De Novo assembly and annotation of the Pacific calico scallop (Argopecten ventricosus) transcriptome for immune-related gene discovery

Abstract

Invertebrates' immune defense mechanisms play a critical role in pathogen recognition and elimination. De novo assembly and annotation of the Argopecten ventricosus transcriptome were performed for the immune-related gene identification. Scallops (height: 4.4 cm) were challenged with inactivated Vibrio parahaemolyticus IPNGS16. The RNA from different tissues was pooled for a single cDNA library construction sequenced by NextSeq 500 platform 2×75 paired‐end chemistry. Before de novo assembling with Trinity, reads were analyzed with FastQC, Trimmomatic, and Prinseq. Assembled sequences were analyzed by CD-HIT-EST and TransDecoder. The corresponding annotation was performed against NCBI-nr, RefSeq protein, and KAAS (KEGG) databases. The Trinity assembly yielded 107,516 contigs. TransDecoder yielded 25,285 sequences as CDSs of which, 16,123 were annotated against the NCBI-nr protein, most of them scored with Crassostrea gigas data. Gene ontology mapped sequences (15,262) were classified in molecular functions (~13,000), cellular components (~11,000), and biological processes (~13,000). The KAAS analysis showed biological categories for metabolism (13%), cellular processes (12%), genetic information processing (10%), organismal systems (19%), environmental information (13%), and human diseases (33 %). Within the organismal systems, 467 immune-related genes (KO) were identified. Sixty-four immune-related genes were annotated/blasted against the NCBI-nr and RefSeq protein databases. An RT-qPCR was performed to analyze the expression level of immune-related genes obtained in the transcriptome analysis in scallops (height 4.5 cm) treated with probiotic bacilli added to culture water. Bacilli significantly increased the expression of the HSP70 and PGRP genes. The gene transcripts analysis of A. ventricosus will better understand its immune response against pathogens in culture systems.