Evaluation of PCR conditions for characterizing bacterial communities with full-length 16S rRNA genes using a portable nanopore sequencer

• Published in: Scientific reports Vol. 10; no. 1; p. 12580
• Main Authors: Fujiyoshi, So, Muto-Fujita, Ai, Maruyama, Fumito
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 28.07.2020; Nature Publishing Group
• Subjects: 631/326/2565/2134; 631/326/2565/855; Bacteria; Bacteria - genetics; Bias; Community structure; Deoxyribonucleic acid; DNA; DNA sequencing; Genes; Genetic engineering; High-Throughput Nucleotide Sequencing - methods; Humanities and Social Sciences; multidisciplinary; Nanopores; Nucleotide sequence; Polymerase chain reaction; Polymerase Chain Reaction - methods; Reptiles & amphibians; RNA, Bacterial - genetics; RNA, Ribosomal, 16S - genetics; rRNA 16S; Science; Science (multidisciplinary); Sequence Analysis, RNA - methods; Taxonomy
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-020-69450-9

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.