Abstract
We have adapted a solution hybrid selection protocol to enrich pathogen DNA in clinical samples dominated by human genetic material. Using mock mixtures of human and Plasmodium falciparum malaria parasite DNA as well as clinical samples from infected patients, we demonstrate an average of approximately 40-fold enrichment of parasite DNA after hybrid selection. This approach will enable efficient genome sequencing of pathogens from clinical samples, as well as sequencing of endosymbiotic organisms such as Wolbachia that live inside diverse metazoan phyla.
Highlights
The falling cost of DNA sequencing means that sample quality, rather than expense, is the blocking issue for many infectious disease genome sequencing projects
Quantitative PCR analysis indicated that whole genome amplification (WGA) does not significantly alter the fraction of malaria DNA present in the sample
The synthetic baits respectively yielded an average of 41-fold and 44-fold parasite DNA enrichment for unamplified and WGA simulated clinical samples in genomic regions targeted by the baits, as measured by Quantitative PCR (qPCR)
Summary
The falling cost of DNA sequencing means that sample quality, rather than expense, is the blocking issue for many infectious disease genome sequencing projects. White blood cell depletion currently requires a significant volume of blood to be drawn from patients (approximately 5 ml), and the blood must be stored at minus 70°C in a special medium to preserve cellular integrity This could preclude sample collection for genome sequencing from many clinical trials due to protocol limitations or lack of equipment in the field. Pathogens that infect or closely associate with nucleated host cells, such as Plasmodium vivax, Trypanosoma cruzi, or Chlamydia trachomatis, are not amenable to purification by white cell depletion Endosymbionts such as Wolbachia, which influence host fertility and other traits in filarial worms, insect disease vectors, and diverse other taxa, may only be cultured in an intracellular system [11], precluding easy isolation of their genomic DNA for sequencing except by elaborate methods [12]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
