Species-resolved sequencing of low-biomass or degraded microbiomes using 2bRAD-M

Zheng Sun,Jia Lv,Jian Xu,Pengfei Zhu,Shi Huang,Gongchao Jing,Meng Zhang,Zhenmin Bao,Xiuping Wang,Lisha Zhou,Shi Wang,Lam Tzehau,Qianya Niu,Jiquan Liu,Helen Zhao,Rongchao Zhang

doi:10.1186/s13059-021-02576-9

Abstract

Microbiome samples with low microbial biomass or severe DNA degradation remain challenging for amplicon-based or whole-metagenome sequencing approaches. Here, we introduce 2bRAD-M, a highly reduced and cost-effective strategy which only sequences ~ 1% of metagenome and can simultaneously produce species-level bacterial, archaeal, and fungal profiles. 2bRAD-M can accurately generate species-level taxonomic profiles for otherwise hard-to-sequence samples with merely 1 pg of total DNA, high host DNA contamination, or severely fragmented DNA from degraded samples. Tests of 2bRAD-M on various stool, skin, environmental, and clinical FFPE samples suggest a successful reconstruction of comprehensive, high-resolution microbial profiles.

Highlights

Metagenome sequencing, widely used to derive the taxonomic profile of microbiome, typically adopts two strategies that target (i) amplicons of phylogenetic “marker genes” (e.g., 16S rRNA for bacteria and archaea, and 18S rRNA or internal transcribed spacer (ITS) for fungi) or (ii) the whole genomes
1 ng is applicable via certain specialized kits [4], WMS usually requires a high amount of DNA as the starting material (≥50 ng preferred, 20 ng at a minimum) and is not efficient in tackling DNA samples that are low in biomass, heavily degraded, or dominated by host DNA [1, 4]
The principle and workflow of 2bRAD-M The principle and appealing features of 2bRAD-M are as follows (Fig. 1): (i) reliable enzyme-digested sequence tags can be derived that are specific to high-resolution taxa yet universally applicable for a broad range or all of bacterial, archaeal, and fungal genomes; (ii) these taxa-specific, iso-length sequence tags can be evenly amplified and sequenced; and (iii) the tag sequences can be mapped to reference genomes to reconstruct faithfully the taxonomic composition

Summary

Introduction

Metagenome sequencing, widely used to derive the taxonomic profile of microbiome, typically adopts two strategies that target (i) amplicons of phylogenetic “marker genes” (e.g., 16S rRNA for bacteria and archaea, and 18S rRNA or internal transcribed spacer (ITS) for fungi) or (ii) the whole genomes (whole metagenome shotgun; WMS). Marker gene analyses can be limited in taxonomic resolution (i.e., at the genus level) and susceptible to PCR bias in composition and abundance estimates [1]; they are usually unable to capture a landscape-like view that includes bacteria, archaea, fungi, and virus due to the lack of universal primers. 1 ng is applicable via certain specialized kits [4], WMS usually requires a high amount of DNA as the starting material (≥50 ng preferred, 20 ng at a minimum) and is not efficient in tackling DNA samples that are low in biomass, heavily degraded, or dominated by host DNA [1, 4]. New methods should be developed that cost-efficiently produce accurate, species-resolution, landscape-like taxonomic profiles for challenging samples like low-biomass, high host-contaminated, and degraded microbiomes

Methods

Results

Discussion

Conclusion