Nearly finished genomes produced using gel microdroplet culturing reveal substantial intraspecies genomic diversity within the human microbiome

Abstract

The majority of microbial genomic diversity remains unexplored. This is largely due to our inability to culture most microorganisms in isolation, which is a prerequisite for traditional genome sequencing. Single-cell sequencing has allowed researchers to circumvent this limitation. DNA is amplified directly from a single cell using the whole-genome amplification technique of multiple displacement amplification (MDA). However, MDA from a single chromosome copy suffers from amplification bias and a large loss of specificity from even very small amounts of DNA contamination, which makes assembling a genome difficult and completely finishing a genome impossible except in extraordinary circumstances. Gel microdrop cultivation allows culturing of a diverse microbial community and provides hundreds to thousands of genetically identical cells as input for an MDA reaction. We demonstrate the utility of this approach by comparing sequencing results of gel microdroplets and single cells following MDA. Bias is reduced in the MDA reaction and genome sequencing, and assembly is greatly improved when using gel microdroplets. We acquired multiple near-complete genomes for two bacterial species from human oral and stool microbiome samples. A significant amount of genome diversity, including single nucleotide polymorphisms and genome recombination, is discovered. Gel microdroplets offer a powerful and high-throughput technology for assembling whole genomes from complex samples and for probing the pan-genome of naturally occurring populations.

Figure 1.

Flow cytometry and light microscopy images of gel microdroplets (GMDs). (A,C) GMDs occupied with a colony are shown circled. (B,D) An unincubated sample that does not contain occupied GMDs. (E) Light microscopy image of GMDs containing a colony.

Figure 2.

Sequencing results of amplified single cells and GMDs. (Blue circles) GMDs; (orange circles) single cells. (A) Assembly size; (B) largest contig; (C) percent contamination. (D) Genome recovery as measured by mapped reads to the best GMD assembly. (*) Statistical significance for that comparison (P < 0.05).

Figure 3.

Distribution of sequencing reads. Reads for single cells and GMDs were mapped to the best GMD assembly. Results for the best single-cell and second best GMD assembly for Streptococcus sp. are shown. (A) The distribution of base pairs having each level of coverage. (Orange) The single-cell distribution; (blue) the GMD distribution. (B) Fold coverage for every position in the genome. The inset zooms in near the origin to highlight the GMD results.

Figure 4.

Location of SNPs within Streptococcus sp. genomes. SNPs for each GMD assembly were located by mapping reads to the concatenated Streptococcus sp. GMD-5 assembly. Contig order was determined by comparison to Streptococcus oralis reference Uo5. SNP density is measured using a sliding 1-kb window with 100-bp increments. The order of samples from outside in is Streptococcus sp. GMD-6, 4, 2, 1, 3, which is in order of least to most number of SNPs relative to the GMD-5 reference.

Figure 5.

Phylogeny of OTUs recovered from GMDs. GMDs occupied by a growing colony of bacteria extracted from feces were sorted for MDA. 16S rDNA was amplified and sequenced. The maximum likelihood tree below represents the diversity of 16S rDNA sequences binned into OTUs at 90% sequence similarity. Related sequences were included to show relationships. Support values are omitted for clarity.