SMRT Sequencing for Parallel Analysis of Multiple Targets and Accurate SNP Phasing.
SMRT Sequencing for Parallel Analysis of Multiple Targets and Accurate SNP Phasing.
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DOI：
10.1534/g3.115.023317
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发表时间：
2015-10-23
期刊：
影响因子：
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通讯作者：
Jinks-Robertson S
中科院分区：
文献类型：
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作者：
Guo X;Lehner K;O'Connell K;Zhang J;Dave SS;Jinks-Robertson S
单分子实时（SMRT）测序产生的读数比其他广泛使用的下一代（下一代）测序方法长得多，但其在全基因组/外显子组分析中的应用受到限制。在这里，我们描述了SMRT测序与条形码结合的使用，以同时分析一个或少量来自多个来源的基因组靶标。在芽殖酵母系统中，SMRT测序用于分析有丝分裂重组过程中产生的链交换中间体，并分析正向突变试验中的遗传变化。然后将一般的条形码-SMRT方法扩展到弥漫性大B细胞淋巴瘤原发性肿瘤和细胞系，其中检测到的变化与先前的Illumina外显子组测序一致。与其他下一代方法相比，SMRT测序提供的一个明显优势是，它立即提供了测序的靶片段中SNP之间的连锁关系。我们的突变/重组研究（以及连锁识别）方法的优势来自其固有的计算简单性，加上缺乏对复杂的统计分析的依赖。
Single-molecule real-time (SMRT) sequencing generates much longer reads than other widely used next-generation (next-gen) sequencing methods, but its application to whole genome/exome analysis has been limited. Here, we describe the use of SMRT sequencing coupled with barcoding to simultaneously analyze one or a small number of genomic targets derived from multiple sources. In the budding yeast system, SMRT sequencing was used to analyze strand-exchange intermediates generated during mitotic recombination and to analyze genetic changes in a forward mutation assay. The general barcoding-SMRT approach was then extended to diffuse large B-cell lymphoma primary tumors and cell lines, where detected changes agreed with prior Illumina exome sequencing. A distinct advantage afforded by SMRT sequencing over other next-gen methods is that it immediately provides the linkage relationships between SNPs in the target segment sequenced. The strength of our approach for mutation/recombination studies (as well as linkage identification) derives from its inherent computational simplicity coupled with a lack of reliance on sophisticated statistical analyses.