MinION Sequencing of colorectal cancer tumour microbiomes-A comparison with amplicon-based and RNA-Sequencing.

William S Taylor,John Pearson,Rachel V Purcell,Allison Miller,Frank A Frizelle,Sebastian Schmeier,Sagarika Banerjee

doi:10.1371/journal.pone.0233170

William S Taylor, John Pearson + Show 5 more

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https://doi.org/10.1371/journal.pone.0233170

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Journal: PloS one	Publication Date: May 20, 2020
Citations: 12	License type: CC BY 4.0

Affiliation: University of Otago, Massey University

Abstract

BackgroundRecent evidence suggests a role for the gut microbiome in the development and progression of many diseases and many studies have been carried out to analyse the microbiome using a variety of methods. In this study, we compare MinION sequencing with meta-transcriptomics and amplicon-based sequencing for microbiome analysis of colorectal tumour tissue samples.MethodsDNA and RNA were extracted from 11 colorectal tumour samples. 16S rRNA amplicon sequencing and MinION sequencing was carried out using genomic DNA, and RNA-Sequencing for meta-transcriptomic analysis. Non-human MinION and RNA-Sequencing reads, and 16S rRNA amplicon sequencing reads were taxonomically classified using a database built from available RefSeq bacterial and archaeal genomes and a k-mer based algorithm in Kraken2. Concordance between the three platforms at different taxonomic levels was tested on a per-sample basis using Spearman’s rank correlation.ResultsThe average number of reads per sample using RNA-Sequencing was greater than 129 times that generated using MinION sequencing. However, the average read length of MinION sequences was more than 13 times that of RNA or 16S rRNA amplicon sequencing. Taxonomic assignment using 16S sequencing was less reliable beyond the genus level, and both RNA-Sequencing and MinION sequencing could detect greater numbers of phyla and genera in the same samples, compared to 16S sequencing. Bacterial species associated with colorectal cancer, Fusobacterium nucleatum, Parvimonas micra, Bacteroides fragilis and Porphyromonas gingivalis, were detectable using MinION, RNA-Sequencing and 16S rRNA amplicon sequencing data.ConclusionsLong-read sequences generated using MinION sequencing can compensate for low numbers of reads for bacterial classification. MinION sequencing can discriminate between bacterial strains and plasmids and shows potential as a cost-effective tool for rapid microbiome sequencing in a clinical setting.

Highlights

The gut microbiome and its relationship to human health and disease is an area of emerging research
DNA and RNA were extracted from 11 colorectal tumour samples. 16S ribosomal RNA (rRNA) amplicon sequencing and MinION sequencing was carried out using genomic DNA, and RNASequencing for meta-transcriptomic analysis
The average read length of MinION sequences was more than 13 times that of RNA or 16S rRNA amplicon sequencing

Summary

Introduction

The gut microbiome and its relationship to human health and disease is an area of emerging research. Colorectal cancer (CRC) is associated with high mortality, and the incidence is increasing, globally [8, 9]. Lifestyle and diet are strong risk factors for the disease, and changes in the gut microbiome have been linked to CRC development and pathogenesis in an increasing number of studies [10]. The assessment of microbiomes in a clinical setting is not widely practised Reasons for this include the lack of access to sequencing technology and clinical training for the interpretation of microbiome sequencing data [11]. Recent evidence suggests a role for the gut microbiome in the development and progression of many diseases and many studies have been carried out to analyse the microbiome using a variety of methods. We compare MinION sequencing with meta-transcriptomics and amplicon-based sequencing for microbiome analysis of colorectal tumour tissue samples.

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