Abstract
Pathogen genome sequencing directly from clinical samples is quickly gaining importance in genetic and medical research studies. However, low DNA yield from blood-borne pathogens is often a limiting factor. The problem worsens in extremely base-biased genomes such as the AT-rich Plasmodium falciparum. We present a strategy for whole-genome amplification (WGA) of low-yield samples from P. falciparum prior to short-read sequencing. We have developed WGA conditions that incorporate tetramethylammonium chloride for improved amplification and coverage of AT-rich regions of the genome. We show that this method reduces amplification bias and chimera formation. Our data show that this method is suitable for as low as 10 pg input DNA, and offers the possibility of sequencing the parasite genome from small blood samples.
Highlights
Detection of emerging genetic variants and other evolutionary features associated with important clinical phenotypes such as increased virulence and drug resistance are central to malaria control strategies
We show that our optimized whole-genome amplification (WGA) conditions are suitable for as low as 10 picograms P. falciparum input DNA, producing high-sequence concordance with unamplified genomic DNA
We have previously shown that addition of tetramethylammonium chloride (TMAC) improves coverage of low GC regions of the genome during PCR,[22] but the same has not been tested with w29, the multiple displacement amplification (MDA) polymerase
Summary
Detection of emerging genetic variants and other evolutionary features associated with important clinical phenotypes such as increased virulence and drug resistance are central to malaria control strategies. Genome sequencing of parasite populations has been identified as an effective tool for detecting genetic changes.[1,2] Despite the current success in the sequencing technology, there remain significant challenges in achieving global genetic surveillance of parasite populations in the field. Most genome-scale analyses, such as whole-genome sequencing, require large amounts of clean genetic material that is often difficult to obtain,[3] and a serious impediment to genetic analysis on many clinical samples. A large number of valuable clinical specimens are collected in the form of small samples that yield low quantity and quality of genetic material.[4,5,6,7] A common method for collecting clinical samples in the field is through heel/finger-pricks.[5,7,8,9,10]
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