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.