Analysis of a small outbreak of Shiga toxin-producing Escherichia coli O157:H7 using long-read sequencing

Compared to short-read sequencing data, long-read sequencing facilitates single contiguous de novo assemblies and characterization of the prophage region of the genome. Here, we describe our methodological approach to using Oxford Nanopore Technology (ONT) sequencing data to quantify genetic relatedness and to look for microevolutionary events in the core and accessory genomes to assess the within-outbreak variation of four genetically and epidemiologically linked isolates. Analysis of both Illumina and ONT sequencing data detected one SNP between the four sequences of the outbreak isolates. The variant calling procedure highlighted the importance of masking homologous sequences in the reference genome regardless of the sequencing technology used. Variant calling also highlighted the systemic errors in ONT base-calling and ambiguous mapping of Illumina reads that results in variations in the genetic distance when comparing one technology to the other. The prophage component of the outbreak strain was analysed, and nine of the 16 prophages showed some similarity to the prophage in the Sakai reference genome, including the stx2a-encoding phage. Prophage comparison between the outbreak isolates identified minor genome rearrangements in one of the isolates, including an inversion and a deletion event. The ability to characterize the accessory genome in this way is the first step to understanding the significance of these microevolutionary events and their impact on the evolutionary history, virulence and potentially the likely source and transmission of this zoonotic, foodborne pathogen.

Download Full-text

Closing Human Reference Genome Gaps: Identifying and Characterizing Gap-Closing Sequences

G3 Genes|Genome|Genetics ◽

10.1534/g3.120.401280 ◽

2020 ◽

Vol 10 (8) ◽

pp. 2801-2809 ◽

Cited By ~ 1

Author(s):

Tingting Zhao ◽

Zhongqu Duan ◽

Georgi Z. Genchev ◽

Hui Lu

Keyword(s):

Reference Genome ◽

De Novo ◽

Sequence Length ◽

Sequencing Data ◽

Human Reference Genome ◽

Satellite Sequences ◽

Long Read ◽

Data Gap ◽

Simple Repeats ◽

Gap Closing

Despite continuous updates of the human reference genome, there are still hundreds of unresolved gaps which account for about 5% of the total sequence length. Given the availability of whole genome de novo assemblies, especially those derived from long-read sequencing data, gap-closing sequences can be determined. By comparing 17 de novo long-read sequencing assemblies with the human reference genome, we identified a total of 1,125 gap-closing sequences for 132 (16.9% of 783) gaps and added up to 2.2 Mb novel sequences to the human reference genome. More than 90% of the non-redundant sequences could be verified by unmapped reads from the Simons Genome Diversity Project dataset. In addition, 15.6% of the non-reference sequences were found in at least one of four non-human primate genomes. We further demonstrated that the non-redundant sequences had high content of simple repeats and satellite sequences. Moreover, 43 (32.6%) of the 132 closed gaps were shown to be polymorphic; such sequences may play an important biological role and can be useful in the investigation of human genetic diversity.

Download Full-text

long-read-tools.org: an interactive catalogue of analysis methods for long-read sequencing data

GigaScience ◽

10.1093/gigascience/giab003 ◽

2021 ◽

Vol 10 (2) ◽

Cited By ~ 1

Author(s):

Shanika L Amarasinghe ◽

Matthew E Ritchie ◽

Quentin Gouil

Keyword(s):

Data Analysis ◽

De Novo ◽

Downstream Processing ◽

Sequencing Data ◽

Base Calling ◽

Analysis Tools ◽

Long Read ◽

Essential Resource ◽

Interactive Browsing ◽

Assembly Error

Abstract Background The data produced by long-read third-generation sequencers have unique characteristics compared to short-read sequencing data, often requiring tailored analysis tools for tasks ranging from quality control to downstream processing. The rapid growth in software that addresses these challenges for different genomics applications is difficult to keep track of, which makes it hard for users to choose the most appropriate tool for their analysis goal and for developers to identify areas of need and existing solutions to benchmark against. Findings We describe the implementation of long-read-tools.org, an open-source database that organizes the rapidly expanding collection of long-read data analysis tools and allows its exploration through interactive browsing and filtering. The current database release contains 478 tools across 32 categories. Most tools are developed in Python, and the most frequent analysis tasks include base calling, de novo assembly, error correction, quality checking/filtering, and isoform detection, while long-read single-cell data analysis and transcriptomics are areas with the fewest tools available. Conclusion Continued growth in the application of long-read sequencing in genomics research positions the long-read-tools.org database as an essential resource that allows researchers to keep abreast of both established and emerging software to help guide the selection of the most relevant tool for their analysis needs.

Download Full-text

GALA: gap-free chromosome-scale assembly with long reads

10.1101/2020.05.15.097428 ◽

2020 ◽

Author(s):

Mohamed Awad ◽

Xiangchao Gan

Keyword(s):

Genome Assembly ◽

Reference Genome ◽

De Novo ◽

Genetic Maps ◽

Sequencing Data ◽

C Elegans ◽

Assembly Method ◽

Long Reads ◽

Long Read ◽

Assembly Technology

AbstractHigh-quality genome assembly has wide applications in genetics and medical studies. However, it is still very challenging to achieve gap-free chromosome-scale assemblies using current workflows for long-read platforms. Here we propose GALA (Gap-free long-read assembler), a chromosome-by-chromosome assembly method implemented through a multi-layer computer graph that identifies mis-assemblies within preliminary assemblies or chimeric raw reads and partitions the data into chromosome-scale linkage groups. The subsequent independent assembly of each linkage group generates a gap-free assembly free from the mis-assembly errors which usually hamper existing workflows. This flexible framework also allows us to integrate data from various technologies, such as Hi-C, genetic maps, a reference genome and even motif analyses, to generate gap-free chromosome-scale assemblies. We de novo assembled the C. elegans and A. thaliana genomes using combined Pacbio and Nanopore sequencing data from publicly available datasets. We also demonstrated the new method’s applicability with a gap-free assembly of a human genome with the help a reference genome. In addition, GALA showed promising performance for Pacbio high-fidelity long reads. Thus, our method enables straightforward assembly of genomes with multiple data sources and overcomes barriers that at present restrict the application of de novo genome assembly technology.

Download Full-text

Contiguous and accurate de novo assembly of metazoan genomes with modest long read coverage

10.1101/029306 ◽

2015 ◽

Cited By ~ 7

Author(s):

Mahul Chakraborty ◽

James G. Baldwin-Brown ◽

Anthony D. Long ◽

J.J. Emerson

Keyword(s):

Best Practices ◽

Dna Isolation ◽

Reference Genome ◽

De Novo ◽

Error Rates ◽

Read Coverage ◽

Sequencing Data ◽

Sequencing Coverage ◽

Long Read ◽

Genome Assemblies

AbstractGenome assemblies that are accurate, complete, and contiguous are essential for identifying important structural and functional elements of genomes and for identifying genetic variation. Nevertheless, most recent genome assemblies remain incomplete and fragmented. While long molecule sequencing promises to deliver more complete genome assemblies with fewer gaps, concerns about error rates, low yields, stringent DNA requirements, and uncertainty about best practices may discourage many investigators from adopting this technology. Here, in conjunction with the platinum standard Drosophila melanogaster reference genome, we analyze recently published long molecule sequencing data to identify what governs completeness and contiguity of genome assemblies. We also present a hybrid meta-assembly approach that achieves remarkable assembly contiguity for both Drosophila and human assemblies with only modest long molecule sequencing coverage. Our results motivate a set of preliminary best practices for obtaining accurate and contiguous assemblies, a “missing manual” that guides key decisions in building high quality de novo genome assemblies, from DNA isolation to polishing the assembly.

Download Full-text

AStrap: identification of alternative splicing from transcript sequences without a reference genome

Bioinformatics ◽

10.1093/bioinformatics/bty1008 ◽

2018 ◽

Vol 35 (15) ◽

pp. 2654-2656 ◽

Cited By ~ 5

Author(s):

Guoli Ji ◽

Wenbin Ye ◽

Yaru Su ◽

Moliang Chen ◽

Guangzao Huang ◽

...

Keyword(s):

Machine Learning ◽

Alternative Splicing ◽

Single Molecule ◽

Reference Genome ◽

De Novo ◽

Supplementary Information ◽

Model Organisms ◽

Sequencing Data ◽

Extensive Evaluation ◽

Reference Genomes

Abstract Summary Alternative splicing (AS) is a well-established mechanism for increasing transcriptome and proteome diversity, however, detecting AS events and distinguishing among AS types in organisms without available reference genomes remains challenging. We developed a de novo approach called AStrap for AS analysis without using a reference genome. AStrap identifies AS events by extensive pair-wise alignments of transcript sequences and predicts AS types by a machine-learning model integrating more than 500 assembled features. We evaluated AStrap using collected AS events from reference genomes of rice and human as well as single-molecule real-time sequencing data from Amborella trichopoda. Results show that AStrap can identify much more AS events with comparable or higher accuracy than the competing method. AStrap also possesses a unique feature of predicting AS types, which achieves an overall accuracy of ∼0.87 for different species. Extensive evaluation of AStrap using different parameters, sample sizes and machine-learning models on different species also demonstrates the robustness and flexibility of AStrap. AStrap could be a valuable addition to the community for the study of AS in non-model organisms with limited genetic resources. Availability and implementation AStrap is available for download at https://github.com/BMILAB/AStrap. Supplementary information Supplementary data are available at Bioinformatics online.

Download Full-text

Genome Assembly of the Popular Korean Soybean Cultivar Hwangkeum

10.1101/2021.04.19.440529 ◽

2021 ◽

Author(s):

Myung-Shin Kim ◽

Taeyoung Lee ◽

Jeonghun Baek ◽

Ji Hong Kim ◽

Changhoon Kim ◽

...

Keyword(s):

Reference Genome ◽

De Novo ◽

Anthocyanin Biosynthesis ◽

Genetic Maps ◽

Soybean Cultivar ◽

Gene Arrangement ◽

Nucleotide Polymorphisms ◽

Sequencing Data ◽

Crop Species ◽

Satellite Repeats

AbstractMassive resequencing efforts have been undertaken to catalog allelic variants in major crop species including soybean, but the scope of the information for genetic variation often depends on short sequence reads mapped to the extant reference genome. Additional de novo assembled genome sequences provide a unique opportunity to explore a dispensable genome fraction in the pan-genome of a species. Here, we report the de novo assembly and annotation of Hwangkeum, a popular soybean cultivar in Korea. The assembly was constructed using PromethION nanopore sequencing data and two genetic maps, and was then error-corrected using Illumina short-reads and PacBio SMRT reads. The 933.12 Mb assembly was annotated 79,870 transcripts for 58,550 genes using RNA-Seq data and the public soybean annotation set. Comparison of the Hwangkeum assembly with the Williams 82 soybean reference genome sequence revealed 1.8 million single-nucleotide polymorphisms, 0.5 million indels, and 25 thousand putative structural variants. However, there was no natural megabase-scale chromosomal rearrangement. Incidentally, by adding two novel groups, we found that soybean contains four clearly separated groups of centromeric satellite repeats. Analyses of satellite repeats and gene content suggested that the Hwangkeum assembly is a high-quality assembly. This was further supported by comparison of the marker arrangement of anthocyanin biosynthesis genes and of gene arrangement at the Rsv3 locus. Therefore, the results indicate that the de novo assembly of Hwangkeum is a valuable additional reference genome resource for characterizing traits for the improvement of this important crop species.

Download Full-text

A unified haplotype-based method for accurate and comprehensive variant calling

10.1101/456103 ◽

2018 ◽

Cited By ~ 3

Author(s):

Daniel P Cooke ◽

David C Wedge ◽

Gerton Lunter

Keyword(s):

De Novo ◽

Variant Calling ◽

Normal Sample ◽

Sequencing Data ◽

Somatic Variation ◽

Data Set ◽

Small Complex ◽

Physical Linkage ◽

Germline Variation ◽

Almost All

Haplotype-based variant callers, which consider physical linkage between variant sites, are currently among the best tools for germline variation discovery and genotyping from short-read sequencing data. However, almost all such tools were designed specifically for detecting common germline variation in diploid populations, and give sub-optimal results in other scenarios. Here we present Octopus, a versatile haplotype-based variant caller that uses a polymorphic Bayesian genotyping model capable of modeling sequencing data from a range of experimental designs within a unified haplotype-aware framework. We show that Octopus accurately calls de novo mutations in parent-offspring trios and germline variants in individuals, including SNVs, indels, and small complex replacements such as microinversions. In addition, using a carefully designed synthetic-tumour data set derived from clean sequencing data from a sample with known germline haplotypes, and observed mutations in large cohort of tumour samples, we show that Octopus accurately characterizes germline and somatic variation in tumours, both with and without a paired normal sample. Sequencing reads and prior information are combined to phase called genotypes of arbitrary ploidy, including those with somatic mutations. Octopus also outputs realigned evidence BAMs to aid validation and interpretation.

Download Full-text

Trio deep-sequencing does not reveal unexpected off-target and on-target mutations in Cas9-edited rhesus monkeys

Nature Communications ◽

10.1038/s41467-019-13481-y ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 8

Author(s):

Xin Luo ◽

Yaoxi He ◽

Chao Zhang ◽

Xiechao He ◽

Lanzhen Yan ◽

...

Keyword(s):

Rhesus Monkeys ◽

De Novo ◽

Preclinical Model ◽

Structural Variants ◽

Sequencing Data ◽

De Novo Mutations ◽

Target Region ◽

Long Read ◽

Target Effect

AbstractCRISPR-Cas9 is a widely-used genome editing tool, but its off-target effect and on-target complex mutations remain a concern, especially in view of future clinical applications. Non-human primates (NHPs) share close genetic and physiological similarities with humans, making them an ideal preclinical model for developing Cas9-based therapies. However, to our knowledge no comprehensive in vivo off-target and on-target assessment has been conducted in NHPs. Here, we perform whole genome trio sequencing of Cas9-treated rhesus monkeys. We only find a small number of de novo mutations that can be explained by expected spontaneous mutations, and no unexpected off-target mutations (OTMs) were detected. Furthermore, the long-read sequencing data does not detect large structural variants in the target region.

Download Full-text

Fully phased human genome assembly without parental data using single-cell strand sequencing and long reads

Nature Biotechnology ◽

10.1038/s41587-020-0719-5 ◽

2020 ◽

Author(s):

David Porubsky ◽

◽

Peter Ebert ◽

Peter A. Audano ◽

Mitchell R. Vollger ◽

...

Keyword(s):

Single Cell ◽

Genome Assembly ◽

De Novo ◽

Error Rates ◽

Sequencing Data ◽

Single Nucleotide Variants ◽

De Novo Genome Assembly ◽

Parental Data ◽

Human Genome Assembly ◽

Long Read

AbstractHuman genomes are typically assembled as consensus sequences that lack information on parental haplotypes. Here we describe a reference-free workflow for diploid de novo genome assembly that combines the chromosome-wide phasing and scaffolding capabilities of single-cell strand sequencing1,2 with continuous long-read or high-fidelity3 sequencing data. Employing this strategy, we produced a completely phased de novo genome assembly for each haplotype of an individual of Puerto Rican descent (HG00733) in the absence of parental data. The assemblies are accurate (quality value > 40) and highly contiguous (contig N50 > 23 Mbp) with low switch error rates (0.17%), providing fully phased single-nucleotide variants, indels and structural variants. A comparison of Oxford Nanopore Technologies and Pacific Biosciences phased assemblies identified 154 regions that are preferential sites of contig breaks, irrespective of sequencing technology or phasing algorithms.

Download Full-text

NanoGalaxy: Nanopore long-read sequencing data analysis in Galaxy

GigaScience ◽

10.1093/gigascience/giaa105 ◽

2020 ◽

Vol 9 (10) ◽

Cited By ~ 1

Author(s):

Willem de Koning ◽

Milad Miladi ◽

Saskia Hiltemann ◽

Astrid Heikema ◽

John P Hays ◽

...

Keyword(s):

Genome Assembly ◽

Bioinformatics Analysis ◽

De Novo ◽

Sequence Data ◽

Ease Of Use ◽

Easy Access ◽

Complex Data ◽

Sequencing Data ◽

Long Read ◽

Sequencing Platforms

Abstract Background Long-read sequencing can be applied to generate very long contigs and even completely assembled genomes at relatively low cost and with minimal sample preparation. As a result, long-read sequencing platforms are becoming more popular. In this respect, the Oxford Nanopore Technologies–based long-read sequencing “nanopore" platform is becoming a widely used tool with a broad range of applications and end-users. However, the need to explore and manipulate the complex data generated by long-read sequencing platforms necessitates accompanying specialized bioinformatics platforms and tools to process the long-read data correctly. Importantly, such tools should additionally help democratize bioinformatics analysis by enabling easy access and ease-of-use solutions for researchers. Results The Galaxy platform provides a user-friendly interface to computational command line–based tools, handles the software dependencies, and provides refined workflows. The users do not have to possess programming experience or extended computer skills. The interface enables researchers to perform powerful bioinformatics analysis, including the assembly and analysis of short- or long-read sequence data. The newly developed “NanoGalaxy" is a Galaxy-based toolkit for analysing long-read sequencing data, which is suitable for diverse applications, including de novo genome assembly from genomic, metagenomic, and plasmid sequence reads. Conclusions A range of best-practice tools and workflows for long-read sequence genome assembly has been integrated into a NanoGalaxy platform to facilitate easy access and use of bioinformatics tools for researchers. NanoGalaxy is freely available at the European Galaxy server https://nanopore.usegalaxy.eu with supporting self-learning training material available at https://training.galaxyproject.org.

Download Full-text