Obtaining high-quality draft genomes from uncultured microbes by cleaning and co-assembly of single-cell amplified genomes

Masato Kogawa,Yohei Nishikawa,Haruko Takeyama,Masahito Hosokawa,Kazuki Mori

doi:10.1038/s41598-018-20384-3

Masato Kogawa, Yohei Nishikawa + Show 3 more

Open Access

https://doi.org/10.1038/s41598-018-20384-3

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Abstract

Single-cell genomics is a straightforward approach to obtain genomes from uncultured microbes. However, sequence reads from a single-cell amplified genome (SAG) contain significant bias and chimeric sequences. Here, we describe Cleaning and Co-assembly of a Single-Cell Amplified Genome (ccSAG), a novel analytical workflow to obtain composite single-cell genomes with elimination of sequence errors. By the integration of ccSAG with a massively parallel single-cell genome amplification platform based on droplet microfluidics, we can generate multiple SAGs and effectively integrate them into the composite genomes quality equivalent to the data obtained from bulk DNA. We obtained two novel draft genomes from single gut microbial cells with high completeness (>96.6%) and extremely low contamination (<1.25%). Moreover, we revealed the presence of single nucleotide polymorphisms in the specific gene by sequence comparison at the single-cell level. Thus, the workflow yields near-complete genomes from uncultured microbes, and enables analyses of genetic heterogeneity within identical strains.

Highlights

A large fraction of microbes cannot be cultured by traditional techniques
In comparison to conventional tools, ccSAG generates composite single-cell genomes with overall quality equivalent to those assembled from bulk DNA
Precleaning and integration of an optimal number of single-cell amplified genomes (SAG) are critical for obtaining a near-complete composite genome with quality equivalent to those obtained from bulk DNA

Summary

Introduction

A large fraction of microbes cannot be cultured by traditional techniques. metagenomics, which does not require cultivation, has become a useful tool to understand microbial diversity. To address persistent issues of chimerism and improve SAG quality from environmental samples, we have developed Cleaning and Co-assembly of a Single-Cell Amplified Genome (ccSAG), a novel, systematic, and generalized workflow to remove potentially chimeric sequences and co-assemble multiple, closely related SAGs de novo into a near-complete genome. We integrated this workflow into a massively parallel single-cell MDA platform based on microfluidic droplets[12] to investigate microbes in the mouse gut. We anticipate that ccSAG will advance single-cell microbiology in meaningful ways, and help illuminate the functional role of microbial dark matters

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