Abstract
BackgroundWhole-genome sequencing (WGS) is becoming increasingly useful to study the biology, epidemiology, and ecology of malaria parasites. Despite ease of sampling, DNA extracted from dried blood spots (DBS) has a high ratio of human DNA compared to parasite DNA, which poses a challenge for downstream genetic analyses. The effects of multiple methods for DNA extraction, digestion of methylated DNA, and amplification were evaluated on the quality and fidelity of WGS data recovered from DBS.MethodsLow parasite density mock DBS samples were created, extracted either with Tween-Chelex or QIAamp, treated with or without McrBC, and amplified with one of three different amplification techniques (two sWGA primer sets and one rWGA). Extraction conditions were evaluated on performance of sequencing depth, percentiles of coverage, and expected SNP concordance.ResultsAt 100 parasites/μL, Chelex-Tween-McrBC samples had higher coverage (5 × depth = 93% genome) than QIAamp extracted samples (5 × depth = 76% genome). The two evaluated sWGA primer sets showed minor differences in overall genome coverage and SNP concordance, with a newly proposed combination of 20 primers showing a modest improvement in coverage over those previously published.ConclusionsOverall, Tween-Chelex extracted samples that were treated with McrBC digestion and are amplified using 6A10AD sWGA conditions had minimal dropout rate, higher percentages of coverage at higher depth, and more accurate SNP concordance than QiaAMP extracted samples. These findings extend the results of previously reported methods, making whole genome sequencing accessible to a larger number of low density samples that are commonly encountered in cross-sectional surveys.
Highlights
Whole-genome sequencing (WGS) is becoming increasingly useful to study the biology, epidemiology, and ecology of malaria parasites
QiaAMP Mini and QiaAMP Investigator spin column kits performed in terms of the percentage of reads that mapped to P. falciparum regardless of parasitaemia
The two selective whole genome amplification (sWGA) primer sets performed with respect to the percentage of reads that mapped to P. falciparum (Fig. 2a) and genotype concordance (Fig. 4)
Summary
Whole-genome sequencing (WGS) is becoming increasingly useful to study the biology, epidemiology, and ecology of malaria parasites. In order to address these challenges, several studies have utilized different enrichment methods such as non-selective whole genome amplification (WGA) [4, 5]; hybrid selection [6,7,8,9]; enzymatic digestion of host DNA using the MspJI family of restriction enzymes [10, 11] and selective whole genome amplification (sWGA) [11,12,13,14]. Collection of leukocyte-depleted dried erythrocyte spots has showed promise to provide parasite enriched samples for WGS [15]. SWGA requires relatively small amounts of starting material and provides relatively high quality data for a low-cost—enabling WGS of field samples that would otherwise contain insufficient genetic material for Plasmodium falciparum [13], Plasmodium vivax [11] and Plasmodium knowlesi [14]. Similar level of coverage was reported in a P. vivax-specific sWGA [11], highlighting the need to improve these protocols for DBS samples
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 callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
