title: Optimized whole-genome amplification strategy for extremely AT-biased template.
abstract: Pathogen genome sequencing directly from clinical samples is quickly gaining importance in genetic and medical research studies. However, low DNA yield from blood-borne pathogens is often a limiting factor. The problem worsens in extremely base-biased genomes such as the AT-rich Plasmodium falciparum. We present a strategy for whole-genome amplification (WGA) of low-yield samples from P. falciparum prior to short-read sequencing. We have developed WGA conditions that incorporate tetramethylammonium chloride for improved amplification and coverage of AT-rich regions of the genome. We show that this method reduces amplification bias and chimera formation. Our data show that this method is suitable for as low as 10 pg input DNA, and offers the possibility of sequencing the parasite genome from small blood samples.
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, UK so1@sanger.ac.uk.
authors.forename: Samuel O
authors.initials: SO
authors.lastname: Oyola
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, UK.
authors.forename: Magnus
authors.initials: M
authors.lastname: Manske
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, UK.
authors.forename: Susana
authors.initials: S
authors.lastname: Campino
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, UK.
authors.forename: Antoine
authors.initials: A
authors.lastname: Claessens
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, UK.
authors.forename: William L
authors.initials: WL
authors.lastname: Hamilton
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, UK.
authors.forename: Mihir
authors.initials: M
authors.lastname: Kekre
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, UK.
authors.forename: Eleanor
authors.initials: E
authors.lastname: Drury
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, UK.
authors.forename: Daniel
authors.initials: D
authors.lastname: Mead
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, UK.
authors.forename: Yong
authors.initials: Y
authors.lastname: Gu
authors.affiliation: MRC Centre for Genomics and Global Health, University of Oxford, Oxford OX3 7BN, UK Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK.
authors.forename: Alistair
authors.initials: A
authors.lastname: Miles
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, UK MRC Centre for Genomics and Global Health, University of Oxford, Oxford OX3 7BN, UK.
authors.forename: Bronwyn
authors.initials: B
authors.lastname: MacInnis
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, UK Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.
authors.forename: Chris
authors.initials: C
authors.lastname: Newbold
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, UK.
authors.forename: Matthew
authors.initials: M
authors.lastname: Berriman
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, UK MRC Centre for Genomics and Global Health, University of Oxford, Oxford OX3 7BN, UK Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK.
authors.forename: Dominic P
authors.initials: DP
authors.lastname: Kwiatkowski
mesh_terms: D016054:DNA, Protozoan / Q000737:chemistry* / Q000235:genetics*; D018503:Genome, Protozoan; D059014:High-Throughput Nucleotide Sequencing; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010963:Plasmodium falciparum / Q000737:chemistry* / Q000235:genetics*
chemical_list: D016054:DNA, Protozoan
keywords: AT-rich; malaria; tetramethylammonium chloride; whole-genome amplification