Optimized Whole-Genome Amplification Strategy for Extremely AT-Biased Template

DNA RESEARCH 21, 661–671, (2014) Advance Access publication on 19 September 2014 doi:10.1093/dnares/dsu028 Optimized Whole-Genome Amplification Strategy for Extremely AT-Biased Template SAMUEL O. Oyola1,*, MAGNUS Manske1, SUSANA Campino1, ANTOINE Claessens1, WILLIAM L. Hamilton1, MIHIR Kekre1, ELEANOR Drury1, DANIEL Mead1, YONG Gu1, ALISTAIR Miles2,3, BRONWYN MacInnis1,2, CHRIS Newbold1,4, MATTHEW Berriman1, and DOMINIC P. Kwiatkowski1,2,3 Wellcome Trust Sanger Institute, Hinxton, UK1; MRC Centre for Genomics and Global Health, University of Oxford, Oxford OX3 7BN, UK2; Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK3 and Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK4 *To whom correspondence should be addressed. Tel. þ44 1223-494-994. Fax. þ44 1223-494-919. Email: Edited by Dr Yuji Kohara (Received 9 June 2014; accepted 22 August 2014) 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. Key words: whole-genome amplification; AT-rich; malaria; tetramethylammonium chloride 1. Introduction Timely detection of emerging genetic variants and other evolutionary features associated with important clinical phenotypes such as increased virulence and drug resistance are central to malaria control strategies. Genome sequencing of parasite populations has been identified as an effective tool for detecting genetic changes.1,2 Despite the current success in the sequencing technology, there remain significant challenges in achieving global genetic surveillance of parasite populations in the field. Most genome-scale analyses, such as whole-genome sequencing, require large amounts of clean genetic material that is often difficult to obtain,3 and therefore a serious impediment to genetic analysis on many clinical samples. A large number of valuable clinical specimens are collected in the form of small samples that yield low quantity and quality of genetic material.4 – 7 A common method for collecting clinical samples in the field is through heel/finger-pricks.5,7 – 10 However, the quantity and quality of parasite genetic material that can be extracted from these small blood samples usually fall below the threshold required by genome sequencing platforms. To alleviate the problem of low DNA quantities, whole-genome amplification (WGA) is now routinely applied in many applications,3,11 but has yet to be optimized for use in genomes of extreme base composition such as Plasmodium falciparum. Two major forms of WGA have been described: multiple displacement amplification (MDA)12,13 and PCR-based amplification methods.14,15 MDA has been the method of choice for a wider range of genome amplification studies, because it produces longer DNA products with extensive genome coverage.16 MDA is based on w29 polymerase, which, in the presence of random hexamers annealed to denatured # The Author 2014. Published by Oxford University Press on behalf of Kazusa DNA Research Institute. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 662 Whole-Genome Amplification for AT-Rich Templates DNA, uses an MDA mechanism to synthesize high-molecular-weight DNA from very minute amounts of input material under isothermal conditions.17,18 The best results, however, have been obtained from genomes with relatively balanced base composition.11,19,20 Amplification of genomes with imbalanced base composition, such as the AT-rich P. falciparum, has remained a challenge.21,22 In this study, we sought to identify and optimize a WGA system suitable for an AT-base-biased genome of P. falciparum. Using standard conditions as outlined for each system, we tested the efficiency of non-MDAand MDA-based methods. Initial findings showed that MDA-based systems produced a more uniform genome coverage than non-MDA methods (data not shown). We have optimized an identified MDA system to produce an improved genome coverage and a reduced base-bias with more accurate genome representation. We show that our optimized WGA conditions are suitable for as low as 10 picograms ( pg) P. falciparum input DNA, producing high-sequence concordance with unamplified genomic DNA. This development promises a significant tool to aid implementation of the global genetic surveillance of parasite populations through small blood sample sequencing. 2. Materials and methods 2.1. DNA samples Plasmodium falciparum 3D7 genomic DNA was a gift from Prof. Chris Newbold (University of Oxford). The clinical isolates were obtained from the Malaria Genetics Group’s Sequencing Sample Repository at the Wellcome Trust Sanger Institute. Other genomic DNA was extracted from 17 progeny clones of P. falciparum strains derived from genetic cross between 7G8xGB423 and a 3D7 strain (3D7_glasgow). 2.2. Whole-genome amplification All non-MDA WGA were performed following individual kit manufacturer’s instructions. MDA-based WGA was performed using either REPLI-g Mini kit (Qiagen) or Genomiphi kit (GE Healthcare). For Genomiphi, the kit manufacturer’s instructions were followed without modification. For the REPLI-g Mini kit, manufacturer’s instructions were followed during preliminary tests. The following modifications were performed in developing optimized conditions for the REPLI-g Mini kit: nuclease-free water and all tubes were UV-treated before use. WGA reactions were performed in 0.2 ml PCR tubes. Buffer D1 stock solution (Qiagen) was reconstituted by adding 500 ml of nuclease-free water, and a working solution was prepared by mixing the stock solution and nuclease-free water in the ratio of 1 : 3.5, respectively. Unmodified Buffer N1 was reconstituted by [Vol. 21, mixing Stop solution (Qiagen) and nuclease-free water in the ratio of 1 : 5.7. Modified buffer N1 was prepared by including tetramethylammonium chloride (TMAC) at a concentration of 300 mM. To denature DNA templates, 5 ml of the DNA solution was mixed with 5 ml of buffer D1 (working solution prepared as described above). The mixture was vort (...truncated)