GENCODE Batch VIII a: determining lncRNA transcript 5' and 3'-ends by RACE-PCR / 454 sequencing. GENCODE Batch VIII a: determining lncRNA transcript 5' and 3'-ends by RACE-PCR / 454 sequencing

As part of the ENCODE consortium the GENCODE project is producing a reference gene set through manual and automated gene prediction. In the current phase of ENCODE we have found strong evidence that many lncRNAs transcript termini are still unknown. This experiment aims to set up an experimental validation strategy to accurately determine the 5' and 3' ends of transcripts, which is based on semi-nested RACE extensions of annotated 5' and 3' ends followed by high throughput sequencing. A total of 400 highly expressed lncRNA transcript models from Gencode 7 which did not have any CAGE/PET support were selected as the test set whereas 25 transcripts with transcript start site (TSS) supported by CAGE tags and transcript termination site (TTS) supported by PET ditags formed the positive control set. Transcript ends were amplified by RACE-PCR from brain and testis RNA samples and sequenced using the Roche 454 platform. The sequencing was performed at the Andalusian Human Genome Sequencing Centre (CASEGH), Seville, Spain.

作为ENCODE（DNA元件百科全书，Encyclopedia of DNA Elements）联盟的组成部分，GENCODE项目正通过手动与自动化基因预测手段构建参考基因集。在当前ENCODE项目阶段，我们已获取充分证据，表明众多长链非编码RNA（long non-coding RNA，lncRNA）的转录本末端仍未被探明。本实验旨在建立一套实验验证策略，以精准测定转录本的5'与3'末端：该策略以对已注释的5'、3'末端进行半嵌套式快速扩增cDNA末端（rapid amplification of cDNA ends，RACE）延伸为基础，随后开展高通量测序。我们从GENCODE 7版本中筛选出400个高表达的长链非编码RNA转录本模型作为测试集，这些模型均未获得帽式基因表达分析（cap analysis of gene expression，CAGE）标签或配对末端双标签（paired-end ditags，PET）的支持；另有25个转录本，其转录起始位点（transcript start site，TSS）得到CAGE标签支持、转录终止位点（transcript termination site，TTS）得到PET双标签支持，作为阳性对照集。研究人员从大脑与睾丸的RNA样本中，通过RACE-PCR扩增转录本末端，并使用罗氏454测序平台完成测序。测序工作由西班牙塞维利亚的安达卢西亚人类基因组测序中心（CASEGH）完成。