Details

Autor(en) / Beteiligte

• 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
• Ouédraogo, Jean Bosco
• Kwiatkowski, Dominic P
• Dimopoulos, George

Titel

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

Ist Teil von

• PloS one, 2011-07, Vol.6 (7), p.e22213-e22213

Ort / Verlag

United States: Public Library of Science

Erscheinungsjahr

2011

Quelle

MEDLINE

Beschreibungen/Notizen

• 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 WBC-removal 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.