Sequencing bias: comparison of different protocols of microRNA library construction.
Sequencing bias: comparison of different protocols of microRNA library construction.
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DOI：
10.1186/1472-6750-10-64
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发表时间：
2010-09-06
影响因子：
3.5
通讯作者：
Li N
中科院分区：
文献类型：
--
作者：
Tian G;Yin X;Luo H;Xu X;Bolund L;Zhang X;Gan SQ;Li N
microRNAs（miRNAs）是一类长度为18-25 nt的小分子RNA，在许多生物学过程中起着重要作用。大多数已知的miRNA都是通过常规克隆和桑格测序方法发现的。下一代测序（NGS）技术使得能够深入表征miRNA的全局库，并且已经开发了用于miRNA文库构建的不同方案。然而，相对表达水平和不同文库制备方案引入的序列之间可能的偏差很少被探索。我们评估了三种不同的miRNA文库制备方案，SOLiD，Illumina版本1和1.5，使用从湖羊和杜泊羊的骨骼肌提取的总RNA样品的克隆或SBS测序，然后通过qRT-PCR验证9种miRNA。我们的结果表明SBS测序数据与Illumina克隆数据高度相关。当与Illumina的数据相比时，SOLiD数据表明长度的分布更分散，靠近3 '和5'端的核苷酸的频率变化更高，含有末端二级结构（ESS）的读段的出现频率更高，并且不映射到已知miRNA的读段的频率更高。qRT-PCR结果显示与SOLiD克隆数据的最佳相关性。湖羊和杜泊羊的qRT-PCR结果与SBS测序结果的倍数差异相关性较好（r = 0.937），而miR-1和miR-206的SOLiD克隆数据与qRT-PCR和SBS测序数据的倍数差异相似。测序深度可以影响miRNA丰度的定量测量，但由于在Illumina克隆和SBS测序数据之间观察到高度相关性，因此由测序深度引起的差异在统计学上不显著。在不同方案获得的数据之间观察到长度分布、序列变异和ESS的偏倚。SOLiD克隆数据与Illumina克隆数据不同，主要是因为衔接子连接的方法不同。qRT-PCR结果与SOLiD数据之间的良好相关性可能是由于基于杂交的方法的相似性。倍数差异分析表明，基于杂交的方法对于定量测量miRNA丰度可能是上级的。由于绵羊的基因组序列不可用，我们的数据可能无法解释整个miRNA在绵羊或其他哺乳动物的天然miRNA表达中的偏倚，无偏倚的人工合成miRNA将有助于评估miRNA文库制备的方法学。
MicroRNAs(miRNAs) are 18-25 nt small RNAs playing critical roles in many biological processes. The majority of known miRNAs were discovered by conventional cloning and a Sanger sequencing approach. The next-generation sequencing (NGS) technologies enable in-depth characterization of the global repertoire of miRNAs, and different protocols for miRNA library construction have been developed. However, the possible bias between the relative expression levels and sequences introduced by different protocols of library preparation have rarely been explored. We assessed three different miRNA library preparation protocols, SOLiD, Illumina versions 1 and 1.5, using cloning or SBS sequencing of total RNA samples extracted from skeletal muscles from Hu sheep and Dorper sheep, and then validated 9 miRNAs by qRT-PCR. Our results show that SBS sequencing data highly correlate with Illumina cloning data. The SOLiD data, when compared to Illumina's, indicate more dispersed distribution of length, higher frequency variation for nucleotides near the 3'- and 5'-ends, higher frequency occurrence for reads containing end secondary structure (ESS), and higher frequency for reads that do not map to known miRNAs. qRT-PCR results showed the best correlation with SOLiD cloning data. Fold difference of Hu sheep and Dorper sheep between qRT-PCR result and SBS sequencing data correlated well (r = 0.937), and fold difference of miR-1 and miR-206 among SOLiD cloning data, qRT-PCR and SBS sequencing data was similar. The sequencing depth can influence the quantitative measurement of miRNA abundance, but the discrepancy caused by it was not statistically significant as high correlation was observed between Illumina cloning and SBS sequencing data. Bias of length distribution, sequence variation, and ESS was observed between data obtained with the different protocols. SOLiD cloning data differ from Illumina cloning data mainly because of distinct methods of adapter ligation. The good correlation between qRT-PCR result and SOLiD data might be due to the similarities of the hybridization-based methods. The fold difference analysis indicated that methods based on hybridization may be superior for quantitative measurement of miRNA abundance. Because of the genome sequence of the sheep is not available, our data may not explain how the entire miRNA bias in the natural miRNAs in sheep or other mammal miRNA expression, unbiased artificially synthesized miRNA will help on evaluating the methodology of miRNA library preparation.
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影响因子：
14.9
作者：
Chen C;Ridzon DA;Broomer AJ;Zhou Z;Lee DH;Nguyen JT;Barbisin M;Xu NL;Mahuvakar VR;Andersen MR;Lao KQ;Livak KJ;Guegler KJ
通讯作者：
Guegler KJ
DOI：
10.3324/haematol.12706
发表时间：
2008-07-01
期刊：
HAEMATOLOGICA-THE HEMATOLOGY JOURNAL
影响因子：
--
作者：
Bruchova, Hana;Merkerova, Michaela;Prchal, Josef T.
通讯作者：
Prchal, Josef T.
影响因子：
14.9
作者：
Griffiths-Jones S;Grocock RJ;van Dongen S;Bateman A;Enright AJ
通讯作者：
Enright AJ
影响因子：
4.8
作者：
Lu, Cheng;Meyers, Blake C.;Green, Pamela J.
通讯作者：
Green, Pamela J.
影响因子：
5.2
作者：
Bar, Merav;Wyman, Stacia K.;Tewari, Muneesh
通讯作者：
Tewari, Muneesh