Numerous uncharacterized and highly divergent microbes which colonize humans are revealed by circulating cell-free DNA

Mark Kowarsky; Joan Camunas-Soler; Michael Kertesz; Iwijn De Vlaminck; Winston Koh; Wenying Pan; Lance Martin; Norma F. Neff; Jennifer Okamoto; Ronald J. Wong; Sandhya Kharbanda; Yasser El-Sayed; Yair Blumenfeld; David K. Stevenson; Gary M. Shaw; Nathan D. Wolfe; Stephen R. Quake

doi:10.1073/pnas.1707009114

New Research In

Contributed by Stephen R. Quake, July 12, 2017 (sent for review April 28, 2017; reviewed by Søren Brunak and Eran Segal)

Significance

Through massive shotgun sequencing of circulating cell-free DNA from the blood of more than 1,000 independent samples, we identified hundreds of new bacteria and viruses which represent previously unidentified members of the human microbiome. Previous studies targeted specific niches such as feces, skin, or the oral cavity, whereas our approach of using blood effectively enables sampling of the entire body and reveals the colonization of niches which have been previously inaccessible. We were thus able to discover that the human body contains a vast and unexpected diversity of microbes, many of which have highly divergent relationships to the known tree of life.

Abstract

Blood circulates throughout the human body and contains molecules drawn from virtually every tissue, including the microbes and viruses which colonize the body. Through massive shotgun sequencing of circulating cell-free DNA from the blood, we identified hundreds of new bacteria and viruses which represent previously unidentified members of the human microbiome. Analyzing cumulative sequence data from 1,351 blood samples collected from 188 patients enabled us to assemble 7,190 contiguous regions (contigs) larger than 1 kbp, of which 3,761 are novel with little or no sequence homology in any existing databases. The vast majority of these novel contigs possess coding sequences, and we have validated their existence both by finding their presence in independent experiments and by performing direct PCR amplification. When their nearest neighbors are located in the tree of life, many of the organisms represent entirely novel taxa, showing that microbial diversity within the human body is substantially broader than previously appreciated.

Footnotes

↵¹Present address: Karius, Redwood City, CA 94065.
↵²To whom correspondence should be addressed. Email: quake{at}stanford.edu.

Author contributions: M. Kowarsky, M. Kertesz, I.D.V., and S.R.Q. designed research; M. Kowarsky, J.C.-S., I.D.V., W.K., W.P., L.M., N.F.N., J.O., R.J.W., S.K., Y.E.-S., Y.B., D.K.S., G.M.S., and S.R.Q. performed research; M. Kowarsky contributed new reagents/analytic tools; R.J.W., S.K., Y.E.-S., Y.B., D.K.S., and G.M.S. recruited patients and collected samples; M. Kowarsky and S.R.Q. analyzed data; and M. Kowarsky, N.D.W., and S.R.Q. wrote the paper.
Reviewers: S.B., Novo Nordisk Foundation Center for Protein Research; and E.S., Weizmann Institute of Science.
Conflict of interest statement: N.D.W. is an employee of Metabiota and founder of Global Viral; S.R.Q. is a founder of Karius. M. Kertesz is an employee and founder of Karius, but all work was performed while at Stanford and before he joined the company. All other authors declare no conflict of interest.
Data deposition: Sequencing data are accessible on the NCBI sequence read archive (accession nos. PRJNA263522, PRJNA222186, PRJNA385009, and PRJNA385180).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1707009114/-/DCSupplemental.

Freely available online through the PNAS open access option.

View Full Text