Integrative Analysis of Nanopore and Illumina Sequencing Reveals Alternative Splicing Complexity in Pig Longissimus Dorsi Muscle

Abstract

Alternative splicing (AS) is a key step in the post-transcriptional regulation of gene expression that can affect intramuscular fat (IMF). In this study, longissimus dorsi muscles from 30 pigs in high- and low- IMF groups were used to perform Oxford Nanopore Technologies (ONT) full-length sequencing and Illumina strand-specific RNA-seq. A total of 43,688 full-length transcripts were identified, with 4,322 novel genes and 30,795 novel transcripts. Using AStalavista, a total of 14,728 AS events were detected in the longissimus dorsi muscle. About 17.79% of the genes produced splicing isoforms, in which exon skipping was the most frequent AS event. By analyzing the expression differences of mRNAs and splicing isoforms, we found that differentially expressed mRNAs with splicing isoforms could participate in skeletal muscle development and fatty acid metabolism, which might determine muscle-related traits. SERBP1, MYL1, TNNT3, and TNNT1 were identified with multiple splicing isoforms, with significant differences in expression. AS events occurring in IFI6 and GADD45G may cause significant differences in gene expression. Other AS events, such as ONT.15153.3, may regulate the function of ART1 by regulating the expression of different transcripts. Moreover, co-expression and protein-protein interaction (PPI) analysis indicated that several genes (MRPL27, AAR2, PYGM, PSMD4, SCNM1, and HNRNPDL) may be related to intramuscular fat. The splicing isoforms investigated in our research provide a reference for the study of alternative splicing regulation of intramuscular fat deposition.

RNA-Seq Study on the Longissimus thoracis Muscle of Italian Large White Pigs Fed Extruded Linseed with or without Antioxidants and Polyphenols

“…Not all clean reads were assigned to that feature, and this is probably because the pig genome was not completely annotated, so a part of the remaining reads was not assigned. However, through these reads, it would be possible to update the annotation of the pig genome [ 22 ].…”

Exploring Multi-Tissue Alternative Splicing and Skeletal Muscle Metabolism Regulation in Obese- and Lean-Type Pigs

“…This may be attributed to the presence of some muscle-specific splicing factors in muscle tissue [49][50][51]. Previous studies have also indicated significant differences in AS events between lean and obese-type pigs in skeletal muscle [52]. Specifically, there were numerous AS events related to mitochondrial function and glucose metabolism in the skeletal muscle of lean-type pigs.…”

Long-read sequencing-based transcriptomic landscape in longissimus dorsi and transcriptome-wide association studies for growth traits of meat rabbits

“…The accuracies of transcript assembly and expression quantification have been further improved due to the later single-molecule long-read RNA sequencing technologies ( 40 ). Therefore, long-read sequencing technologies are expecting to be increasingly used in transcriptome studies ( 41 ), such as in cattle ( 42 ), pigs ( 43 ), and chicken ( 44 ). In rabbit, Chen et al ( 24 ) first used PacBio long-read sequencing technology for exploring transcriptomic landscape and revealed a large proportion of novel genes and transcripts using a pooling sample of multiple organ tissues sampled at different ages.…”