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An effective method to purify Plasmodium falciparum DNA directly from clinical blood samples for whole genome high-throughput sequencing

Auburn, Sarah, Campino, Susana, Clark, Taane G., Djimde, Abdoulaye A., Zongo, Issaka, Pinches, Robert, Manske, Magnus, Mangano, Valentina, Alcock, Daniel, Anastasi, Elisa, Maslen, Gareth, MacInnis, Bronwyn, Rockett, Kirk, Modiano, David, Newbold, Christopher I., Doumbo, Ogobara K., Oue´draogo, Jean Bosco and Kwiatkowski, Dominic P. (2011). An effective method to purify Plasmodium falciparum DNA directly from clinical blood samples for whole genome high-throughput sequencing. PLoS ONE,6(7):e22213.

Document type: Journal Article
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IRMA ID kmckayxPUB40
Title An effective method to purify Plasmodium falciparum DNA directly from clinical blood samples for whole genome high-throughput sequencing
Author Auburn, Sarah
Campino, Susana
Clark, Taane G.
Djimde, Abdoulaye A.
Zongo, Issaka
Pinches, Robert
Manske, Magnus
Mangano, Valentina
Alcock, Daniel
Anastasi, Elisa
Maslen, Gareth
MacInnis, Bronwyn
Rockett, Kirk
Modiano, David
Newbold, Christopher I.
Doumbo, Ogobara K.
Oue´draogo, Jean Bosco
Kwiatkowski, Dominic P.
Journal Name PLoS ONE
Publication Date 2011
Volume Number 6
Issue Number 7
ISSN 1932-6203   (check CDU catalogue open catalogue search in new window)
Start Page e22213
Total Pages 8
Place of Publication United States
Publisher Public Library of Science
HERDC Category C1 - Journal Article (DEST)
Abstract Highly parallel sequencing technologies permit cost-effective whole genome sequencing of hundreds of Plasmodium parasites. The ability to sequence clinical Plasmodium samples, extracted directly from patient blood without a culture step, presents a unique opportunity to sample the diversity of ‘‘natural’’ parasite populations in high resolution clinical and epidemiological studies. A major challenge to sequencing clinical Plasmodium samples is the abundance of human DNA, which may substantially reduce the yield of Plasmodium sequence.

We tested a range of human white blood cell (WBC) depletion methods on P. falciparum-infected patient samples in search of a method displaying an optimal balance of WBCremoval efficacy, cost, simplicity, and applicability to low resource settings. In the first of a two-part study, combinations of three different WBC depletion methods were tested on 43 patient blood samples in Mali. A two-step combination of Lymphoprep plus Plasmodipur best fitted our requirements, although moderate variability was observed in human DNA quantity. This approach was further assessed in a larger sample of 76 patients from Burkina Faso. WBC-removal efficacy remained high (,30% human DNA in .70% samples) and lower variation was observed in human DNA quantities.

In order to assess the Plasmodium sequence yield at different human DNA proportions, 59 samples with up to 60% human DNA contamination were sequenced on the Illumina Genome Analyzer platform. An average ,40-fold coverage of the genome was observed per lane for samples with #30% human DNA. Even in low resource settings, using a simple two-step combination of Lymphoprep plus Plasmodipur, over 70% of clinical sample preparations should exhibit sufficiently low human DNA quantities to enable ,40-fold sequence coverage of the P. falciparum genome using a single lane on the Illumina Genome Analyzer platform. This approach should greatly facilitate large-scale clinical and epidemiologic studies of P. falciparum.
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