Full-length novel MHC class I allele discovery by next-generation sequencing: two platforms are better than one.
Full-length novel MHC class I allele discovery by next-generation sequencing: two platforms are better than one.
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DOI：
10.1007/s00251-013-0744-3
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发表时间：
2014-01
期刊：
影响因子：
3.2
通讯作者：
O'Connor, David H.
中科院分区：
文献类型：
--
作者：
Dudley, Dawn M.;Karl, Julie A.;Creager, Hannah M.;Bohn, Patrick S.;Wiseman, Roger W.;O'Connor, David H.
关键词：
深度测序通过实现高通量、经济和全面的基因分型，彻底改变了非人灵长类动物的主要组织相容性复合体（MHC）I类分析。产生试剂如MHC：肽四聚体所需的全长MHC I类cDNA序列不能通过短读深测序直接获得。我们结合了两个新一代测序平台的数据，在中国恒河猴中发现了新的全长MHC I类mRNA/cDNA转录本。我们首先通过Roche/454焦磷酸测序使用跨越编码肽结合区的MHC I类基因座的密集多态性外显子2至4的530 bp扩增子对猕猴进行基因分型。然后，我们以高严格性将跨越全长转录物的短配对末端250 bp Illumina序列读段映射到每个530 bp扩增子，并使用配对末端信息重建全长等位基因序列。我们从6个中国恒河猴的65个全长序列的特征。总体而言，在这6只动物中区分的等位基因中约有70%含有新的序列信息，包括29种新的转录本。这种方法的灵活性应该使全长MHC I类等位基因基因分型可用于任何感兴趣的非人灵长类动物群体。我们目前正在优化这种方法的全长表征其他高度多态性，重复位点，如MHC II类DRB和杀手免疫球蛋白样受体。我们预计，这种方法将有助于快速扩展和接近完成的多态位点，如MHC I类序列库，在几年内。
Deep sequencing has revolutionized major histocompatibility complex (MHC) class I analysis of nonhuman primates by enabling high-throughput, economical, and comprehensive genotyping. Full-length MHC class I cDNA sequences, which are required to generate reagents such as MHC:peptide tetramers, cannot be directly obtained by short read deep sequencing. We combined data from two next-generation sequencing platforms to discover novel full-length MHC class I mRNA/cDNA transcripts in Chinese rhesus macaques. We first genotyped macaques by Roche/454 pyrosequencing using a 530 bp amplicon spanning the densely polymorphic exons 2 through 4 of the MHC class I loci that encode the peptide-binding region. We then mapped short paired-end 250 bp Illumina sequence reads spanning the full-length transcript to each 530 bp amplicon at high stringency and used paired-end information to reconstruct full-length allele sequences. We characterized 65 full-length sequences from 6 Chinese rhesus macaques. Overall, approximately 70% of the alleles distinguished in these 6 animals contained new sequence information, including 29 novel transcripts. The flexibility of this approach should make full-length MHC class I allele genotyping accessible for any nonhuman primate population of interest. We are currently optimizing this method for full-length characterization of other highly polymorphic, duplicated loci such as the MHC class II DRB and killer immunoglobulin-like receptors. We anticipate that this method will facilitate rapid expansion and near completion of sequence libraries of polymorphic loci, such as MHC class I, within a few years.
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影响因子：
14.9
作者：
Robinson J;Halliwell JA;McWilliam H;Lopez R;Marsh SG
通讯作者：
Marsh SG
影响因子：
3.7
作者：
Chen S;Lai C;Wu X;Lu Y;Han D;Guo W;Fu L;Andrieu JM;Lu W
通讯作者：
Lu W
DOI：
10.1534/g3.113.006254
发表时间：
2013-07-08
期刊：
G3 (Bethesda, Md.)
影响因子：
--
作者：
Karl JA;Bohn PS;Wiseman RW;Nimityongskul FA;Lank SM;Starrett GJ;O'Connor DH
通讯作者：
O'Connor DH
影响因子：
3.2
作者：
de Groot, Natasja G.;Otting, Nel;Robinson, James;Blancher, Antoine;Lafont, Bernard A. P.;Marsh, Steven G. E.;O'Connor, David H.;Shiina, Takashi;Walter, Lutz;Watkins, David I.;Bontrop, Ronald E.
通讯作者：
Bontrop, Ronald E.
影响因子：
2.6
作者：
Stocchi, Laura;Cascella, Raffaella;Giardina, Emiliano
通讯作者：
Giardina, Emiliano