Validation of two ribosomal RNA removal methods for microbial metatranscriptomics
Validation of two ribosomal RNA removal methods for microbial metatranscriptomics
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DOI：
10.1038/nmeth.1507
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发表时间：
2010-10-01
期刊：
影响因子：
48
通讯作者：
Hugenholtz, Philip
中科院分区：
文献类型：
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作者：
He, Shaomei;Wurtzel, Omri;Hugenholtz, Philip
rRNA在转录组中的优势是微生物分离株和群落cDNA基于序列分析的主要技术挑战。已经应用了几种方法来从（Meta）转录组中耗尽rRNA，但没有系统的研究这些方法中的任何一种引入的潜在偏倚。在这里，我们验证了两个最常用的方法，消减杂交和核酸外切酶消化，以及这些治疗的组合，在两个合成的五个微生物metatranscriptomes使用大规模平行测序的有效性和保真度。我们发现，去除rRNA的有效性是这些治疗的社区组成和RNA完整性的函数。单独的消减杂交引入了相对转录丰度的最小偏差，而核酸外切酶，特别是组合处理大大损害了mRNA丰度的保真度。Illumina测序本身也可以通过在运行之间引入G+C偏倚来损害定量数据分析。
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.