The Wellcome Trust Sanger Institute, Cambridge, UK.
Nat Protoc. 2011 Oct 20;6(11):1736-47. doi: 10.1038/nprot.2011.399.
The unifying feature of second-generation sequencing technologies is that single template strands are amplified clonally onto a solid surface prior to the sequencing reaction. To convert template strands into a compatible state for attachment to this surface, a multistep library preparation is required, which typically culminates in amplification by the PCR. PCR is an inherently biased process, which decreases the efficiency of data acquisition. Flowcell reverse transcription sequencing is a method of transcriptome sequencing for Illumina sequencers in which the reverse transcription reaction is performed on the flowcell by using unamplified, adapter-ligated mRNA as a template. This approach removes PCR biases and duplicates, generates strand-specific paired-end data and is highly reproducible. The procedure can be performed quickly, taking 2 d to generate clusters from mRNA.
第二代测序技术的统一特点是，在测序反应之前，将单个模板链克隆扩增到固体表面上。为了将模板链转换为适合附着在该表面的状态，需要进行多步文库制备，通常最终通过 PCR 进行扩增。PCR 是一个固有偏倚的过程，降低了数据获取的效率。Flowcell 反转录测序是一种用于 Illumina 测序仪的转录组测序方法，其中反转录反应在 Flowcell 上进行，使用未扩增的、连接了接头的 mRNA 作为模板。这种方法消除了 PCR 偏倚和重复，生成了链特异性的配对末端数据，并且高度可重复。该过程可以快速进行，从 mRNA 生成簇只需 2 天。
