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.
Highlights
Illumina sequencing is routinely used to study microbial genomics because of its low cost and the high accuracy of the sequence reads generated
The de novo assembly of short reads (100–300 base pairs) results in fragmented assemblies because repetitive sequences in bacterial genomes are invariably longer than the length of a short read and the span of paired-end reads
In the past few years, long-read sequencing technologies have been developed by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) (Ameur et al, 2018)
Summary
Illumina sequencing is routinely used to study microbial genomics because of its low cost and the high accuracy of the sequence reads generated. De novo Assembly Using Single MinION Run. accurate draft assemblies can provide some information for comparative analyses, determining the complete genome sequence is still essential. PacBio and ONT technologies can generate long reads in tens of kilobase pairs, making it possible to obtain a complete assembly (Koren and Phillippy, 2014; Liao et al, 2015; Bayliss et al, 2017; Wick et al, 2017a,b; Bainomugisa et al, 2018; Li et al, 2018; Schmid et al, 2018). Compared with the PacBio technology, the ONT MinION is affordable and portable and enables realtime analysis, which render it more attractive for in-field and clinical deployment (Jain et al, 2016; Ameur et al, 2018). The applications of the ONT MinION range from microbial genome assembly to cancer variant discovery and transcript isoform identification (Magi et al, 2017)
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