Optimization of whole-genome sequencing of Plasmodium falciparum from low-density dried blood spot samples

Malaria Journal

Rene Sit¹  Sofonias K. Tessema²  Elias M. Duarte²  Noam B. Teyssier²  Anna Chen²  Bryan Greenhouse³ 

[1] Chan Zuckerberg Biohub, San Francisco, CA, USA;Department of Medicine, EPPIcenter, University of California, San Francisco, CA, USA;Department of Medicine, EPPIcenter, University of California, San Francisco, CA, USA;Chan Zuckerberg Biohub, San Francisco, CA, USA;

关键词: Malaria;    P. falciparum;    Dried blood spots;    Tween-Chelex;    McrBC;    Selective whole genome amplification;    Whole genome sequencing;   

DOI  :  10.1186/s12936-021-03630-4

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY