cDNA Library Enrichment of Full Length Transcripts for SMRT Long Read Sequencing.
cDNA Library Enrichment of Full Length Transcripts for SMRT Long Read Sequencing.
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DOI：
10.1371/journal.pone.0157779
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Schneeberger K
中科院分区：
文献类型：
--
作者：
Cartolano M;Huettel B;Hartwig B;Reinhardt R;Schneeberger K
基因组组装的实用性不仅取决于组装基因组序列的质量，还取决于基因注释的质量。Pacific Biosciences的Iso-Seq技术是对真核生物基因模型准确注释的有力支持，因为它允许直接读出全长cDNA序列，而不需要嘈杂的基于短读的转录物组装。我们建议将telprime全长cDNA扩增试剂盒应用于Pacific Biosciences的Iso-Seq技术，以丰富cDNA文库中真正的全长转录本。我们提供的证据表明，在鉴定拟南芥转录起始和结束位点方面，TeloPrime优于常用的SMARTer PCR cDNA Synthesis Kit。此外，我们发现基于teloprime的Pacific Biosciences Iso-Seq可以成功地应用于面包小麦（Triticum aestivum）的多倍体基因组，不仅可以有效地对基因模型进行标注，还可以识别新的转录位点、基因同源物、剪接异构体和以前未识别的基因位点。
The utility of genome assemblies does not only rely on the quality of the assembled genome sequence, but also on the quality of the gene annotations. The Pacific Biosciences Iso-Seq technology is a powerful support for accurate eukaryotic gene model annotation as it allows for direct readout of full-length cDNA sequences without the need for noisy short read-based transcript assembly. We propose the implementation of the TeloPrime Full Length cDNA Amplification kit to the Pacific Biosciences Iso-Seq technology in order to enrich for genuine full-length transcripts in the cDNA libraries. We provide evidence that TeloPrime outperforms the commonly used SMARTer PCR cDNA Synthesis Kit in identifying transcription start and end sites in Arabidopsis thaliana. Furthermore, we show that TeloPrime-based Pacific Biosciences Iso-Seq can be successfully applied to the polyploid genome of bread wheat (Triticum aestivum) not only to efficiently annotate gene models, but also to identify novel transcription sites, gene homeologs, splicing isoforms and previously unidentified gene loci.