title: An effective method to purify Plasmodium falciparum DNA directly from clinical blood samples for whole genome high-throughput sequencing.
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 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.
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom. sa3@sanger.ac.uk
authors.forename: Sarah
authors.initials: S
authors.lastname: Auburn
authors.forename: Susana
authors.initials: S
authors.lastname: Campino
authors.forename: Taane G
authors.initials: TG
authors.lastname: Clark
authors.forename: Abdoulaye A
authors.initials: AA
authors.lastname: Djimde
authors.forename: Issaka
authors.initials: I
authors.lastname: Zongo
authors.forename: Robert
authors.initials: R
authors.lastname: Pinches
authors.forename: Magnus
authors.initials: M
authors.lastname: Manske
authors.forename: Valentina
authors.initials: V
authors.lastname: Mangano
authors.forename: Daniel
authors.initials: D
authors.lastname: Alcock
authors.forename: Elisa
authors.initials: E
authors.lastname: Anastasi
authors.forename: Gareth
authors.initials: G
authors.lastname: Maslen
authors.forename: Bronwyn
authors.initials: B
authors.lastname: Macinnis
authors.forename: Kirk
authors.initials: K
authors.lastname: Rockett
authors.forename: David
authors.initials: D
authors.lastname: Modiano
authors.forename: Christopher I
authors.initials: CI
authors.lastname: Newbold
authors.forename: Ogobara K
authors.initials: OK
authors.lastname: Doumbo
authors.forename: Jean Bosco
authors.initials: JB
authors.lastname: Ouédraogo
authors.forename: Dominic P
authors.initials: DP
authors.lastname: Kwiatkowski
mesh_terms: D002050:Burkina Faso; D016054:DNA, Protozoan / Q000097:blood* / Q000302:isolation & purification*; D016678:Genome / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007962:Leukocytes / Q000166:cytology; D008212:Lymphocyte Depletion; D008302:Mali; D018512:Parasitemia / Q000097:blood / Q000469:parasitology; D010963:Plasmodium falciparum / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: D016054:DNA, Protozoan