Suitability of Illumina deep mRNA sequencing for reliable gene expression profiling in a non-model conifer species (Pseudotsuga menziesii)
Suitability of Illumina deep mRNA sequencing for reliable gene expression profiling in a non-model conifer species (Pseudotsuga menziesii)
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Illumina 深度 mRNA 测序对非模式针叶树物种（Pseudotsuga menziesii）进行可靠基因表达谱分析的适用性
DOI：
10.1007/s11295-013-0656-2
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发表时间：
2013
影响因子：
2.4
通讯作者：
Ensminger I
中科院分区：
文献类型：
--
作者：
Hess M;Wildhagen H;Ensminger I
Pseudotsuga menziesii（花旗松）是研究局部适应和转录组对环境响应的种内变异的影响的理想模型系统。尽管如此，缺乏用于基因表达谱分析的基因组资源和标准化微阵列平台一直是检验转录组组织和变异假设的限制。直到最近，深度 mRNA 测序才成为克服当前局限性的有希望的替代方案。然而，为了从 mRNA 测序数据中进行无偏差的基因表达谱分析，需要有关转录本丰度分布的信息。由于成年针叶树针叶组织无法获得此信息，因此我们推断了转录本丰度分布，并测试了测序深度对 50 年树龄花旗松针叶组织转录本可靠检测和定量的影响。我们在 mRNA 测序文库和之前发布的花旗松推定独特转录本 (PUT) 中获得了类似的 GO-slim 类别分布，这些转录本被用作比对参考。然而，花旗松文库和花旗松 PUT 中的 GO-slim 分布与从拟南芥叶组织获得的 mRNA 深度测序文库报告的 GO-slim 分布不同。显然，一些与光合作用相关蛋白质相关的高丰度 PUT 限制了增加测序深度的好处。模拟和经验数据表明，从 5 到 1500 万个对齐读取增加 3 倍，导致超过用于稳健转录本定量的 100 个对齐读取阈值的 PUT 数量大约是两倍。
Pseudotsuga menziesii(Douglas-fir) is an ideal model system to study the effect of local adaptation and intraspecific variation in transcriptome responses to the environment. Nonetheless, the lack of genomic resources and standardized microarray platforms for gene expression profiling has been a limitation to test the hypothesis on transcriptome organization and variation. Only recently, deep mRNA sequencing has become a promising alternative to overcome the present limitations. However, information on the transcript abundance distribution is needed for unbiased gene expression profiling from mRNA sequencing data. Since this information is not available for adult conifer needle tissue, we inferred the transcript abundance distribution and tested the effect of sequencing depth on the reliable detection and quantification of transcripts from the needle tissue of 50-year-old Douglas-fir trees. We obtained a similar distribution of GO-slim categories in our mRNA-sequencing libraries and in previously published putative unique transcripts (PUTs) for Douglas-fir, that were used as alignment reference. However, the GO-slim distribution in the Douglas-fir libraries and the Douglas-fir PUTs differed from the GO-slim distributions reported from mRNA deep sequencing libraries obtained fromArabidopsis thalianaleaf tissue. Apparently, several highly abundant PUTs associated with proteins involved in photosynthesis were limiting the benefits of increased sequencing depth. Simulations and empirical data indicated that a 3-fold increase from 5 to 15 million aligned reads results in about twice the number of PUTs that surpass the 100 aligned reads threshold that was used for robust transcript quantification.
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影响因子：
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