Pooled sequencing and rare variant association tests for identifying the determinants of emerging drug resistance in malaria parasites.
Pooled sequencing and rare variant association tests for identifying the determinants of emerging drug resistance in malaria parasites.
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DOI：
10.1093/molbev/msu397
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发表时间：
2015-04
影响因子：
10.7
通讯作者：
Anderson TJ
中科院分区：
文献类型：
--
作者：
Cheeseman IH;McDew-White M;Phyo AP;Sriprawat K;Nosten F;Anderson TJ
我们探索了合并测序的潜力，以快速和经济地确定由于东南亚疟疾寄生虫种群中出现的青蒿素（ART）耐药性而进行的选择性扫描。抗逆转录病毒治疗耐药的定义是从抗逆转录病毒治疗患者的血液中缓慢清除寄生虫，并且强烈暗示kelch基因（chr. 13）中的突变发挥作用。我们构建了从泰国-缅甸边境收集的70个缓慢清除（抗性）和70个快速清除（敏感）感染的一式三份池，并将其测序到高（约150倍）读取深度。从池中的等位基因频率估计显示出几乎完美的相关性（林的一致性= 0.98）与等位基因频率在93个单核苷酸多态性直接从个人感染，给我们信心，这种方法的准确性。通过绘制耐药和药物敏感寄生虫库之间的全基因组差异（FST），我们确定了两个大（>150 kb）区域（在chrs. 13和14）和17个较小的候选基因组区域。为了识别这些基因组区域内的单个基因，我们重新测序了另外38个寄生虫基因组（16个缓慢清除和22个快速清除），并进行了罕见变异关联测试。这证实了kelch是ART耐药的主要分子标记（P = 6.03 × 10−6）。这种双层方法非常强大，因为合并测序可以快速缩小感兴趣的基因组区域，而这些区域内的靶向罕见变异关联测试可以查明耐药性的遗传基础。我们表明，我们的方法是强大的经常性突变和软选择性扫描的产生，这是预测在病原体种群与大的有效人口规模是常见的，并可能混淆更传统的基因定位方法。
We explored the potential of pooled sequencing to swiftly and economically identify selective sweeps due to emerging artemisinin (ART) resistance in a South-East Asian malaria parasite population. ART resistance is defined by slow parasite clearance from the blood of ART-treated patients and mutations in the kelch gene (chr. 13) have been strongly implicated to play a role. We constructed triplicate pools of 70 slow-clearing (resistant) and 70 fast-clearing (sensitive) infections collected from the Thai–Myanmar border and sequenced these to high (∼150-fold) read depth. Allele frequency estimates from pools showed almost perfect correlation (Lin’s concordance = 0.98) with allele frequencies at 93 single nucleotide polymorphisms measured directly from individual infections, giving us confidence in the accuracy of this approach. By mapping genome-wide divergence (FST) between pools of drug-resistant and drug-sensitive parasites, we identified two large (>150 kb) regions (on chrs. 13 and 14) and 17 smaller candidate genome regions. To identify individual genes within these genome regions, we resequenced an additional 38 parasite genomes (16 slow and 22 fast-clearing) and performed rare variant association tests. These confirmed kelch as a major molecular marker for ART resistance (P = 6.03 × 10−6). This two-tier approach is powerful because pooled sequencing rapidly narrows down genome regions of interest, while targeted rare variant association testing within these regions can pinpoint the genetic basis of resistance. We show that our approach is robust to recurrent mutation and the generation of soft selective sweeps, which are predicted to be common in pathogen populations with large effective population sizes, and may confound more traditional gene mapping approaches.
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DOI：
10.1098/rstb.2000.0716
发表时间：
2000-11-29
影响因子：
6.3
作者：
Barton, NH
通讯作者：
Barton, NH
影响因子：
15.8
作者：
Carrara VI;Lwin KM;Phyo AP;Ashley E;Wiladphaingern J;Sriprawat K;Rijken M;Boel M;McGready R;Proux S;Chu C;Singhasivanon P;White N;Nosten F
通讯作者：
Nosten F
影响因子：
1.2
作者：
Cingolani, Pablo;Platts, Adrian;Ruden, Douglas M.
通讯作者：
Ruden, Douglas M.
影响因子：
4.9
作者：
Hebert, Francois Olivier;Renaut, Sebastien;Bernatchez, Louis
通讯作者：
Bernatchez, Louis
影响因子：
46.9
作者：
Ghorbal, Mehdi;Gorman, Molly;Lopez-Rubio, Jose-Juan
通讯作者：
Lopez-Rubio, Jose-Juan