Genotyping-in-Thousands by sequencing (GT-seq): A cost effective SNP genotyping method based on custom amplicon sequencing
Genotyping-in-Thousands by sequencing (GT-seq): A cost effective SNP genotyping method based on custom amplicon sequencing
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DOI：
10.1111/1755-0998.12357
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发表时间：
2015-07-01
影响因子：
7.7
通讯作者：
Narum, Shawn R.
中科院分区：
文献类型：
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作者：
Campbell, Nathan R.;Harmon, Stephanie A.;Narum, Shawn R.
千人测序基因分型(GT-SEQ)是一种方法，它使用多路聚合酶链式反应产物的下一代测序，从相对较小的靶向单核苷酸多态(SNPs)小组(50-500个)中产生基因型，这些SNPs用于单个Illumina HiSeq车道中的数千个个体。该方法在两个热循环步骤中只使用未标记的寡聚和聚合酶链式反应母体混合物来扩增靶向SNP基因座。在这个过程中，测序适配器和双条形码序列标签被整合到扩增片段中，从而使数千个个体被汇集到一个测序文库中。测序后，使用其独特的条形码序列组合，将来自单个样本的读数分割成单独的文件。基因分型是用一个简单的Perl脚本执行的，该脚本计算每个等位基因的扩增特定序列，并使用等位基因比率来确定基因类型。我们通过对一个文库中的一组192个SNP标记的2068个钢头鲑鱼样本进行基因分型来演示这项技术，这些标记在一个Illumina HiSeq车道上进行了测序。在相同的192个基因座上，GT-seq与先前收集的TaqMan基因型的符合率为99.9%，但GT-seq的呼叫率(96.4%)略低于Taqman(99.0%)。在192个SNPs中，187个在90%的个体样本中被分型，只有3个SNPs在
Genotyping-in-Thousands by sequencing (GT-seq) is a method that uses next-generation sequencing of multiplexed PCR products to generate genotypes from relatively small panels (50-500) of targeted single-nucleotide polymorphisms (SNPs) for thousands of individuals in a single Illumina HiSeq lane. This method uses only unlabelled oligos and PCR master mix in two thermal cycling steps for amplification of targeted SNP loci. During this process, sequencing adapters and dual barcode sequence tags are incorporated into the amplicons enabling thousands of individuals to be pooled into a single sequencing library. Post sequencing, reads from individual samples are split into individual files using their unique combination of barcode sequences. Genotyping is performed with a simple perl script which counts amplicon-specific sequences for each allele, and allele ratios are used to determine the genotypes. We demonstrate this technique by genotyping 2068 individual steelhead trout (Oncorhynchus mykiss) samples with a set of 192 SNP markers in a single library sequenced in a single Illumina HiSeq lane. Genotype data were 99.9% concordant to previously collected TaqMan genotypes at the same 192 loci, but call rates were slightly lower with GT-seq (96.4%) relative to Taqman (99.0%). Of the 192 SNPs, 187 were genotyped in 90% of the individual samples and only 3 SNPs were genotyped in