Whole-genome sequences ofChlamydia trachomatisdirectly from clinical samples without culture

The use of whole-genome sequencing as a tool for the study of infectious bacteria is of growing clinical interest.Chlamydia trachomatisis responsible for sexually transmitted infections and the blinding disease trachoma, which affect hundreds of millions of people worldwide. Recombination is widespread within the genome ofC. trachomatis, thus whole-genome sequencing is necessary to understand the evolution, diversity, and epidemiology of this pathogen. Culture ofC. trachomatishas, until now, been a prerequisite to obtain DNA for whole-genome sequencing; however, asC. trachomatisis an obligate intracellular pathogen, this procedure is technically demanding and time consuming. Discarded clinical samples represent a large resource for sequencing the genomes of pathogens, yet clinical swabs frequently contain very low levels ofC. trachomatisDNA and large amounts of contaminating microbial and human DNA. To determine whether it is possible to obtain whole-genome sequences from bacteria without the need for culture, we have devised an approach that combines immunomagnetic separation (IMS) for targeted bacterial enrichment with multiple displacement amplification (MDA) for whole-genome amplification. Using IMS-MDA in conjunction with high-throughput multiplexed Illumina sequencing, we have produced the first whole bacterial genome sequences direct from clinical samples. We also show that this method can be used to generate genome data from nonviable archived samples. This method will prove a useful tool in answering questions relating to the biology of many difficult-to-culture or fastidious bacteria of clinical concern.