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. 2021 Oct 15;18(sup1):232-243.
doi: 10.1080/15476286.2021.1974208. Epub 2021 Sep 29.

MustSeq, an alternative approach for multiplexible strand-specific 3' end sequencing of mRNA transcriptome confers high efficiency and practicality

Liyao Mai  1 , Yinbin Qiu  1 , Zhiwei Lian  1 , Caiming Chen  1 , Linlin Wang  1 , Yao Yin  1 , Siqi Wang  1 , Xiang Yang  1   2 , Yazi Li  1 , Wanwan Peng  1 , Chaochao Luo  1 , Xinghua Pan  1   2   3   4
Affiliations

MustSeq, an alternative approach for multiplexible strand-specific 3' end sequencing of mRNA transcriptome confers high efficiency and practicality

Liyao Mai et al. RNA Biol. .

Abstract

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 1st strand cDNA by random-priming initiation, immediately after reverse transcription for each RNA extract using sample-barcoded poly-dT primers, then 3' ends-enriching PCR is applied to construct the library. Unlike the conventional RNA seq, MustSeq avoids procedures such as mRNA isolation, fragmentation and RNA 5'-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 3' 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.

Keywords: 3ʹ end sequencing; RNA-Seq; differentially expressed genes; high throughput; methodology; transcriptomics.

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Conflict of interest statement

We declare that we have a patent in connection with the work on filing process.

Figures

Figure 1.
Figure 1.
Workflow of RNA library construction with MustSeq
Figure 2.
Figure 2.
Distribution and reproducibility of MustSeq
Figure 3.
Figure 3.
Quantification of the transcriptome for K562 sequenced with MustSeq in comparison with TruSeq and QuantSeq
Figure 4.
Figure 4.
Transcriptomic analysis of 4 cancer cell lines sequenced with MustSeq in comparison with TruSeq
Figure 5.
Figure 5.
Transcriptomic analysis of mouse hepatocarcinoma (HCC) and healthy liver
See this image and copyright information in PMC

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