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

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 safe...

Full description

Saved in:

Bibliographic Details
Published in:	Malaria journal Vol. 15; no. 1; p. 597
Main Authors:	Oyola, Samuel O, Ariani, Cristina V, Hamilton, William L, Kekre, Mihir, Amenga-Etego, Lucas N, Ghansah, Anita, Rutledge, Gavin G, Redmond, Seth, Manske, Magnus, Jyothi, Dushyanth, Jacob, Chris G, Otto, Thomas D, Rockett, Kirk, Newbold, Chris I, Berriman, Matthew, Kwiatkowski, Dominic P
Format:	Journal Article
Language:	English
Published:	England BioMed Central Ltd 20-12-2016 BioMed Central
Subjects:	Analysis Blood - parasitology Care and treatment Desiccation DNA Primers - genetics DNA sequencing DNA, Protozoan - chemistry DNA, Protozoan - genetics DNA, Protozoan - isolation & purification Drug resistance Epidemiology Genetic aspects Genome, Protozoan Humans Malaria Nucleic Acid Amplification Techniques - methods Plasmodium falciparum Plasmodium falciparum - genetics Plasmodium falciparum - isolation & purification Sequence Analysis, DNA Specimen Handling - methods Selective whole genome amplification Malaria Field samples Whole genome sequencing Dried blood spot
Online Access:	Get full text
Tags:	Add Tag No Tags, Be the first to tag this record!

Description
Summary:	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. 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. 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. 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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1475-2875 1475-2875
DOI:	10.1186/s12936-016-1641-7