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.