Sequencing

11th Annual Sequencing, Finishing, and Analysis in the Future Meeting
SEQUENCING THE ELEPHANT IN THE ROOM: RAPID
RESOLUTION OF WHOLE GENOME SEQUENCES
FROM UNCULTURED BACTERIA IN THE HUMAN
VAGINAL MICROBIOME
Friday, 3rd June 09:30 La Fonda Ballroom Talk (OS‐7.01)
Laura Sycuro 1 , Andrew Wiser 1 , Josh Burton 2 , Ivan Liachko 3 , Jonathan Golob 1 ,
Maitreya Dunham 2 , Jay Shendure 2 , David Fredricks 1
1 Fred Hutch Cancer Research Center, 2 University of Washington, 3 Phase Genomics
It is widely assumed that most species comprising the human microbiome have now been catalogued through 16S
rRNA gene sequencing. However, many species remain uncultured and without additional genomic data from these
organisms, our ability to consistently detect and functionally characterize them is handicapped all too often, they are
the proverbial elephant in the room.
The human vaginal microbiome contains dozens of uncultured species, some of which are highly prevalent in
women with bacterial vaginosis (BV) and increasingly implicated in preterm birth. We selected a single BV(+)
vaginal sample with a high density of uncultured species for metagenomic sequencing and bacterial genome
segregation using intracellular chromosome linkage analysis (Hi‐C). Reconstruction of 16S rRNA genes from our
shotgun reads (EMIRGE) predicted 27 species with relative abundance (RA) above 0.1% (total abundance = 99.5%),
11 of which are uncultured (total abundance = 72.3%). From a series of metagenomic assemblies (maximal
assembly size = 59 Mb, n50 = 17 kb), we successfully segregated draft genome sequences for 23 of the predicted
species, including all taxa >0.2% RA, 8 of the 11 uncultured species, and two distinct strains of Atopobium vaginae.
Most of our Hi‐C‐derived genomes approached the quality of isolate genome sequences (median completeness and
contamination of all 23 genomes assessed with CheckM = 85% and 1%, respectively).
Draft genomes were submitted to genome finishing and characterization pipelines, dubbed CoAlEScE and AnPhIRL,
that we are developing for high throughput processing of bacterial genomes obtained from metagenomes. CoAlEScE
was used to collapse redundant genomes clustered by Hi‐C from different assemblies, resulting in significantly
improved genome contiguity. Using assembled 16S rRNA and cpn60 marker genes, as well as whole genome
comparative functionalities within AnPhIRL (based on pplacer and MiSi methodologies), we provisionally classified
the 8 uncultured species as representatives of a novel phylum, 2 novel classes, 1 novel family, 1 novel genus, and 3
novel species. Since five of these species were moderately abundant (each 1‐10% RA) and a sixth dominated the
community (>50% RA), our findings underscore the extent to which uncultured and largely undescribed biological
diversity within the vaginal microbiome could impact community function and reproductive health. Indeed, 3 of the
uncultured species for which we successfully resolved a genome sequence, including both species representing
novel classes, have never before been named or otherwise consistently identified across studies; nonetheless, all 3
were previously detected using full‐length 16S amplification and sequencing in vaginal samples from women
experiencing BV and/or preterm birth.
This work demonstrates the feasibility of rapidly populating reference genome databases with high quality, and in some
cases strain‐resolved genomes of uncultured bacteria using Hi‐C linkage anal‐ ysis coupled with automated in silico genome
finishing. Newly generated genomes of uncultured BV‐associated bacteria that may contribute to pregnancy
complications will provide linkages be‐ tween phylogeny and function that are needed to better understand the
microbiome’s role in human reproduction.
132