Direct Amplification, Sequencing and Profiling of Chlamydia trachomatis Strains in Single and Mixed Infection Clinical Samples

• Type of Material: Article
• Authors: Sandeep J. Joseph, Emory University; Ben Li, Emory University; Tanvi Ghonasgi, Children's Hospital Oakland Research Institute; Chad P. Haase, Emory University; Zhaohui Qin, Emory University; Deborah Dean, Children's Hospital Oakland Research Institute; Timothy D Read, Emory University
• Language: English
• Date: 2014
• Publisher: Public Library of Science
• Publication Version: Final Published Version
• Copyright Statement: © 2014 Joseph et al.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0099290
• Title of Journal or Parent Work: PLoS ONE
• ISSN: 1932-6203
• Volume: 9
• Issue: 6
• Start Page: e99290
• End Page: e99290
• Grant/Funding Information: This project made use of the Emory Genomics Center, which used equipment funded by the Georgia Research Alliance and Atlanta Clinical and Translational Sciences Institute.; This work was supported by development funds from Emory University School of Medicine (to TDR), and Public Health Service grant R01AI098843 from the National Institutes of Health (to DD and TDR).
• Research Categories: Health Sciences, Public Health; Health Sciences, General

Abstract

Sequencing bacterial genomes from DNA isolated directly from clinical samples offers the promise of rapid and precise acquisition of informative genetic information. In the case of Chlamydia trachomatis, direct sequencing is particularly desirable because it obviates the requirement for culture in mammalian cells, saving time, cost and the possibility of missing low abundance strains. In this proof of concept study, we developed methodology that would allow genome-scale direct sequencing, using a multiplexed microdroplet PCR enrichment technology to amplify a 100 kb region of the C. trachomatis genome with 500 1.1–1.3 kb overlapping amplicons (5-fold amplicon redundancy). We integrated comparative genomic data into a pipeline to preferentially select conserved sites for amplicon design. The 100 kb target region could be amplified from clinical samples, including remnants from diagnostics tests, originating from the cervix, urethra and urine, For rapid analysis of these data, we developed a framework for whole-genome based genotyping called binstrain. We used binstrain to estimate the proportion of SNPs originating from 14 C. trachomatis reference serotype genomes in each sample. Direct DNA sequencing methods such as the one described here may have an important role in understanding the biology of C. trachomatis mixed infections and the natural genetic variation of the species within clinically relevant ecological niches.