Whole genome amplification and sequencing of low cell numbers directly from a bacteria spiked blood model

Abstract

Whilst next generation sequencing is frequently used to whole genome sequence bacteria from cultures, its rarely applied directly to clinical samples. Therefore, this study addresses the issue of applying NGS microbial diagnostics directly to blood samples. To demonstrate the potential of direct from blood sequencing a bacteria spiked blood model was developed. Horse blood was spiked with clinical samples of E. coli and S. aureus, and a process developed to isolate bacterial cells whilst removing the majority of host DNA. One sample of each isolate was then amplified using {phi}29 multiple displacement amplification (MDA) and sequenced. The total processing time, from sample to amplified DNA ready for sequencing was 3.5 hours, significantly faster than the 18-hour overnight culture step which is typically required. Both bacteria showed 100% survival through the processing. The direct from sample sequencing resulted in greater than 92% genome coverage of the pathogens whilst limiting the sequencing of host genome (less than 7% of all reads). Analysis of de novo assembled reads allowed accurate genotypic antibiotic resistance prediction. The sample processing is easily applicable to multiple sequencing platforms. Overall this model demonstrates potential to rapidly generate whole genome bacterial data directly from blood.