Characterization of Shallow Whole-Metagenome Shotgun Sequencing as a High-Accuracy and Low-Cost Method by Complicated Mock Microbiomes

Abstract

Characterization of the bacterial composition and functional repertoires of microbiome samples is the most common application of metagenomics. Although deep whole-metagenome shotgun sequencing (WMS) provides high taxonomic resolution, it is generally cost-prohibitive for large longitudinal investigations. Until now, 16S rRNA gene amplicon sequencing (16S) has been the most widely used approach and usually cooperates with WMS to achieve cost-efficiency. However, the accuracy of 16S results and its consistency with WMS data have not been fully elaborated, especially by complicated microbiomes with defined compositional information. Here, we constructed two complex artificial microbiomes, which comprised more than 60 human gut bacterial species with even or varied abundance. Utilizing real fecal samples and mock communities, we provided solid evidence demonstrating that 16S results were of poor consistency with WMS data, and its accuracy was not satisfactory. In contrast, shallow whole-metagenome shotgun sequencing (shallow WMS, S-WMS) with a sequencing depth of 1 Gb provided outputs that highly resembled WMS data at both genus and species levels and presented much higher accuracy taxonomic assignments and functional predictions than 16S, thereby representing a better and cost-efficient alternative to 16S for large-scale microbiome studies.

Taxonomic and functional alterations in the salivary microbiota of children with and without severe early childhood caries (S-ECC) at the age of 3

“… Jiang et al (2016) reported an increase of Actinomyces graevenitzii in the salivary microbiota of caries children by applying 16S rRNA sequencing, while no increase but mild decrease of Actinomyces graevenitzii was observed in our study. The probable explanation is that 16S rRNA sequencing and shotgun sequencing has different consistency and accuracy, especially at the species level ( Xu et al, 2021 ).…”

High resolution shotgun metagenomics: the more data, the better?

“…Shallow shotgun metagenomic (SSM) sequencing has been the subject of only a few studies so far [11][12][13] and focused mainly on the correlation between SSM and 16S rRNA amplicon sequencing. They were done with artificial datasets or executed with reads-based bioinformatics methods, avoiding the de novo co-assembly process inherent to HRSM workflows.…”

The Interaction between the Gut Microbiome and Bile Acids in Cardiometabolic Diseases

“…Ideally, these extensive cohort studies should be multi-ethnic as ethnicity has been shown to play an important role in gut microbial profiles [12,13]. Moreover, microbial sequencing techniques should focus more on shotgun metagenomic sequencing to better understand the functional profile of the microbial community, rather than being stuck at the compositional level [111]. This is becoming more realistic as shotgun metagenomic sequencing and mass spectrometry have become more affordable.…”