New methods for next generation sequencing based microRNA expression profiling.
New methods for next generation sequencing based microRNA expression profiling.
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DOI：
10.1186/1471-2164-11-716
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发表时间：
2010-12-20
期刊：
影响因子：
4.4
通讯作者：
't Hoen PA
中科院分区：
文献类型：
--
作者：
Buermans HP;Ariyurek Y;van Ommen G;den Dunnen JT;'t Hoen PA
MicroRNA是一种小的非编码RNA转录物，调节转录后基因的表达。下一代测序技术产生的数百万个短序列读段使该技术明确适用于已知和新型microRNA的分析。描述了对用于SOLiD测序平台的小RNA表达试剂盒（SREK，Ambion）文库制备方法的修改，以生成与Illumina基因组分析仪兼容的microRNA测序文库。高质量的测序文库可以成功地从低至100 ng的小RNA富集RNA制备。开发了一种易于使用的基于Perl的分析管道，称为E-miR，以在几个自动化步骤中处理测序数据，包括数据格式转换、3'衔接子去除、基因组比对和非编码RNA转录物的注释。使用样品制备和E-miR管道来鉴定16期鸡胚胎中的37个心脏富集的microRNA。心脏和胚胎之间的Isomir表达谱与所有miRNA高度相关，表明组织或细胞特异性miRNA修饰不会发生。总之，我们的替代样品制备方法可以成功地应用于为Illumina基因组分析仪生成高质量的miRNA测序文库。
MicroRNAs are small non-coding RNA transcripts that regulate post-transcriptional gene expression. The millions of short sequence reads generated by next generation sequencing technologies make this technique explicitly suitable for profiling of known and novel microRNAs. A modification to the small-RNA expression kit (SREK, Ambion) library preparation method for the SOLiD sequencing platform is described to generate microRNA sequencing libraries that are compatible with the Illumina Genome Analyzer. High quality sequencing libraries can successfully be prepared from as little as 100 ng small RNA enriched RNA. An easy to use perl-based analysis pipeline called E-miR was developed to handle the sequencing data in several automated steps including data format conversion, 3' adapter removal, genome alignment and annotation to non-coding RNA transcripts. The sample preparation and E-miR pipeline were used to identify 37 cardiac enriched microRNAs in stage 16 chicken embryos. Isomir expression profiles between the heart and embryo were highly correlated for all miRNAs suggesting that tissue or cell specific miRNA modifications do not occur. In conclusion, our alternative sample preparation method can successfully be applied to generate high quality miRNA sequencing libraries for the Illumina genome analyzer.
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