MustSeq, an alternative approach for multiplexible strand-specific 3' end sequencing of mRNA transcriptome confers high efficiency and practicality
MustSeq, an alternative approach for multiplexible strand-specific 3' end sequencing of mRNA transcriptome confers high efficiency and practicality
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MustSeq 是一种用于 mRNA 转录组多重链特异性 3' 末端测序的替代方法，具有高效率和实用性
DOI：
10.1080/15476286.2021.1974208
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发表时间：
2021-09-30
期刊：
影响因子：
4.1
通讯作者：
Pan，Xinghua
中科院分区：
文献类型：
--
作者：
Mai，Liyao;Qiu，Yinbin;Pan，Xinghua
RNA-seq已被广泛用于揭示生命过程变异的分子机制。我们开发了一种替代方法 MustSeq，该方法通过随机引发引发沿着单个第一链 cDNA 生成多个第二链，在使用样品条形码聚 dT 引物对每个 RNA 提取物进行逆转录后立即进行，然后应用 3ʹ 末端富集 PCR 来构建文库。与传统的RNA seq不同，MustSeq避免了mRNA分离、片段化和RNA 5′端捕获等程序，能够早期汇集多个样本，并且只需要全长测序的二十分之一的测序读数。我们将 MustSeq 与 TruSeq 和 NEBNext RNA-seq（两种传统的全长方法）以及 QuantSeq（一种工业 3ʹ 末端方法）进行比较，展示了 MustSeq 的功能和特性。在癌细胞系中，MustSeq 检测到的 CDS 外显子以及基因、lncRNA 和 GO term 的 read 分布比 QuantSeq 更接近 TruSeq。在小鼠肝癌和健康肝脏中，MustSeq 丰富了与 NEBNext 相同的途径，并揭示了癌变的分子特征。总体而言，MustSeq 是一种稳健且准确的 RNA-seq 方法，可实现高效的文库构建、测序和分析，对于分析大量样本的差异表达基因特别有价值。 MustSeq将大大加速bulk RNA-seq在不同领域的应用，并有可能适用于单细胞RNA-seq。
RNA-seq has been widely used to reveal the molecular mechanism of variants of life process. We have developed an alternative method, MustSeq, which generates multiple second strands along a single 1ststrand cDNA by random-priming initiation, immediately after reverse transcription for each RNA extract using sample-barcoded poly-dT primers, then 3ʹ ends-enriching PCR is applied to construct the library. Unlike the conventional RNA seq, MustSeq avoids procedures such as mRNA isolation, fragmentation and RNA 5ʹ-end capture, enables early pooling of multiple samples, and requires only one twentieth of sequencing reads of full-length sequencing. We demonstrate the power and features of MustSeq comparing with TruSeq and NEBNext RNA-seq, two conventional full-length methods and QuantSeq, an industrial 3ʹ end method. In cancer cell lines, the reads distribution of CDS-exon as well as genes, lncRNAs and GO terms detected by MustSeq are closer than QuantSeq to TruSeq. In mouse hepatocarcinoma and healthy livers, MustSeq enriches the same pathways as by NEBNext, and reveals the molecular profile of carcinogenesis. Overall MustSeq is a robust and accurate RNA-seq method allowing efficient library construction, sequencing and analysis, particularly valuable for analysis of differentially expressed genes with a large number of samples. MustSeq will greatly accelerate the application of bulk RNA-seq on different fields, and potentially applicable for single cell RNA-seq.