Sequencing-based variant detection in the polyploid crop oilseed rape.
Sequencing-based variant detection in the polyploid crop oilseed rape.
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DOI：
10.1186/1471-2229-13-111
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发表时间：
2013-08-06
影响因子：
5.3
通讯作者：
Bancroft I
中科院分区：
文献类型：
--
作者：
Wells R;Trick M;Fraser F;Soumpourou E;Clissold L;Morgan C;Pauquet J;Bancroft I
遗传变异的检测和利用是作物改良的基础。然而，我们许多最重要的作物基因组的多倍体性质是一个障碍，特别是对基因内变异的分析。为了克服这一点，我们的目标是开发基于扩增子测序的方法，该方法涉及将条形码扩增标签（BAT）掺入PCR产物中。开发了一种方案，以在合并之前用5' 6-碱基寡核苷酸条形码延伸标记PCR产物，用于使用标准Illumina衔接子进行测序文库产生。开发了一种计算方法，用于产物的去卷积以及序列变体的稳健检测和评分。使用该方法，在B中筛选靶向基因序列的扩增子。napus作图群体和所得到的24个标记的等位基因评分串连锁映射到基因组的预期区域。此外，使用一维8倍合并，4608行a B。对10个甘蓝型油菜突变群体进行了GL 2.b直向同源基因和FAD 2直向同源基因共扩增产物的诱导突变筛选，分别鉴定出10个和41个突变体。在多重变异筛选中，利用条形码标签通过Illumina测序对合并的PCR产物进行去卷积，提供了一种用于SNP基因分型和突变检测的成本有效的方法，并且潜在地提供了用于致病变化的标记物，甚至在多倍体物种中。将这种方法与现有的Illumina多路复用工作流程相结合，可以在单次测序运行中以最低的文库生产成本廉价有效地分析数千条线。
The detection and exploitation of genetic variation underpins crop improvement. However, the polyploid nature of the genomes of many of our most important crops represents a barrier, particularly for the analysis of variation within genes. To overcome this, we aimed to develop methodologies based on amplicon sequencing that involve the incorporation of barcoded amplification tags (BATs) into PCR products. A protocol was developed to tag PCR products with 5’ 6-base oligonucleotide barcode extensions before pooling for sequencing library production using standard Illumina adapters. A computational method was developed for the de-convolution of products and the robust detection and scoring of sequence variants. Using this methodology, amplicons targeted to gene sequences were screened across a B. napus mapping population and the resulting allele scoring strings for 24 markers linkage mapped to the expected regions of the genome. Furthermore, using one-dimensional 8-fold pooling, 4608 lines of a B. napus mutation population were screened for induced mutations in a locus-specific amplicon (an orthologue of GL2.b) and mixed product of three co-amplified loci (orthologues of FAD2), identifying 10 and 41 mutants respectively. The utilisation of barcode tags to de-convolute pooled PCR products in multiplexed, variation screening via Illumina sequencing provides a cost effective method for SNP genotyping and mutation detection and, potentially, markers for causative changes, even in polyploid species. Combining this approach with existing Illumina multiplexing workflows allows the analysis of thousands of lines cheaply and efficiently in a single sequencing run with minimal library production costs.
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影响因子：
46.9
作者：
Loman, Nicholas J.;Misra, Raju V.;Pallen, Mark J.
通讯作者：
Pallen, Mark J.
影响因子：
14.9
作者：
Oleykowski, CA;Mullins, CRB;Yeung, AT
通讯作者：
Yeung, AT
影响因子：
5.4
作者：
Yang, Qingyong;Fan, Chuchuan;Zhou, Yongming
通讯作者：
Zhou, Yongming
影响因子：
7
作者：
Li, Heng;Ruan, Jue;Durbin, Richard
通讯作者：
Durbin, Richard
甘蓝型油菜的功能基因组学资源：EMS 诱变群体的开发以及 TILLING 发现的 FAE1 点突变。
影响因子：
9.4
作者：
Wang, Nian;Wang, Yajie;Meng, Jinling
通讯作者：
Meng, Jinling