AQRNA-seq 定量绘制细胞小 RNA 图谱。

Quantitative mapping of the cellular small RNA landscape with AQRNA-seq.

机构信息

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA.

Bristol Myers Squibb, Seattle, WA, USA.

出版信息

Nat Biotechnol. 2021 Aug;39(8):978-988. doi: 10.1038/s41587-021-00874-y. Epub 2021 Apr 15.

Current next-generation RNA-sequencing (RNA-seq) methods do not provide accurate quantification of small RNAs within a sample, due to sequence-dependent biases in capture, ligation and amplification during library preparation. We present a method, absolute quantification RNA-sequencing (AQRNA-seq), that minimizes biases and provides a direct, linear correlation between sequencing read count and copy number for all small RNAs in a sample. Library preparation and data processing were optimized and validated using a 963-member microRNA reference library, oligonucleotide standards of varying length, and RNA blots. Application of AQRNA-seq to a panel of human cancer cells revealed >800 detectable miRNAs that varied during cancer progression, while application to bacterial transfer RNA pools, with the challenges of secondary structure and abundant modifications, revealed 80-fold variation in tRNA isoacceptor levels, stress-induced site-specific tRNA fragmentation, quantitative modification maps, and evidence for stress-induced, tRNA-driven, codon-biased translation. AQRNA-seq thus provides a versatile means to quantitatively map the small RNA landscape in cells.

目前的下一代 RNA 测序（RNA-seq）方法由于在文库制备过程中捕获、连接和扩增过程中的序列依赖性偏差，无法对样品中的小 RNA 进行准确的定量。我们提出了一种方法，绝对定量 RNA 测序（AQRNA-seq），该方法最大限度地减少了偏差，并为样品中小 RNA 的测序读长与拷贝数之间提供了直接的线性相关性。通过使用 963 个成员的 microRNA 参考文库、不同长度的寡核苷酸标准品和 RNA 印迹，对文库制备和数据处理进行了优化和验证。AQRNA-seq 在一组人类癌细胞中的应用揭示了在癌症进展过程中存在 >800 个可检测 miRNA，而在细菌转移 RNA 池中的应用，由于二级结构和丰富修饰的挑战，揭示了 tRNA 同工受体水平的 80 倍变化、应激诱导的特定 tRNA 片段化、定量修饰图谱以及应激诱导、tRNA 驱动的密码子偏向翻译的证据。因此，AQRNA-seq 为定量绘制细胞中小 RNA 图谱提供了一种通用的方法。