg/ml), β-lactam/β-lactamase inhibitors and cephalosporins (amoxicillin/clavulanic acid MIC ≥ 256 μg/mL; ceftriaxone MIC ≥ 8 μg/L). Genomic analysis of BQ11 identified acquisition of a novel transposon carrying the penicillin-binding protein (PBP) Pbp2c, responsible for resistance to penicillin and cephalosporins. When strain BQ11 was exposed to meropenem, selective pressure drove amplification of the transposon in a tandem array and led to a corresponding change from a low-level to a high-level meropenem-resistant phenotype. Conclusions We have identified a novel mechanism of inducible antibiotic resistance whereby isolates that appear to be carbapenem susceptible on initial testing can develop in vivo resistance to carbapenems with repeated exposure. This phenomenon could have significant implications for the treatment of C. diphtheriae infection, and may lead to clinical failure.

scholarly journals Single-molecule long-read sequencing reveals a conserved selection mechanism determining intact long RNA and miRNA profiles in sperm

2020 ◽  
Author(s):  
Yu H. Sun ◽  
Anqi Wang ◽  
Chi Song ◽  
Rajesh K. Srivastava ◽  
Kin Fai Au ◽  
...  
Keyword(s):  
Single Molecule ◽  
Ribosomal Proteins ◽  
Selection Process ◽  
Future Research ◽  
Selection Mechanism ◽  
Bioinformatics Pipeline ◽  
Long Reads ◽  
Evolutionarily Conserved ◽  
Long Read ◽  
Early Trauma

AbstractSperm contributes diverse RNAs to the zygote. While sperm small RNAs have been shown to be shaped by paternal environments and impact offspring phenotypes, we know little about long RNAs in sperm, including mRNAs and long non-coding RNAs. Here, by integrating PacBio single-molecule long reads with Illumina short reads, we found 2,778 sperm intact long transcript (SpILT) species in mouse. The SpILTs profile is evolutionarily conserved between rodents and primates. mRNAs encoding ribosomal proteins are enriched in SpILTs, and in mice they are sensitive to early trauma. Mouse and human SpILT profiles are determined by a post-transcriptional selection process during spermiogenesis, and are co-retained in sperm with base pair-complementary miRNAs. In sum, we have developed a bioinformatics pipeline to define intact transcripts, added SplLTs into the “sperm RNA code” for use in future research and potential diagnosis, and uncovered selection mechanism(s) controlling sperm RNA profiles.

scholarly journals Cytogenomic identification and long-read single molecule real-time (SMRT) sequencing of a Bardet–Biedl Syndrome 9 (BBS9) deletion

2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Jennifer Reiner ◽  
Laura Pisani ◽  
Wanqiong Qiao ◽  
Ram Singh ◽  
Yao Yang ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Smrt Sequencing ◽  
Bardet Biedl Syndrome ◽  
Long Read

scholarly journals QAlign: Aligning nanopore reads accurately using current-level modeling

2019 ◽  
Author(s):  
Dhaivat Joshi ◽  
Shunfu Mao ◽  
Sreeram Kannan ◽  
Suhas Diggavi
Keyword(s):  
Reference Genome ◽  
Genomic Analysis ◽  
Vital Role ◽  
High Error Rate ◽  
Sequencing Technology ◽  
Long Reads ◽  
A Genome ◽  
Long Read ◽  
Nanopore Sequencer ◽  
Sequencing Process

AbstractMotivationEfficient and accurate alignment of DNA / RNA sequence reads to each other or to a reference genome / transcriptome is an important problem in genomic analysis. Nanopore sequencing has emerged as a major sequencing technology and many long-read aligners have been designed for aligning nanopore reads. However, the high error rate makes accurate and efficient alignment difficult. Utilizing the noise and error characteristics inherent in the sequencing process properly can play a vital role in constructing a robust aligner. In this paper, we design QAlign, a pre-processor that can be used with any long-read aligner for aligning long reads to a genome / transcriptome or to other long reads. The key idea in QAlign is to convert the nucleotide reads into discretized current levels that capture the error modes of the nanopore sequencer before running it through a sequence aligner.ResultsWe show that QAlign is able to improve alignment rates from around 80% up to 90% with nanopore reads when aligning to the genome. We also show that QAlign improves the average overlap quality by 9.2%, 2.5% and 10.8% in three real datasets for read-to-read alignment. Read-to-transcriptome alignment rates are improved from 51.6% to 75.4% and 82.6% to 90% in two real datasets.Availabilityhttps://github.com/joshidhaivat/QAlign.git

scholarly journals Variant Phasing and Haplotypic Expression from Single-molecule Long-read Sequencing in Maize

2019 ◽  
Author(s):  
Bo Wang ◽  
Elizabeth Tseng ◽  
Primo Baybayan ◽  
Kevin Eng ◽  
Michael Regulski ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genomic Analysis ◽  
Full Length ◽  
Specific Gene ◽  
Population Genetic Analysis ◽  
Functional Genomic Analysis ◽  
Long Read ◽  
Allele Specific ◽  
Regulatory Effects ◽  
Hybrid Lines

AbstractHaplotype phasing of genetic variants in maize is important for interpretation of the genome, population genetic analysis and functional genomic analysis of allelic activity. Accordingly, accurate methods for phasing the full-length isoforms are essential for functional genomics studies. We performed an isoform-level phasing study in maize, using two inbred lines and their reciprocal crosses, based on the single-molecule full-length cDNA sequencing. To phase and analyze the full-length transcripts between hybrids and parents, we developed a tool called IsoPhase. Using this tool, we validated the majority of SNPs called against matching short-read data and identified cases of allele-specific, gene-level and isoform-level expression. Our results revealed that maize parental lines and hybrid lines exhibit different splicing activities. After phasing 6,907 genes in two reciprocal hybrids using embryo, endosperm and root tissues, we annotated the SNPs and identified large-effect genes. In addition, based on single-molecule sequencing, we identified parent-of-origin isoforms in maize hybrids, distinct novel isoforms in maize parent and hybrid lines, and imprinted genes from different tissues. Finally, we characterized variation in cis- and trans-regulatory effects. Our study provides measures of haplotypic expression that could increase accuracy in studies of allelic expression.

scholarly journals PacBio library preparation using blunt-end adapter ligation produces significant artefactual fusion DNA sequences

2018 ◽  
Author(s):  
Paul Griffith ◽  
Castle Raley ◽  
David Sun ◽  
Yongmei Zhao ◽  
Zhonghe Sun ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Sequences ◽  
Turnaround Time ◽  
Library Preparation ◽  
Adapter Ligation ◽  
Sequence Production ◽  
Long Reads ◽  
Complex Structural Variation ◽  
Long Read ◽  
Complex Structural

AbstractPacific Biosciences’ (PacBio) RS II sequencer, utilizing Single-Molecule, Real-Time (SMRT) technology, has revolutionized next-generation sequencing by providing an accurate long-read platform. PacBio single-molecule long reads have been used to delineate complex spliceoforms, detect mutations in highly homologous sequences, identify mRNA chimeras and chromosomal translocations, accurately haplotype phasing over multiple kilobase distances and aid in assembly of genomes with complex structural variation. The PacBio protocol for preparation of sequencing templates employs blunt-end hairpin adapter ligation, which enables a short turnaround time for sequence production. However, we have found a significant portion of sequencing yield contains chimeric reads resulting from blunt-end ligation of multiple template molecules to each other prior to adapter ligation. These artefactual fusion DNA sequences pose a major challenge to analysis and can lead to false-positive detection of fusion events. We assessed the frequency of artefactual fusion when using blunt-end adapter ligation and compared it to an alternative method using A/T overhang adapter ligation. The A/T overhang adapter ligation method showed a vast improvement in limiting artefactual fusion events and is now our recommended procedure for adapter ligation during PacBio library preparation.

scholarly journals High-Quality Genome Resource of Xanthomonas hyacinthi Generated via Long-Read Sequencing

Plant Disease ◽  
2020 ◽  
Vol 104 (4) ◽  
pp. 1011-1012
Author(s):  
Stephen P. Cohen ◽  
Emily K. Luna ◽  
Jillian M. Lang ◽  
Janet Ziegle ◽  
Christine Chang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Complete Genome ◽  
Plant Disease ◽  
Basic Research ◽  
Ornamental Plant ◽  
Smrt Sequencing ◽  
High Quality ◽  
Long Read ◽  
High Quality Genome ◽  
Bacterial Plant Pathogen

The bacterial plant pathogen Xanthomonas hyacinthi is the causal agent of yellow disease of Hyacinthus and other ornamental plant genera. There is no available complete genome for X. hyacinthi, limiting basic research for this pathogen. Here, we release a high-quality complete genome sequence for the X. hyacinthi type strain, CFBP 1156. Single-molecule real-time (SMRT) sequencing with a mean coverage of 306× revealed two contigs of 4,918,645 and 44,381 bp in size. This was the first characterized plant-disease-causing species of Xanthomonas and this genome provides a resource to better understand the biology of yellow disease of hyacinth.

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