抑制消减杂交
生物
计算生物学
大规模并行测序
核糖体RNA
转录组
核糖核酸
基因组
遗传学
DNA测序
基因
互补DNA
基因表达
cDNA文库
作者
Shaomei He,Omri Wurtzel,Kanwar Pal Singh,Jeff Froula,Suzan Yilmaz,Susannah G. Tringe,Zhong Wang,Feng Chen,Erika Lindquist,Rotem Sorek,Philip Hugenholtz
出处
期刊:Nature Methods
[Springer Nature]
日期:2010-09-19
卷期号:7 (10): 807-812
被引量:195
摘要
Compared in this Analysis are two widely used procedures for ribosomal RNA removal in metatranscriptomic samples, and the authors present recommendations to prevent misleading analyses of microbial communities. The predominance of rRNAs in the transcriptome is a major technical challenge in sequence-based analysis of cDNAs from microbial isolates and communities. Several approaches have been applied to deplete rRNAs from (meta)transcriptomes, but no systematic investigation of potential biases introduced by any of these approaches has been reported. Here we validated the effectiveness and fidelity of the two most commonly used approaches, subtractive hybridization and exonuclease digestion, as well as combinations of these treatments, on two synthetic five-microorganism metatranscriptomes using massively parallel sequencing. We found that the effectiveness of rRNA removal was a function of community composition and RNA integrity for these treatments. Subtractive hybridization alone introduced the least bias in relative transcript abundance, whereas exonuclease and in particular combined treatments greatly compromised mRNA abundance fidelity. Illumina sequencing itself also can compromise quantitative data analysis by introducing a G+C bias between runs.
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