Abstract
MinION (Oxford Nanopore Technologies), a portable nanopore sequencer, was introduced in 2014 as a new DNA sequencing technology. MinION is now widely used because of its low initial start-up costs relative to existing DNA sequencers, good portability, easy-handling, real-time analysis and long-read output. However, differences in the experimental conditions used for 16S rRNA-based PCR can bias bacterial community assessments in samples. Therefore, basic knowledge about reliable experimental conditions is needed to ensure the appropriate use of this technology. Our study concerns the reliability of techniques for obtaining accurate and quantitative full-length 16S rRNA amplicon sequencing data for bacterial community structure assessment using MinION. We compared five PCR conditions using three independent mock microbial community standard DNAs and established appropriate, standardized, better PCR conditions among the trials. We then sequenced two mock communities and six environmental samples using Illumina MiSeq for comparison. Modifying the PCR conditions improved the sequencing quality; the optimized conditions were 35 cycles of 95 °C for 1 min, 60 °C for 1 min and 68 °C for 3 min. Our results provide important information for researchers to determine bacterial community using MinION accurately.
Highlights
Most microbes in the natural environment have not yet been cultured, but recent molecular technological advances make it possible to study them without cultivation
We investigated whether fractional changes in taxonomic assignment and bacterial community composition exist in the comparison of PCR conditions using the MinION sequencer (Oxford Nanopore Technologies) with mock community and environmental samples
We compared the results from the bacterial community samples from MinION with those from the MiSeq sequencer (Illumina)
Summary
Most microbes in the natural environment have not yet been cultured, but recent molecular technological advances make it possible to study them without cultivation. An increasing number of studies have reported their concerns about on-site and real-time measurements using MinION22–24 These MinION-based gene sequencing techniques have provided new insight into microbial community structures much more rapidly and than ever before. Cost-effective marker gene amplicon sequencing is often preferred over metagenomic sequencing for microbial community analysis because it enables the assessment of uncultivable organisms With this background, the aim of this study was to evaluate MinION PCR conditions through three approaches: (1) sequencing the full-length bacterial 16S rRNA gene from a single bacterial species to examine our bioinformatics pipeline; (2) sequencing the amplicon of full-length bacterial 16S rRNA gene from three different types of bacterial mock community DNAs under five different PCR conditions; and (3) sequencing the amplicon of full-length 16S rRNA genes from six environmental samples to compare the results with those of bacterial 16S rRNA V3–V4 regions sequenced using MiSeq
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