4.7 Article

Long read isoform sequencing reveals hidden transcriptional complexity between cattle subspecies

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BMC GENOMICS
卷 24, 期 1, 页码 -

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BMC
DOI: 10.1186/s12864-023-09212-9

关键词

Iso-Seq; RNA-seq; Cattle; Differential Isoform expression; Transcriptome; Multi-mapped reads; Sequence duplication; Subspecies; Alternative splicing; Long read sequencing

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The Iso-Seq method of full-length cDNA sequencing is suitable for quantifying DEGs, DETs, and DTU. However, the higher cost of Iso-Seq limits its comparison with RNA-seq. In this study, RNA-seq and deep Iso-Seq data were compared in fetal liver samples from two cattle subspecies. The results showed higher concordance within technology than between technologies. Iso-Seq identified more DEGs and revealed greater transcript abundance and usage differences between subspecies. Factors influencing DEG identification included size selection, transcript abundance, multi-mapping of RNA-seq reads, and gene coordinates. Some DEGs called by RNA-seq alone were likely sequence duplication artifacts. Iso-Seq uncovered hidden transcriptional complexity in DEGs, DETs, and DTU genes missed by RNA-seq.
The Iso-Seq method of full-length cDNA sequencing is suitable to quantify differentially expressed genes (DEGs), transcripts (DETs) and transcript usage (DTU). However, the higher cost of Iso-Seq relative to RNA-seq has limited the comparison of both methods. Transcript abundance estimated by RNA-seq and deep Iso-Seq data for fetal liver from two cattle subspecies were compared to evaluate concordance. Inter-sample correlation of gene- and transcript-level abundance was higher within technology than between technologies. Identification of DEGs between the cattle subspecies depended on sequencing method with only 44 genes identified by both that included 6 novel genes annotated by Iso-Seq. There was a pronounced difference between Iso-Seq and RNA-seq results at transcript-level wherein Iso-Seq revealed several magnitudes more transcript abundance and usage differences between subspecies. Factors influencing DEG identification included size selection during Iso-Seq library preparation, average transcript abundance, multi-mapping of RNA-seq reads to the reference genome, and overlapping coordinates of genes. Some DEGs called by RNA-seq alone appear to be sequence duplication artifacts. Among the 44 DEGs identified by both technologies some play a role in immune system, thyroid function and cell growth. Iso-Seq revealed hidden transcriptional complexity in DEGs, DETs and DTU genes between cattle subspecies previously missed by RNA-seq.

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