2.8 Transcriptome sequencing
Specimens of mature Meretrix meretrix and Ruditapes philippinarum were collected from coastal area in Nantong, Jiangsu. They and Corbicula fluminea were dissected and fixed in RNAlater. The RNA was extracted using TRIzol (Thermo Fisher, USA), and the libraries were generated using a NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following the instruction manual. Three biological repeats were set for each species. All samples were sequenced on an Illumina HiSeq X Ten platform (Illumina, Inc., San Diego, CA, USA). The clean reads for C. fluminea were mapped to the Asian Clam genome using Tophat (version 2.0) (Ghosh, & Chan, 2016), and only reads with a perfect match or one mismatch were used for further analysis. For the transcriptome data of M. meretrix and R. philippinarum, a de novo transcriptome assembly was conducted by Trinity (version 2.1.1) (Haas et al., 2013), and CD-HIT (Fu, Niu, Zhu, Wu, & Li, 2012) was used to reduce sequence redundancy. Finally, we presented Kallisto (Bray, Pimentel, Melsted, & Pachter, 2016), an RNA-seq quantification program that mapped clean reads to known transcripts for quantification and standardization. Gene expression levels were estimated by TPM.