Sensitive, Highly Multiplexed Sequencing of Microhaplotypes From the Plasmodium falciparum Heterozygome.
Sensitive, Highly Multiplexed Sequencing of Microhaplotypes From the Plasmodium falciparum Heterozygome.
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DOI：
10.1093/infdis/jiaa527
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发表时间：
2022-04-01
期刊：
影响因子：
--
通讯作者：
Greenhouse B
中科院分区：
文献类型：
--
作者：
Tessema SK;Hathaway NJ;Teyssier NB;Murphy M;Chen A;Aydemir O;Duarte EM;Simone W;Colborn J;Saute F;Crawford E;Aide P;Bailey JA;Greenhouse B
靶向下一代测序提供了一致的，信息丰富的基因组区域的深度覆盖，以表征多克隆恶性疟原虫感染的潜力。然而，鉴定和测序这些基因组区域的方法目前是有限的。开发了一种生物信息学管道和多重方法，以识别并同时测序100个靶标，并应用于莫桑比克的干血斑（DBS）对照和田间分离株。为了进行比较，针对相同的对照生成全基因组测序数据。使用公开可用的基因组，鉴定了4465个适合靶向测序的高多样性基因组区域，代表恶性疟原虫异源基因组。本研究共筛选出沿着7个耐药基因座的93个微单倍型，这些微单倍型具有较高的多样性（平均期望杂合度= 0.7）。 测序方法实现了非常高的覆盖率（中位数99%），特异性（99.8%）和灵敏度（单倍型为90%，100个寄生虫/微升的干血斑中的样本频率为5%）。计算机模拟分析显示，微单倍型提供了比双等位基因单核苷酸多态性条形码高得多的分辨率来区分相关与不相关的多克隆感染。本文概述的生物信息学和实验室方法为恶性疟原虫基因组的高效、低成本、高通量询问提供了灵活的工具，并且可以定制以同时解决各种流行病学环境中的多个感兴趣的问题。创建了一种新的生物信息学管道，以选择和表征恶性疟原虫基因组中最多样化和最易处理的短距离序列（微单倍型），然后采用基于聚合酶链反应的多路复用方法同时扩增和测序这些微单倍型。
Targeted next-generation sequencing offers the potential for consistent, deep coverage of information-rich genomic regions to characterize polyclonal Plasmodium falciparum infections. However, methods to identify and sequence these genomic regions are currently limited. A bioinformatic pipeline and multiplex methods were developed to identify and simultaneously sequence 100 targets and applied to dried blood spot (DBS) controls and field isolates from Mozambique. For comparison, whole-genome sequencing data were generated for the same controls. Using publicly available genomes, 4465 high-diversity genomic regions suited for targeted sequencing were identified, representing the P. falciparum heterozygome. For this study, 93 microhaplotypes with high diversity (median expected heterozygosity = 0.7) were selected along with 7 drug resistance loci. The sequencing method achieved very high coverage (median 99%), specificity (99.8%), and sensitivity (90% for haplotypes with 5% within sample frequency in dried blood spots with 100 parasites/µL). In silico analyses revealed that microhaplotypes provided much higher resolution to discriminate related from unrelated polyclonal infections than biallelic single-nucleotide polymorphism barcodes. The bioinformatic and laboratory methods outlined here provide a flexible tool for efficient, low-cost, high-throughput interrogation of the P. falciparum genome, and can be tailored to simultaneously address multiple questions of interest in various epidemiological settings. A novel bioinformatics pipeline was created to select and characterize the most diverse and tractable short-range sequences (microhaplotypes) in the Plasmodium falciparum genome, followed by a robust polymerase chain reaction–based multiplexing method to simultaneously amplify and sequence these microhaplotypes.
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影响因子：
4.4
作者：
Lerch A;Koepfli C;Hofmann NE;Messerli C;Wilcox S;Kattenberg JH;Betuela I;O'Connor L;Mueller I;Felger I
通讯作者：
Felger I
DOI：
10.1093/infdis/jiy223
发表时间：
2018-08-14
期刊：
The Journal of infectious diseases
影响因子：
--
作者：
Aydemir O;Janko M;Hathaway NJ;Verity R;Mwandagalirwa MK;Tshefu AK;Tessema SK;Marsh PW;Tran A;Reimonn T;Ghani AC;Ghansah A;Juliano JJ;Greenhouse BR;Emch M;Meshnick SR;Bailey JA
通讯作者：
Bailey JA
影响因子：
1.5
作者：
Kumar, Shiva;Mudeppa, Devaraja G.;Rathod, Pradipsinh K.
通讯作者：
Rathod, Pradipsinh K.
DOI：
10.1073/pnas.1505691112
发表时间：
2015-06-02
影响因子：
11.1
作者：
Daniels, Rachel F.;Schaffner, Stephen F.;Wirth, Dyann F.
通讯作者：
Wirth, Dyann F.
影响因子：
12.3
作者：
Cerqueira GC;Cheeseman IH;Schaffner SF;Nair S;McDew-White M;Phyo AP;Ashley EA;Melnikov A;Rogov P;Birren BW;Nosten F;Anderson TJC;Neafsey DE
通讯作者：
Neafsey DE