Dataset Information

Completing Circular Bacterial Genomes With Assembly Complexity by Using a Sampling Strategy From a Single MinION Run With Barcoding.

ABSTRACT: The Oxford Nanopore MinION is an affordable and portable DNA sequencer that can produce very long reads (tens of kilobase pairs), which enable de novo bacterial genome assembly. Although many algorithms and tools have been developed for base calling, read mapping, de novo assembly, and polishing, an automated pipeline is not available for one-stop analysis for circular bacterial genome reconstruction. In this paper, we present the pipeline CCBGpipe for completing circular bacterial genomes. Raw current signals are demultiplexed and base called to generate sequencing data. Sequencing reads are de novo assembled several times by using a sampling strategy to produce circular contigs that have a sequence in common between their start and end. The circular contigs are polished by using raw signals and sequencing reads; then, duplicated sequences are removed to form a linear representation of circular sequences. The circularized contigs are finally rearranged to start at the start position of dnaA/repA or a replication origin based on the GC skew. CCBGpipe implemented in Python is available at https://github.com/jade-nhri/CCBGpipe. Using sequencing data produced from a single MinION run, we obtained 48 circular sequences, comprising 12 chromosomes and 36 plasmids of 12 bacteria, including Acinetobacter nosocomialis, Acinetobacter pittii, and Staphylococcus aureus. With adequate quantities of sequencing reads (80×), CCBGpipe can provide a complete and automated assembly of circular bacterial genomes.

SUBMITTER: Liao YC

PROVIDER: S-EPMC6737777 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Completing Circular Bacterial Genomes With Assembly Complexity by Using a Sampling Strategy From a Single MinION Run With Barcoding.

Liao Yu-Chieh YC Cheng Hung-Wei HW Wu Han-Chieh HC Kuo Shu-Chen SC Lauderdale Tsai-Ling Yang TY Chen Feng-Jui FJ

Frontiers in microbiology 20190904

The Oxford Nanopore MinION is an affordable and portable DNA sequencer that can produce very long reads (tens of kilobase pairs), which enable <i>de novo</i> bacterial genome assembly. Although many algorithms and tools have been developed for base calling, read mapping, <i>de novo</i> assembly, and polishing, an automated pipeline is not available for one-stop analysis for circular bacterial genome reconstruction. In this paper, we present the pipeline CCBGpipe for completing circular bacterial ...[more]

PMID: 31551994

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Project description:BACKGROUND:Short-read sequencing technologies have made microbial genome sequencing cheap and accessible. However, closing genomes is often costly and assembling short reads from genomes that are repetitive and/or have extreme %GC content remains challenging. Long-read, single-molecule sequencing technologies such as the Oxford Nanopore MinION have the potential to overcome these difficulties, although the best approach for harnessing their potential remains poorly evaluated. RESULTS:We sequenced nine bacterial genomes spanning a wide range of GC contents using Illumina MiSeq and Oxford Nanopore MinION sequencing technologies to determine the advantages of each approach, both individually and combined. Assemblies using only MiSeq reads were highly accurate but lacked contiguity, a deficiency that was partially overcome by adding MinION reads to these assemblies. Even more contiguous genome assemblies were generated by using MinION reads for initial assembly, but these assemblies were more error-prone and required further polishing. This was especially pronounced when Illumina libraries were biased, as was the case for our strains with both high and low GC content. Increased genome contiguity dramatically improved the annotation of insertion sequences and secondary metabolite biosynthetic gene clusters, likely because long-reads can disambiguate these highly repetitive but biologically important genomic regions. CONCLUSIONS:Genome assembly using short-reads is challenged by repetitive sequences and extreme GC contents. Our results indicate that these difficulties can be largely overcome by using single-molecule, long-read sequencing technologies such as the Oxford Nanopore MinION. Using MinION reads for assembly followed by polishing with Illumina reads generated the most contiguous genomes with sufficient accuracy to enable the accurate annotation of important but difficult to sequence genomic features such as insertion sequences and secondary metabolite biosynthetic gene clusters. The combination of Oxford Nanopore and Illumina sequencing can therefore cost-effectively advance studies of microbial evolution and genome-driven drug discovery.

Dataset Information

Completing Circular Bacterial Genomes With Assembly Complexity by Using a Sampling Strategy From a Single MinION Run With Barcoding.

Publications

Completing Circular Bacterial Genomes With Assembly Complexity by Using a Sampling Strategy From a Single MinION Run With Barcoding.

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