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

Malaria Journal

Research

Seth Redmond¹  Kirk Rockett²  Lucas N. Amenga-Etego³  Anita Ghansah⁴  Chris I. Newbold⁵  Gavin G. Rutledge⁶  Matthew Berriman⁶  Magnus Manske⁶  Cristina V. Ariani⁶  Thomas D. Otto⁶  Dushyanth Jyothi⁶  Mihir Kekre⁶  Chris G. Jacob⁶  William L. Hamilton⁷  Samuel O. Oyola⁸  Dominic P. Kwiatkowski⁹ 

[1] Broad Institute, 415 Main St, 02142, Cambridge, MA, USA;MRC Centre for Genomics and Global Health, University of Oxford, OX3 7BN, Oxford, UK;Wellcome Trust Centre for Human Genetics, University of Oxford, OX3 7BN, Oxford, UK;Navrongo Health Research Centre, Post Office Box 114, Navrongo, Ghana;Noguchi Memorial Institute for Medical Research, University of Ghana, P. O. Box LG 581, Legon, Accra, Ghana;Weatherall Institute of Molecular Medicine, University of Oxford, OX3 9DS, Oxford, UK;Wellcome Trust Sanger Institute, CB10 1SA, Hinxton, UK;Wellcome Trust Sanger Institute, CB10 1SA, Hinxton, UK;Addenbrooke’s Hospital, University of Cambridge School of Clinical Medicine, Hills Rd, CB2 0SP, Cambridge, UK;Wellcome Trust Sanger Institute, CB10 1SA, Hinxton, UK;International Livestock Research Institute, Box 30709, Nairobi, Kenya;Wellcome Trust Sanger Institute, CB10 1SA, Hinxton, UK;MRC Centre for Genomics and Global Health, University of Oxford, OX3 7BN, Oxford, UK;Wellcome Trust Centre for Human Genetics, University of Oxford, OX3 7BN, Oxford, UK;

关键词: Malaria;    Dried blood spot;    Selective whole genome amplification;    Field samples;    Whole genome sequencing;   

DOI  :  10.1186/s12936-016-1641-7

 received in 2016-08-02, accepted in 2016-11-28,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundTranslating 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.MethodsParasite 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.ResultsUsing 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.ConclusionThe 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.

【 授权许可】

CC BY   
© The Author(s) 2016