PacBio single molecule long-read sequencing provides insight into the complexity and diversity of the Pinctada fucata martensii transcriptome

Abstract

Background The pearl oyster Pinctada fucata martensii is an economically valuable shellfish for seawater pearl production, and production of pearls depends on its growth. To date, the molecular mechanisms of the growth of this species remain poorly understood. The transcriptome sequencing has been considered to understanding of the complexity of mechanisms of the growth of P. f. martensii. The recently released genome sequences of P. f. martensii , as well as emerging Pacific Bioscience (PacBio) single-molecular sequencing technologies, provide an opportunity to thoroughly investigate these molecular mechanisms. Results Herein, the full-length transcriptome was analysed by combining PacBio single-molecule long-read sequencing (PacBio sequencing) and Illumina sequencing. A total of 20.65 Gb of clean data were generated, including 574,561 circular consensus reads, among which 443,944 full-length non-chimeric (FLNC) sequences were identified. Through transcript clustering analysis of FLNC reads, 32,755 consensus isoforms were identified, including 32,095 high-quality consensus sequences. After removing redundant reads, 16,388 transcripts were obtained, and 641 fusion transcripts were derived by performing fusion transcript prediction of consensus sequences. Alternative splicing analysis of the 16,388 transcripts was performed after accounting for redundancy, and 9097 gene loci were detected, including 1607 new gene loci and 14,946 newly discovered transcripts. The original boundary of 11,235 genes on the chromosomes was corrected, 12,025 complete open reading frame sequences and 635 long non-coding RNAs (LncRNAs) were predicted, and functional annotation of 13,482 new transcripts was achieved. Two thousand three hundred eighteen alternative splicing events were detected. A total of 228 differentially expressed transcripts (DETs) were identified between the largest (L) and smallest (S) pearl oysters. Compared with the S, the L showed 99 and 129 significantly up-and down-regulated DETs, respectively. Six of these DETs were further confirmed by quantitative real-time RT-PCR (RT-qPCR) in independent experiment. Conclusions Our results significantly improve existing gene models and genome annotations, optimise the genome structure, and in-depth understanding of the complexity and diversity of the differential growth patterns of P. f. martensii .

SMRT sequencing of the full-length transcriptome of Gekko gecko

“…Chinese medicinal animal, obtaining a full-length transcriptome and understanding the structure of genes in G. gecko is a primary step in studying gene function, which is very important, yet it is still unknown. SMRT sequencing provides new knowledge of full-length sequences, which is confirmed to be useful for performing gene annotation and interpreting gene functions, especially for species lacking a reference genome [12,31]. In the present study, we obtained 882,273 CCSs, identified 746,317 FLNC, and then yielded 212,964 corrected isoforms with an average read length of 4,153 bp.…”

“…For decades, a substantial number of RNA-seq studies have been conducted to understand gene expression and molecular mechanisms, moreover, RNAseq is particularly widely used for nonmodel species that lack a reference genome [6][7][8][9], it provides insights into mRNA splicing and gene expression and has been used to screen candidate genes; however, the gene structure and full-length sequence are limited [10,11]. In addition, the extent of alternative splicing (AS) and transcriptome diversity remain largely unknown due to its short read length [12]. Recently, the single molecule real-time (SMRT) sequencing technique revolutionized the limitation of short read sequences and fragmentation, and postsequencing assembly are not needed.…”

“…Moreover, SMRT sequencing provides accurate fulllength transcripts, and average sequence read that up to 50 kb have been reported [13,14]. Therefore, SMRT sequencing represents an effective tool that has been widely and successfully used to annotate and analyze full-length transcripts among mammals, marine animals, aquatic animals and insects [15], such as Tachypleus tridentatus [16], Pinctada fucata martensii [12], Sogatella furcifera [17], and Odontotermes formosanu [18][19][20]. However, no studies have investigated on G. gecko.…”

Research Trends of Development on Pearl Bivalve Mollusks Based on a Bibliometric Network Analysis in the Past 25 Years

“…Through integrated transcriptome and metabolome analyses, Hao et al (2019) assessed the metabolic and transcript changes between the fast-growing and slowgrowing P. f. martensii groups and determined the mechanism underlying this phenomenon. Pacific Bioscience (PacBio) singlemolecular sequencing technologies, an emerging transcriptome sequencing method, enables researchers to understand the complexity and diversity of the molecular mechanisms of the growth of P. f. martensii (Zhang et al, 2020). Using a next-generation sequencing method and RNA-seq technology, Vaihiti et al performed gonad transcriptome analysis of the hermaphroditic P. margaritifera from several gonadic samples of males and females at different development stages.…”

Transcriptomic analysis to elucidate the effects of high stocking density on grass carp (Ctenopharyngodon idella)

“…Stress-related molecules have been studied using Illumina sequencing. PacBio SMRT sequencing can provide information on transcript diversity, including alternative splicing (AS), alternative polyadenylation (APA), and long noncoding RNAs (lncRNAs) [ 7 ]. AS can generate various mRNAs and significantly enhance regulatory capacities and proteomic complexities [ 8 ].…”