Whole genome sequencing of Plasmodium falciparum from dried blood spots using selective whole genome amplification

Oyola, S. O. et al. (2016) Whole genome sequencing of Plasmodium falciparum from dried blood spots using selective whole genome amplification. Malaria Journal, 15(1), 597. (doi: 10.1186/s12936-016-1641-7) (PMID:27998271) (PMCID:PMC5175302)

Abstract

BACKGROUND: Translating genomic technologies into healthcare applications for the malaria parasite Plasmodium falciparum has been limited by the technical and logistical difficulties of obtaining high quality clinical samples from the field. Sampling by dried blood spot (DBS) finger-pricks can be performed safely and efficiently with minimal resource and storage requirements compared with venous blood (VB). Here, the use of selective whole genome amplification (sWGA) to sequence the P. falciparum genome from clinical DBS samples was evaluated, and the results compared with current methods that use leucodepleted VB. METHODS: Parasite DNA with high (>95%) human DNA contamination was selectively amplified by Phi29 polymerase using short oligonucleotide probes of 8-12 mers as primers. These primers were selected on the basis of their differential frequency of binding the desired (P. falciparum DNA) and contaminating (human) genomes. RESULTS: Using sWGA method, clinical samples from 156 malaria patients, including 120 paired samples for head-to-head comparison of DBS and leucodepleted VB were sequenced. Greater than 18-fold enrichment of P. falciparum DNA was achieved from DBS extracts. The parasitaemia threshold to achieve >5× coverage for 50% of the genome was 0.03% (40 parasites per 200 white blood cells). Over 99% SNP concordance between VB and DBS samples was achieved after excluding missing calls. CONCLUSION: The sWGA methods described here provide a reliable and scalable way of generating P. falciparum genome sequence data from DBS samples. The current data indicate that it will be possible to get good quality sequence on most if not all drug resistance loci from the majority of symptomatic malaria patients. This technique overcomes a major limiting factor in P. falciparum genome sequencing from field samples, and paves the way for large-scale epidemiological applications.

• Item Type: Articles
• Additional Information: This research was supported by the Wellcome Trust through the Wellcome Trust Sanger Institute (098051), the Resource Centre for Genomic Epidemiol‑ ogy of Malaria (090770/Z/09/Z) and the Wellcome Trust Centre for Human Genetics (090532/Z/09/Z). The Centre for Genomics and Global Health is supported by the Medical Research Council (G0600718). GGR is supported by the Medical Research Council (MR/J004111/1).
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Otto, Professor Thomas
• Authors: Oyola, S. O., Ariani, C. V., Hamilton, W. L., Kekre, M., Amenga-Etego, L. N., Ghansah, A., Rutledge, G. G., Redmond, S., Manske, M., Jyothi, D., Jacob, C. G., Otto, T. D., Rockett, K., Newbold, C. I., Berriman, M., and Kwiatkowski, D. P.
• College/School: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
• Journal Name: Malaria Journal
• Publisher: BioMed Central
• ISSN: 1475-2875
• ISSN (Online): 1475-2875
• Copyright Holders: Copyright © 2016 The Authors
• First Published: First published in Malaria Journal 15:597
• Publisher Policy: Reproduced under a Creative Commons License