title: Direct amplification, sequencing and profiling of Chlamydia trachomatis strains in single and mixed infection clinical samples.
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.
authors: Department of Medicine, Division of Infectious Diseases, Emory University School of Medicine, Atlanta, Georgia, United States of America.
authors: Sandeep J
authors: SJ
authors: Joseph
authors: Department of Biostatistics and Bioinformatics, Rollins School of Public Health, Emory University, Atlanta, Georgia, United States of America.
authors: Ben
authors: B
authors: Li
authors: Center for Immunobiology and Vaccine Development, Children's Hospital Oakland Research Institute, Oakland, California, United States of America.
authors: Tanvi
authors: T
authors: Ghonasgi
authors: Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia, United States of America.
authors: Chad P
authors: CP
authors: Haase
authors: Department of Biostatistics and Bioinformatics, Rollins School of Public Health, Emory University, Atlanta, Georgia, United States of America.
authors: Zhaohui S
authors: ZS
authors: Qin
authors: Center for Immunobiology and Vaccine Development, Children's Hospital Oakland Research Institute, Oakland, California, United States of America; Department of Medicine, University of California San Francisco, San Francisco, California, United States of America; Joint Graduate Program in Bioengineering, University of California San Francisco, San Francisco, California, United States of America; University of California Berkeley, Berkeley, California, United States of America.
authors: Deborah
authors: D
authors: Dean
authors: Department of Medicine, Division of Infectious Diseases, Emory University School of Medicine, Atlanta, Georgia, United States of America; Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia, United States of America.
authors: Timothy D
authors: TD
authors: Read
mesh_terms: D002690:Chlamydia Infections / Q000382:microbiology*; D002692:Chlamydia trachomatis / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*