A high-throughput multiplexing and selection strategy to complete bacterial genomes

Abstract Background Bacterial whole-genome sequencing based on short-read technologies often results in a draft assembly formed by contiguous sequences. The introduction of long-read sequencing technologies permits those contiguous sequences to be unambiguously bridged into complete genomes. However, the elevated costs associated with long-read sequencing frequently limit the number of bacterial isolates that can be long-read sequenced. Here we evaluated the recently released 96 barcoding kit from Oxford Nanopore Technologies (ONT) to generate complete genomes on a high-throughput basis. In addition, we propose an isolate selection strategy that optimizes a representative selection of isolates for long-read sequencing considering as input large-scale bacterial collections. Results Despite an uneven distribution of long reads per barcode, near-complete chromosomal sequences (assembly contiguity = 0.89) were generated for 96 Escherichia coli isolates with associated short-read sequencing data. The assembly contiguity of the plasmid replicons was even higher (0.98), which indicated the suitability of the multiplexing strategy for studies focused on resolving plasmid sequences. We benchmarked hybrid and ONT-only assemblies and showed that the combination of ONT sequencing data with short-read sequencing data is still highly desirable (i) to perform an unbiased selection of isolates for long-read sequencing, (ii) to achieve an optimal genome accuracy and completeness, and (iii) to include small plasmids underrepresented in the ONT library. Conclusions The proposed long-read isolate selection ensures the completion of bacterial genomes that span the genome diversity inherent in large collections of bacterial isolates. We show the potential of using this multiplexing approach to close bacterial genomes on a high-throughput basis.

Download Full-text

A high-throughput multiplexing and selection strategy to complete bacterial genomes

10.1101/2021.06.14.448320 ◽

2021 ◽

Author(s):

Sergio Arredondo-Alonso ◽

Anna K. Pöntinen ◽

François Cléon ◽

Rebecca A. Gladstone ◽

Anita C. Schürch ◽

...

Keyword(s):

High Throughput ◽

Selection Strategy ◽

Bacterial Isolates ◽

Sequencing Data ◽

Bacterial Genomes ◽

Short Read ◽

Short Read Sequencing ◽

Long Read ◽

Isolate Selection ◽

Selection Of

Background: Bacterial whole-genome sequencing based on short-read sequencing data often results in a draft assembly formed by contiguous sequences. The introduction of long-read sequencing technologies permits to unambiguously bridge those contiguous sequences into complete genomes. However, the elevated costs associated with long-read sequencing frequently limit the number of bacterial isolates that can be long-read sequenced. Here we evaluated the recently released 96 barcoding kit from Oxford Nanopore Technologies (ONT) to generate complete genomes on a high-throughput basis. In addition, we propose a long-read isolate selection strategy that optimizes a representative selection of isolates from large-scale bacterial collections. Results: Despite an uneven distribution of long-reads per barcode, near-complete chromosomal sequences (assembly contiguity = 0.89) were generated for 96 Escherichia coli isolates with associated short-read sequencing data. The assembly contiguity of the plasmid replicons was even higher (0.98) which indicated the suitability of the multiplexing strategy for studies focused on resolving plasmid sequences. We benchmarked hybrid and ONT-only assemblies and showed that the combination of ONT sequencing data with short-read sequencing data is still highly desirable: (i) to perform an unbiased selection of isolates for long-read sequencing, (ii) to achieve an optimal genome accuracy and completeness, and (iii) to include small plasmids underrepresented in the ONT library. Conclusions: The proposed long-read isolate selection ensures completing bacterial genomes of isolates that span the genome diversity inherent in large collections of bacterial isolates. We show the potential of using this multiplexing approach to close bacterial genomes on a high-throughput basis.

Download Full-text

Rapid Mycobacterium tuberculosis spoligotyping from uncorrected long reads using Galru

10.1101/2020.05.31.126490 ◽

2020 ◽

Author(s):

Andrew J. Page ◽

Nabil-Fareed Alikhan ◽

Michael Strinden ◽

Thanh Le Viet ◽

Timofey Skvortsov

Keyword(s):

Mycobacterium Tuberculosis ◽

State Of The Art ◽

Sequence Data ◽

Human Pathogen ◽

Sequencing Data ◽

Short Read ◽

Short Read Sequencing ◽

Long Reads ◽

Long Read

AbstractSpoligotyping of Mycobacterium tuberculosis provides a subspecies classification of this major human pathogen. Spoligotypes can be predicted from short read genome sequencing data; however, no methods exist for long read sequence data such as from Nanopore or PacBio. We present a novel software package Galru, which can rapidly detect the spoligotype of a Mycobacterium tuberculosis sample from as little as a single uncorrected long read. It allows for near real-time spoligotyping from long read data as it is being sequenced, giving rapid sample typing. We compare it to the existing state of the art software and find it performs identically to the results obtained from short read sequencing data. Galru is freely available from https://github.com/quadram-institute-bioscience/galru under the GPLv3 open source licence.

Download Full-text

Complete Genome Sequence of Rubrobacter xylanophilus Strain AA3-22, Isolated from Arima Onsen in Japan

Microbiology Resource Announcements ◽

10.1128/mra.00818-19 ◽

2019 ◽

Vol 8 (34) ◽

Cited By ~ 1

Author(s):

Natsuki Tomariguchi ◽

Kentaro Miyazaki

Keyword(s):

Genome Sequence ◽

Complete Genome Sequence ◽

Complete Genome ◽

Hot Spring ◽

Sequencing Data ◽

Short Read ◽

Content Type ◽

Short Read Sequencing ◽

Oxford Nanopore ◽

Long Read

Rubrobacter xylanophilus strain AA3-22, belonging to the phylum Actinobacteria, was isolated from nonvolcanic Arima Onsen (hot spring) in Japan. Here, we report the complete genome sequence of this organism, which was obtained by combining Oxford Nanopore long-read and Illumina short-read sequencing data.

Download Full-text

LinkedSV for detection of mosaic structural variants from linked-read exome and genome sequencing data

10.1101/409789 ◽

2018 ◽

Cited By ~ 2

Author(s):

Li Fang ◽

Charlly Kao ◽

Michael V Gonzalez ◽

Fernanda A Mafra ◽

Renata Pellegrino da Silva ◽

...

Keyword(s):

Exome Sequencing ◽

Read Depth ◽

Structural Variants ◽

Sequencing Data ◽

High Coverage ◽

Short Read ◽

Short Read Sequencing ◽

Sequencing Studies ◽

Long Read ◽

Local Assembly

AbstractLinked-read sequencing provides long-range information on short-read sequencing data by barcoding reads originating from the same DNA molecule, and can improve the detection and breakpoint identification for structural variants (SVs). We present LinkedSV for SV detection on linked-read sequencing data. LinkedSV considers barcode overlapping and enriched fragment endpoints as signals to detect large SVs, while it leverages read depth, paired-end signals and local assembly to detect small SVs. Benchmarking studies demonstrates that LinkedSV outperforms existing tools, especially on exome data and on somatic SVs with low variant allele frequencies. We demonstrate clinical cases where LinkedSV identifies disease causal SVs from linked-read exome sequencing data missed by conventional exome sequencing, and show examples where LinkedSV identifies SVs missed by high-coverage long-read sequencing. In summary, LinkedSV can detect SVs missed by conventional short-read and long-read sequencing approaches, and may resolve negative cases from clinical genome/exome sequencing studies.

Download Full-text

Systematic analysis of dark and camouflaged genes: disease-relevant genes hiding in plain sight

10.1101/514497 ◽

2019 ◽

Cited By ~ 1

Author(s):

Mark T. W. Ebbert ◽

Tanner D. Jensen ◽

Karen Jansen-West ◽

Jonathon P. Sens ◽

Joseph S. Reddy ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Sequencing Data ◽

Systematic Analysis ◽

Protein Coding ◽

Short Read ◽

Sequencing Project ◽

Short Read Sequencing ◽

Sequencing Technologies ◽

Long Read

AbstractBackgroundThe human genome contains ‘dark’ gene regions that cannot be adequately assembled or aligned using standard short-read sequencing technologies, preventing researchers from identifying mutations within these gene regions that may be relevant to human disease. Here, we identify regions that are ‘dark by depth’ (few mappable reads) and others that are ‘camouflaged’ (ambiguous alignment), and we assess how well long-read technologies resolve these regions. We further present an algorithm to resolve most camouflaged regions (including in short-read data) and apply it to the Alzheimer’s Disease Sequencing Project (ADSP; 13142 samples), as a proof of principle.ResultsBased on standard whole-genome lllumina sequencing data, we identified 37873 dark regions in 5857 gene bodies (3635 protein-coding) from pathways important to human health, development, and reproduction. Of the 5857 gene bodies, 494 (8.4%) were 100% dark (142 protein-coding) and 2046 (34.9%) were ≥5% dark (628 protein-coding). Exactly 2757 dark regions were in protein-coding exons (CDS) across 744 genes. Long-read sequencing technologies from 10x Genomics, PacBio, and Oxford Nanopore Technologies reduced dark CDS regions to approximately 45.1%, 33.3%, and 18.2% respectively. Applying our algorithm to the ADSP, we rescued 4622 exonic variants from 501 camouflaged genes, including a rare, ten-nucleotide frameshift deletion in CR1, a top Alzheimer’s disease gene, found in only five ADSP cases and zero controls.ConclusionsWhile we could not formally assess the CR1 frameshift mutation in Alzheimer’s disease (insufficient sample-size), we believe it merits investigating in a larger cohort. There remain thousands of potentially important genomic regions overlooked by short-read sequencing that are largely resolved by long-read technologies.

Download Full-text

NextSV: a meta-caller for structural variants from low-coverage long-read sequencing data

10.1101/092544 ◽

2016 ◽

Author(s):

Li Fang ◽

Jiang Hu ◽

Depeng Wang ◽

Kai Wang

Keyword(s):

Whole Genome ◽

Ashkenazi Jewish ◽

Structural Variants ◽

Sequencing Data ◽

Short Read ◽

Short Read Sequencing ◽

Human Genomes ◽

Long Read ◽

Personal Genomes ◽

Low Coverage

AbstractBackgroundStructural variants (SVs) in human genomes are implicated in a variety of human diseases. Long-read sequencing delivers much longer read lengths than short-read sequencing and may greatly improve SV detection. However, due to the relatively high cost of long-read sequencing, it is unclear what coverage is needed and how to optimally use the aligners and SV callers.ResultsIn this study, we developed NextSV, a meta-caller to perform SV calling from low coverage long-read sequencing data. NextSV integrates three aligners and three SV callers and generates two integrated call sets (sensitive/stringent) for different analysis purposes. We evaluated SV calling performance of NextSV under different PacBio coverages on two personal genomes, NA12878 and HX1. Our results showed that, compared with running any single SV caller, NextSV stringent call set had higher precision and balanced accuracy (F1 score) while NextSV sensitive call set had a higher recall. At 10X coverage, the recall of NextSV sensitive call set was 93.5% to 94.1% for deletions and 87.9% to 93.2% for insertions, indicating that ~10X coverage might be an optimal coverage to use in practice, considering the balance between the sequencing costs and the recall rates. We further evaluated the Mendelian errors on an Ashkenazi Jewish trio dataset.ConclusionsOur results provide useful guidelines for SV detection from low coverage whole-genome PacBio data and we expect that NextSV will facilitate the analysis of SVs on long-read sequencing data.

Download Full-text

DACCOR - Detection, charACterization, and reconstruction of Repetitive regions in bacterial genomes

10.7287/peerj.preprints.3480v1 ◽

2017 ◽

Author(s):

Alexander Seitz ◽

Friederike Hanssen ◽

Kay Nieselt

Keyword(s):

Base Pair ◽

Repetitive Sequence ◽

Reference Genome ◽

De Novo ◽

Treponema Pallidum ◽

Sequencing Data ◽

Bacterial Genomes ◽

Short Read ◽

Short Reads ◽

Short Read Sequencing

The reconstruction of genomes using mapping based approaches with short reads experiences difficulties when resolving repetitive regions. These repetitive regions in genomes result in low mapping qualities of the respective reads, which in turn lead to many unresolved bases of the genotypers. Currently, the reconstruction of these regions is often based on modified references in which the repetitive regions are masked. However, for many references such masked genomes are not available or are based on repetitive regions of other genomes. Our idea is to identify repetitive regions in the reference genome de novo. These regions can then be used to reconstruct them separately using short read sequencing data. Afterwards the reconstructed repetitive sequence can be inserted into the reconstructed genome. We present the program DACCOR, which performs these steps automatically. Our results show an increased base pair resolution of the repetitive regions in the reconstruction of Treponema pallidum samples, resulting in fewer unresolved bases.

Download Full-text

High-throughput Interpretation of Killer-cell Immunoglobulin-like Receptor Short-read Sequencing Data with PING

10.1101/2021.03.24.436770 ◽

2021 ◽

Author(s):

Wesley Marin ◽

Ravi Dandekar ◽

Danillo G. Augusto ◽

Tasneem Yusufali ◽

Bianca Heyn ◽

...

Keyword(s):

High Resolution ◽

High Throughput ◽

Copy Number ◽

Killer Cell ◽

Gene Copy Number ◽

Gene Copy ◽

Sequencing Data ◽

Short Read ◽

Short Read Sequencing ◽

Kir Genes

The killer-cell immunoglobulin-like receptor ( KIR) complex on chromosome 19 encodes receptors that modulate the activity of natural killer cells, and variation in these genes has been linked to infectious and autoimmune disease, as well as having bearing on pregnancy and transplant outcomes. The medical relevance and high variability of KIR genes makes short-read sequencing an attractive technology for interrogating the region, providing a high-throughput, high-fidelity sequencing method that is cost-effective. However, because this gene complex is characterized by extensive nucleotide polymorphism, structural variation including gene fusions and deletions, and a high level of homology between genes, its interrogation at high resolution has been thwarted by bioinformatic challenges, with most studies limited to examining presence or absence of specific genes. Here, we present the PING (Pushing Immunogenetics to the Next Generation) pipeline, which incorporates empirical data, novel alignment strategies and a custom alignment processing workflow to enable high-throughput KIR sequence analysis from short-read data. PING provides KIR gene copy number classification functionality for all KIR genes through use of a comprehensive alignment reference. The gene copy number determined per individual enables an innovative genotype determination workflow using genotype-matched references. Together, these methods address the challenges imposed by the structural complexity and overall homology of the KIR complex. To determine copy number and genotype determination accuracy, we applied PING to European and African validation cohorts and a synthetic dataset. PING demonstrated exceptional copy number determination performance across all datasets and robust genotype determination performance. Finally, an investigation into discordant genotypes for the synthetic dataset provides insight into misaligned reads, advancing our understanding in interpretation of short-read sequencing data in complex genomic regions. PING promises to support a new era of studies of KIR polymorphism, delivering high-resolution KIR genotypes that are highly accurate, enabling high-quality, high-throughput KIR genotyping for disease and population studies.

Download Full-text

LinkedSV for detection of mosaic structural variants from linked-read exome and genome sequencing data

Nature Communications ◽

10.1038/s41467-019-13397-7 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Li Fang ◽

Charlly Kao ◽

Michael V. Gonzalez ◽

Fernanda A. Mafra ◽

Renata Pellegrino da Silva ◽

...

Keyword(s):

Exome Sequencing ◽

Read Depth ◽

Structural Variants ◽

Sequencing Data ◽

High Coverage ◽

Short Read ◽

Short Read Sequencing ◽

Sequencing Studies ◽

Long Read ◽

Local Assembly

AbstractLinked-read sequencing provides long-range information on short-read sequencing data by barcoding reads originating from the same DNA molecule, and can improve detection and breakpoint identification for structural variants (SVs). Here we present LinkedSV for SV detection on linked-read sequencing data. LinkedSV considers barcode overlapping and enriched fragment endpoints as signals to detect large SVs, while it leverages read depth, paired-end signals and local assembly to detect small SVs. Benchmarking studies demonstrate that LinkedSV outperforms existing tools, especially on exome data and on somatic SVs with low variant allele frequencies. We demonstrate clinical cases where LinkedSV identifies disease-causal SVs from linked-read exome sequencing data missed by conventional exome sequencing, and show examples where LinkedSV identifies SVs missed by high-coverage long-read sequencing. In summary, LinkedSV can detect SVs missed by conventional short-read and long-read sequencing approaches, and may resolve negative cases from clinical genome/exome sequencing studies.

Download Full-text

Complete Genome Sequence of Vibrio rotiferianus Strain AM7

Microbiology Resource Announcements ◽

10.1128/mra.01591-19 ◽

2020 ◽

Vol 9 (21) ◽

Author(s):

Kentaro Miyazaki ◽

Apirak Wiseschart ◽

Kusol Pootanakit ◽

Kei Kitahara

Keyword(s):

Genome Sequence ◽

Complete Genome Sequence ◽

Complete Genome ◽

The Novel ◽

Sequencing Data ◽

Short Read ◽

Content Type ◽

Short Read Sequencing ◽

Oxford Nanopore ◽

Long Read

ABSTRACT We isolated the novel strain Vibrio rotiferianus AM7 from the shell of an abalone. In this article, we report the complete genome sequence of this organism, which was obtained by combining Oxford Nanopore long-read and Illumina short-read sequencing data.

Download Full-text