一种用于测序低输入翻译组样本的文库制备方法的比较分析。
A comparative analysis of library prep approaches for sequencing low input translatome samples.
机构信息
Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.
Department of Otorhinolaryngology-Head & Neck Surgery, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.
出版信息
BMC Genomics. 2018 Sep 21;19(1):696. doi: 10.1186/s12864-018-5066-2.
BACKGROUND
Cell type-specific ribosome-pulldown has become an increasingly popular method for analysis of gene expression. It allows for expression analysis from intact tissues and monitoring of protein synthesis in vivo. However, while its utility has been assessed, technical aspects related to sequencing of these samples, often starting with a smaller amount of RNA, have not been reported. In this study, we evaluated the performance of five library prep protocols for ribosome-associated mRNAs when only 250 pg-4 ng of total RNA are used.
RESULTS
We obtained total and RiboTag-IP RNA, in three biological replicates. We compared 5 methods of library preparation for Illumina Next Generation sequencing: NuGEN Ovation RNA-Seq system V2 Kit, TaKaRa SMARTer Stranded Total RNA-Seq Kit, TaKaRa SMART-Seq v4 Ultra Low Input RNA Kit, Illumina TruSeq RNA Library Prep Kit v2 and NEBNext® Ultra™ Directional RNA Library Prep Kit using slightly modified protocols each with 4 ng of total RNA. An additional set of samples was processed using the TruSeq kit with 70 ng, as a 'gold standard' control and the SMART-Seq v4 with 250 pg of total RNA. TruSeq-processed samples had the best metrics overall, with similar results for the 4 ng and 70 ng samples. The results of the SMART-Seq v4 processed samples were similar to TruSeq (Spearman correlation > 0.8) despite using lower amount of input RNA. All RiboTag-IP samples had an increase in the intronic reads compared with the corresponding whole tissue, suggesting that the IP captures some immature mRNAs. The SMARTer-processed samples had a higher representation of ribosomal and non-coding RNAs leading to lower representation of protein coding mRNA. The enrichment or depletion of IP samples compared to corresponding input RNA was similar across all kits except for SMARTer kit.
CONCLUSION
RiboTag-seq can be performed successfully with as little as 250 pg of total RNA when using the SMART-Seq v4 kit and 4 ng when using the modified protocols of other library preparation kits. The SMART-Seq v4 and TruSeq kits resulted in the highest quality libraries. RiboTag IP RNA contains some immature transcripts.
背景
细胞特异性核糖体下拉技术已成为分析基因表达的一种越来越流行的方法。它允许从完整的组织中进行表达分析，并监测体内的蛋白质合成。然而，尽管已经评估了其效用，但与这些样品测序相关的技术方面，通常从较少量的 RNA 开始，尚未得到报道。在这项研究中，我们评估了在仅使用 250pg-4ng 总 RNA 的情况下，五种核糖体相关 mRNA 文库制备方案的性能。
结果
我们获得了三个生物学重复的总 RNA 和 RiboTag-IP RNA。我们比较了 5 种用于 Illumina 下一代测序的文库制备方法：NuGEN Ovation RNA-Seq 系统 V2 试剂盒、TaKaRa SMARTer Stranded Total RNA-Seq 试剂盒、TaKaRa SMART-Seq v4 Ultra Low Input RNA 试剂盒、Illumina TruSeq RNA 文库制备试剂盒 v2 和 NEBNext® Ultra™ Directional RNA 文库制备试剂盒，每种方法均使用略有修改的方案，每个方案使用 4ng 总 RNA。一组额外的样品使用 TruSeq 试剂盒处理，用 70ng 作为“金标准”对照，用 250pg 总 RNA 处理 SMART-Seq v4。TruSeq 处理的样品总体上具有最佳的指标，4ng 和 70ng 样品的结果相似。尽管使用的输入 RNA 量较低，但 SMART-Seq v4 处理的样品的结果与 TruSeq 相似（Spearman 相关性>0.8）。所有 RiboTag-IP 样品与相应的整个组织相比，内含子读数增加，表明 IP 捕获了一些不成熟的 mRNA。SMARTer 处理的样品中核糖体和非编码 RNA 的比例较高，导致编码蛋白 mRNA 的比例较低。与相应的输入 RNA 相比，所有试剂盒的 IP 样品的富集或耗尽情况相似，除了 SMARTer 试剂盒。
结论
当使用 SMART-Seq v4 试剂盒时，使用 250pg 总 RNA 或使用其他文库制备试剂盒的修改方案时使用 4ng 总 RNA 时，可以成功进行 RiboTag-seq。SMART-Seq v4 和 TruSeq 试剂盒产生了最高质量的文库。RiboTag IP RNA 包含一些不成熟的转录本。
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