Integrative analysis of Iso-Seq and RNA-seq data reveals transcriptome complexity and differentially expressed transcripts in sheep tail fat

Zehu Yuan; Ling Ge; Jingyi Sun; Weibo Zhang; Shanhe Wang; Xiukai Cao; Wei Sun

doi:10.7717/peerj.12454

Integrative analysis of Iso-Seq and RNA-seq data reveals transcriptome complexity and differentially expressed transcripts in sheep tail fat

PeerJ. 2021 Nov 3:9:e12454. doi: 10.7717/peerj.12454. eCollection 2021.

Authors

Zehu Yuan¹, Ling Ge², Jingyi Sun³, Weibo Zhang², Shanhe Wang², Xiukai Cao¹, Wei Sun^{1

2}

Affiliations

¹ Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education, Yangzhou University, Yangzhou, China.
² College of Animal Science and Technology, Yangzhou University, Yangzhou, China.
³ College of Veterinary Medicine, Yangzhou University, Yangzhou, China.

Abstract

Background: Nowadays, both customers and producers prefer thin-tailed fat sheep. To effectively breed for this phenotype, it is important to identify candidate genes and uncover the genetic mechanism related to tail fat deposition in sheep. Accumulating evidence suggesting that post-transcriptional modification events of precursor-messenger RNA (pre-mRNA), including alternative splicing (AS) and alternative polyadenylation (APA), may regulate tail fat deposition in sheep. Differentially expressed transcripts (DETs) analysis is a way to identify candidate genes related to tail fat deposition. However, due to the technological limitation, post-transcriptional modification events in the tail fat of sheep and DETs between thin-tailed and fat-tailed sheep remains unclear.

Methods: In the present study, we applied pooled PacBio isoform sequencing (Iso-Seq) to generate transcriptomic data of tail fat tissue from six sheep (three thin-tailed sheep and three fat-tailed sheep). By comparing with reference genome, potential gene loci and novel transcripts were identified. Post-transcriptional modification events, including AS and APA, and lncRNA in sheep tail fat were uncovered using pooled Iso-Seq data. Combining Iso-Seq data with six RNA-sequencing (RNA-Seq) data, DETs between thin- and fat-tailed sheep were identified. Protein protein interaction (PPI) network, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were implemented to investigate the potential functions of DETs.

Results: In the present study, we revealed the transcriptomic complexity of the tail fat of sheep, result in 9,001 potential novel gene loci, 17,834 AS events, 5,791 APA events, and 3,764 lncRNAs. Combining Iso-Seq data with RNA-Seq data, we identified hundreds of DETs between thin- and fat-tailed sheep. Among them, 21 differentially expressed lncRNAs, such as ENSOART00020036299, ENSOART00020033641, ENSOART00020024562, ENSOART00020003848 and 9.53.1 may regulate tail fat deposition. Many novel transcripts were identified as DETs, including 15.527.13 (DGAT2), 13.624.23 (ACSS2), 11.689.28 (ACLY), 11.689.18 (ACLY), 11.689.14 (ACLY), 11.660.12 (ACLY), 22.289.6 (SCD), 22.289.3 (SCD) and 22.289.14 (SCD). Most of the identified DETs have been enriched in GO and KEGG pathways related to extracellular matrix (ECM). Our result revealed the transcriptome complexity and identified many candidate transcripts in tail fat, which could enhance the understanding of molecular mechanisms behind tail fat deposition.

Keywords: DET; Iso-seq; Sheep; Tail fat.

Grants and funding

This research was funded by the National Natural Science Foundation of China (31872333, 32172689), National Natural Science Foundation of China-CGIAR (32061143036), Major New Varieties of Agricultural Projects in Jiangsu Province (PZCZ201739), The Projects of Domesticated Animals Platform of the Ministry of Science, Key Research and Development Plan (modern agriculture) in Jiangsu Province (BE2018354), Jiangsu Agricultural Science and Technology Innovation Fund (CX(18)2003), and Natural Science Foundation of Jiangsu Province (BK20210811). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.