A new hybrid approach for MHC genotyping: high-throughput NGS and long read MinION nanopore sequencing, with application to the non-model vertebrate Alpine chamois (Rupicapra rupicapra).
A new hybrid approach for MHC genotyping: high-throughput NGS and long read MinION nanopore sequencing, with application to the non-model vertebrate Alpine chamois (Rupicapra rupicapra).
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DOI：
10.1038/s41437-018-0070-5
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发表时间：
2018-10
期刊：
影响因子：
3.8
通讯作者：
Bertorelle G
中科院分区：
文献类型：
--
作者：
Fuselli S;Baptista RP;Panziera A;Magi A;Guglielmi S;Tonin R;Benazzo A;Bauzer LG;Mazzoni CJ;Bertorelle G
主要组织相容性复合体 (MHC) 充当免疫系统和传染病之间的界面。对极其可变的 MHC 位点进行准确表征和基因分型具有挑战性，尤其是在没有参考序列的情况下。我们设计了长程 PCR、Illumina 短读长和 Oxford Nanopore MinION 长读长方法的组合，以捕获意大利高山羚羊 (Rupicapra rupicapra) 种群中 MHC II DRB 基因座的遗传变异。我们利用长程 PCR 生成 DRB 基因座的 9 Kb 片段。来自六个不同个体的扩增子被片段化、标记，并同时使用 Illumina MiSeq 进行测序。使用 MinION 设备对其中一个扩增子进行测序，该设备产生覆盖整个扩增片段的长读数。结合短读长和长读长的管道解析了几个短串联重复序列和同聚物，并产生了从头参考，然后将其用于对所有个体的短读长进行定位和基因分型。组装的 DRB 基因座显示出高水平的多态性和重组断点的存在。我们的结果表明，基于扩增子的 NGS 方法与单分子 MinION 纳米孔测序相结合，可以在没有参考序列的情况下有效地实现多个个体中复杂基因组区域的组装和基因分型。
The major histocompatibility complex (MHC) acts as an interface between the immune system and infectious diseases. Accurate characterization and genotyping of the extremely variable MHC loci are challenging especially without a reference sequence. We designed a combination of long-range PCR, Illumina short-reads, and Oxford Nanopore MinION long-reads approaches to capture the genetic variation of the MHC II DRB locus in an Italian population of the Alpine chamois (Rupicapra rupicapra). We utilized long-range PCR to generate a 9 Kb fragment of the DRB locus. Amplicons from six different individuals were fragmented, tagged, and simultaneously sequenced with Illumina MiSeq. One of these amplicons was sequenced with the MinION device, which produced long reads covering the entire amplified fragment. A pipeline that combines short and long reads resolved several short tandem repeats and homopolymers and produced a de novo reference, which was then used to map and genotype the short reads from all individuals. The assembled DRB locus showed a high level of polymorphism and the presence of a recombination breakpoint. Our results suggest that an amplicon-based NGS approach coupled with single-molecule MinION nanopore sequencing can efficiently achieve both the assembly and the genotyping of complex genomic regions in multiple individuals in the absence of a reference sequence.
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影响因子：
10.7
作者：
Froeschke, G;Sommer, S
通讯作者：
Sommer, S
影响因子：
7.7
作者：
Biedrzycka, Aleksandra;Sebastian, Alvaro;Radwan, Jacek
通讯作者：
Radwan, Jacek
DOI：
10.1093/bioinformatics/bts199
发表时间：
2012-06-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Kearse M;Moir R;Wilson A;Stones-Havas S;Cheung M;Sturrock S;Buxton S;Cooper A;Markowitz S;Duran C;Thierer T;Ashton B;Meintjes P;Drummond A
通讯作者：
Drummond A
影响因子：
--
作者：
Jain M;Tyson JR;Loose M;Ip CLC;Eccles DA;O'Grady J;Malla S;Leggett RM;Wallerman O;Jansen HJ;Zalunin V;Birney E;Brown BL;Snutch TP;Olsen HE;MinION Analysis and Reference Consortium
通讯作者：
MinION Analysis and Reference Consortium
影响因子：
38.3
作者：
Clarke, James;Wu, Hai-Chen;Bayley, Hagan
通讯作者：
Bayley, Hagan