Effective preparation of Plasmodium vivax field isolates for high-throughput whole genome sequencing.
Effective preparation of Plasmodium vivax field isolates for high-throughput whole genome sequencing.
复制标题
DOI：
10.1371/journal.pone.0053160
复制
发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Price RN
中科院分区：
文献类型：
--
作者：
Auburn S;Marfurt J;Maslen G;Campino S;Ruano Rubio V;Manske M;Machunter B;Kenangalem E;Noviyanti R;Trianty L;Sebayang B;Wirjanata G;Sriprawat K;Alcock D;Macinnis B;Miotto O;Clark TG;Russell B;Anstey NM;Nosten F;Kwiatkowski DP;Price RN
间日疟原虫的全基因组测序（WGS）是有问题的，由于依赖于临床分离株，通常是低寄生虫血症和样品量。此外，临床分离株含有宿主DNA的显著污染背景，这混淆了定位靶间日疟原虫DNA的短读段序列的努力。在这里，我们讨论了一种方法，以显着提高间日疟原虫WGS对自然（非适应）患者分离株的成功率。使用来自印度尼西亚、泰国和旅行者的37个患者分离株，我们评估了单独使用基于CF 11的白色血细胞过滤以及与短期离体嗜酸性粒细胞成熟联合使用的应用。虽然CF11过滤减少了8个印度尼西亚分离株中的人类DNA污染，但额外的短期培养使间日疟原虫DNA产量从中位数0.15 ng µl−1浓缩红细胞（pRBC）增加到6.2 ng µl− 1（p = 0.001），并将人类DNA百分比从中位数33.9%降低到6.22%（p = 0.008）。 此外，来自泰国的CF11后和培养样本的中位间日疟原虫DNA产率为2.34 ng µl−1 pRBC和2.65%人DNA。在使用2步法制备的22株间日疟原虫患者分离株中，我们在Illumina GAII和HiSeq平台上证明了高覆盖深度（中位数654 X覆盖率）和广度（≥89%）。与富含A+ T的恶性疟原虫基因组相反，在间日疟原虫基因组的编码区和非编码区之间的覆盖深度中观察到可忽略的偏差。这种均匀的覆盖率将极大地促进整个基因组中SNP和拷贝数变异的检测，从而能够公正地探索间日疟原虫种群的自然多样性。
Whole genome sequencing (WGS) of Plasmodium vivax is problematic due to the reliance on clinical isolates which are generally low in parasitaemia and sample volume. Furthermore, clinical isolates contain a significant contaminating background of host DNA which confounds efforts to map short read sequence of the target P. vivax DNA. Here, we discuss a methodology to significantly improve the success of P. vivax WGS on natural (non-adapted) patient isolates. Using 37 patient isolates from Indonesia, Thailand, and travellers, we assessed the application of CF11-based white blood cell filtration alone and in combination with short term ex vivo schizont maturation. Although CF11 filtration reduced human DNA contamination in 8 Indonesian isolates tested, additional short-term culture increased the P. vivax DNA yield from a median of 0.15 to 6.2 ng µl−1 packed red blood cells (pRBCs) (p = 0.001) and reduced the human DNA percentage from a median of 33.9% to 6.22% (p = 0.008). Furthermore, post-CF11 and culture samples from Thailand gave a median P. vivax DNA yield of 2.34 ng µl−1 pRBCs, and 2.65% human DNA. In 22 P. vivax patient isolates prepared with the 2-step method, we demonstrate high depth (median 654X coverage) and breadth (≥89%) of coverage on the Illumina GAII and HiSeq platforms. In contrast to the A+T-rich P. falciparum genome, negligible bias was observed in coverage depth between coding and non-coding regions of the P. vivax genome. This uniform coverage will greatly facilitate the detection of SNPs and copy number variants across the genome, enabling unbiased exploration of the natural diversity in P. vivax populations.
登录
查看更多内容
影响因子：
3.8
作者：
Carlton JM;Sina BJ;Adams JH
通讯作者：
Adams JH
影响因子：
64.8
作者：
Carlton, Jane M.;Adams, John H.;Silva, Joana C.;Bidwell, Shelby L.;Lorenzi, Hernan;Caler, Elisabet;Crabtree, Jonathan;Angiuoli, Samuel V.;Merino, Emilio F.;Amedeo, Paolo;Cheng, Qin;Coulson, Richard M. R.;Crabb, Brendan S.;del Portillo, Hernando A.;Essien, Kobby;Feldblyum, Tamara V.;Fernandez-Becerra, Carmen;Gilson, Paul R.;Gueye, Amy H.;Guo, Xiang;Kang'a, Simon;Kooij, Taco W. A.;Korsinczky, Michael;Meyer, Esmeralda V. -S.;Nene, Vish;Paulsen, Ian;White, Owen;Ralph, Stuart A.;Ren, Qinghu;Sargeant, Tobias J.;Salzberg, Steven L.;Stoeckert, Christian J.;Sullivan, Steven A.;Yamamoto, Marcio M.;Hoffman, Stephen L.;Wortman, Jennifer R.;Gardner, Malcolm J.;Galinski, Mary R.;Barnwell, John W.;Fraser-Liggett, Claire M.
通讯作者：
Fraser-Liggett, Claire M.
影响因子：
14.9
作者：
Leinonen R;Akhtar R;Birney E;Bower L;Cerdeno-Tárraga A;Cheng Y;Cleland I;Faruque N;Goodgame N;Gibson R;Hoad G;Jang M;Pakseresht N;Plaister S;Radhakrishnan R;Reddy K;Sobhany S;Ten Hoopen P;Vaughan R;Zalunin V;Cochrane G
通讯作者：
Cochrane G
影响因子：
12.3
作者：
Melnikov A;Galinsky K;Rogov P;Fennell T;Van Tyne D;Russ C;Daniels R;Barnes KG;Bochicchio J;Ndiaye D;Sene PD;Wirth DF;Nusbaum C;Volkman SK;Birren BW;Gnirke A;Neafsey DE
通讯作者：
Neafsey DE
影响因子：
--
作者：
Padley, D;Moody, AH;Saldanha, J
通讯作者：
Saldanha, J