Parallel tagged sequencing on the 454 platform
Parallel tagged sequencing on the 454 platform
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DOI：
10.1038/nprot.2007.520
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Hofreiter, Michael
中科院分区：
文献类型：
--
作者：
Meyer, Matthias;Stenzel, Udo;Hofreiter, Michael
平行标记测序（PTS）是一种分子条形码化方法，旨在适应最近开发的高通量454平行测序技术，用于多个样品。与其他条形码方法不同，PTS可以应用于任何类型的双链DNA（dsDNA）样品，包括鸟枪DNA文库和PCR产物池，并且不需要扩增或凝胶纯化步骤。该方法依赖于通过连接和链置换将样品特异性条形码化衔接子（其包括序列标签和限制性位点）附接至平端修复的DNA样品。在汇集多个条形码化样品后，通过去磷酸化和限制性消化将没有序列标签的分子有效地从测序中排除，并且使用标签序列，可以追踪每个DNA序列的来源。该方案允许在单次454 GS FLX运行中对300个或更多个完整的线粒体基因组进行测序，或者仅在一个第16板区域上对25个6-kb质粒序列进行测序。大多数反应可以在96孔反应板上以多通道设置进行，允许在几天内处理多达数百个样品。
Parallel tagged sequencing (PTS) is a molecular barcoding method designed to adapt the recently developed high-throughput 454 parallel sequencing technology for use with multiple samples. Unlike other barcoding methods, PTS can be applied to any type of double-stranded DNA (dsDNA) sample, including shotgun DNA libraries and pools of PCR products, and requires no amplification or gel purification steps. The method relies on attaching sample-specific barcoding adapters, which include sequence tags and a restriction site, to blunt-end repaired DNA samples by ligation and strand-displacement. After pooling multiple barcoded samples, molecules without sequence tags are effectively excluded from sequencing by dephosphorylation and restriction digestion, and using the tag sequences, the source of each DNA sequence can be traced. This protocol allows for sequencing 300 or more complete mitochondrial genomes on a single 454 GS FLX run, or twenty-five 6-kb plasmid sequences on only one 16th plate region. Most of the reactions can be performed in a multichannel setup on 96-well reaction plates, allowing for processing up to several hundreds of samples in a few days.