Dataset Information

De Novo Structural Variations of Escherichia coli Detected by Nanopore Long-Read Sequencing.

ABSTRACT: Spontaneous mutations power evolution, whereas large-scale structural variations (SVs) remain poorly studied, primarily because of the lack of long-read sequencing techniques and powerful analytical tools. Here, we explore the SVs of Escherichia coli by running 67 wild-type (WT) and 37 mismatch repair (MMR)-deficient (ΔmutS) mutation accumulation lines, each experiencing more than 4,000 cell divisions, by applying Nanopore long-read sequencing and Illumina PE150 sequencing and verifying the results by Sanger sequencing. In addition to precisely repeating previous mutation rates of base-pair substitutions and insertion and deletion (indel) mutation rates, we do find significant improvement in insertion and deletion detection using long-read sequencing. The long-read sequencing and corresponding software can particularly detect bacterial SVs in both simulated and real data sets with high accuracy. These lead to SV rates of 2.77 × 10-4 (WT) and 5.26 × 10-4 (MMR-deficient) per cell division per genome, which is comparable with previous reports. This study provides the SV rates of E. coli by applying long-read sequencing and SV detection programs, revealing a broader and more accurate picture of spontaneous mutations in bacteria.

SUBMITTER: Zhou X

PROVIDER: S-EPMC10292909 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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De Novo Structural Variations of Escherichia coli Detected by Nanopore Long-Read Sequencing.

Zhou Xia X Pan Jiao J Wang Yaohai Y Lynch Michael M Long Hongan H Zhang Yu Y

Genome biology and evolution 20230601 6

Spontaneous mutations power evolution, whereas large-scale structural variations (SVs) remain poorly studied, primarily because of the lack of long-read sequencing techniques and powerful analytical tools. Here, we explore the SVs of Escherichia coli by running 67 wild-type (WT) and 37 mismatch repair (MMR)-deficient (ΔmutS) mutation accumulation lines, each experiencing more than 4,000 cell divisions, by applying Nanopore long-read sequencing and Illumina PE150 sequencing and verifying the resu ...[more]

PMID: 37293824

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Project description:BACKGROUND:Human cytomegalovirus (HCMV) has a double-stranded DNA genome of approximately 235 Kbp that is structurally complex including extended GC-rich repeated regions. Genomic recombination events are frequent in HCMV cultures but have also been observed in vivo. Thus, the assembly of HCMV whole genomes from technologies producing shorter than 500 bp sequences is technically challenging. Here we improved the reconstruction of HCMV full genomes by means of a hybrid, de novo genome-assembly bioinformatics pipeline upon data generated from the recently released MinION MkI B sequencer from Oxford Nanopore Technologies. RESULTS:The MinION run of the HCMV (strain TB40/E) library resulted in ~?47,000 reads from a single R9 flowcell and in ~?100× average read depth across the virus genome. We developed a novel, self-correcting bioinformatics algorithm to assemble the pooled HCMV genomes in three stages. In the first stage of the bioinformatics algorithm, long contigs (N50?=?21,892) of lower accuracy were reconstructed. In the second stage, short contigs (N50?=?5686) of higher accuracy were assembled, while in the final stage the high quality contigs served as template for the correction of the longer contigs resulting in a high-accuracy, full genome assembly (N50?=?41,056). We were able to reconstruct a single representative haplotype without employing any scaffolding steps. The majority (98.8%) of the genomic features from the reference strain were accurately annotated on this full genome construct. Our method also allowed the detection of multiple alternative sub-genomic fragments and non-canonical structures suggesting rearrangement events between the unique (UL /US) and the repeated (T/IRL/S) genomic regions. CONCLUSIONS:Third generation high-throughput sequencing technologies can accurately reconstruct full-length HCMV genomes including their low-complexity and highly repetitive regions. Full-length HCMV genomes could prove crucial in understanding the genetic determinants and viral evolution underpinning drug resistance, virulence and pathogenesis.

Project description:BackgroundLong-read sequencing (LRS) techniques have been very successful in identifying structural variants (SVs). However, the high error rate of LRS made the detection of small variants (substitutions and short indels < 20 bp) more challenging. The introduction of PacBio HiFi sequencing makes LRS also suited for detecting small variation. Here we evaluate the ability of HiFi reads to detect de novo mutations (DNMs) of all types, which are technically challenging variant types and a major cause of sporadic, severe, early-onset disease.MethodsWe sequenced the genomes of eight parent-child trios using high coverage PacBio HiFi LRS (~ 30-fold coverage) and Illumina short-read sequencing (SRS) (~ 50-fold coverage). De novo substitutions, small indels, short tandem repeats (STRs) and SVs were called in both datasets and compared to each other to assess the accuracy of HiFi LRS. In addition, we determined the parent-of-origin of the small DNMs using phasing.ResultsWe identified a total of 672 and 859 de novo substitutions/indels, 28 and 126 de novo STRs, and 24 and 1 de novo SVs in LRS and SRS respectively. For the small variants, there was a 92 and 85% concordance between the platforms. For the STRs and SVs, the concordance was 3.6 and 0.8%, and 4 and 100% respectively. We successfully validated 27/54 LRS-unique small variants, of which 11 (41%) were confirmed as true de novo events. For the SRS-unique small variants, we validated 42/133 DNMs and 8 (19%) were confirmed as true de novo event. Validation of 18 LRS-unique de novo STR calls confirmed none of the repeat expansions as true DNM. Confirmation of the 23 LRS-unique SVs was possible for 19 candidate SVs of which 10 (52.6%) were true de novo events. Furthermore, we were able to assign 96% of DNMs to their parental allele with LRS data, as opposed to just 20% with SRS data.ConclusionsHiFi LRS can now produce the most comprehensive variant dataset obtainable by a single technology in a single laboratory, allowing accurate calling of substitutions, indels, STRs and SVs. The accuracy even allows sensitive calling of DNMs on all variant levels, and also allows for phasing, which helps to distinguish true positive from false positive DNMs.

Dataset Information

De Novo Structural Variations of Escherichia coli Detected by Nanopore Long-Read Sequencing.

Publications

De Novo Structural Variations of Escherichia coli Detected by Nanopore Long-Read Sequencing.

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