Unionid454seq
Unionid transcriptome project, cDNA sequences
Initial transcriptomes were developed for three accepted species of unionid freshwater mussels.  Specimens of the endangered Alasmidonta heterodon were collected from the Connecticut River. Alamidonta varicosa were sampled from Croyden Brook, a tributary of the Connecticut River.  Elliptio complanata were sampled from Pine Creek, a tributary of the Susquehanna River.  RNA was isolated from 50-100 mg of fresh or RNA Later- preserved gill, mantle, foot, and hepatopancreas tissue from each of five specimens. The tissues were subjected to a Trizol extraction following methods described by P. Chomczynski and N. Sacchi, Single Step Method of RNA Isolation by Acid Guanidinium Thiocyanate Phenol Chloroform Extraction (Analytical Biochemistry, vol. 162, p.156).  Removal of ribosomal RNA was performed using Lock-Nucleic Acids (Invitrogen RiboMinus Eukaryote Kit for RNA-Seq #A10837-08).  The cleaned mRNAs from each species were subjected to quantification and qualification using a Bioanalyzer 2100 Lab-on-a-Chip (Agilent Technologies).  mRNAs for each species were sent to the University of Iowa DNA Core Sequencing Facility for cDNA library preparation using the cDNA Rapid Library Preparation Method for GS FLX Titanium Series (October 2009). 200ng of sample RNA was fragmented and then prepared for double- stranded cDNA Synthesis using the cDNA Synthesis System Kit (Roche) followed by fragment end repair using New England Biolabs NEBNext End Repair Module. The RL Multiplex IDentifiers (MID) Adaptor was ligated to the sample following end repair.  Small fragment removal was performed using AMPure Beads (Agencourt). Each library was quantified and then prepared for emulsion PCR.  Massively parallel sequencing was performed on the Roche 454 GS FLX Titanium Series sequencer. A total of 1,175,226 reads with an average read length of 401bp were obtained, which equated to 472Mb. Sequence assembly was performed for each species separately and combined using the 454 GS de novo Assembler.  Assembled contigs were searched for microsatellite motifs using MSATCOMMANDER.  Additional assembly combinations and single nucleotide polymorphisms were identified using CLC Bio''s Genomics Workbench. Contigs were searched for significant homology by running a local BLAST search against relevant genomes. Manual curation was performed by using a 70-70 approach (70% or above, coverage of the ORF and at least 70% homology). In the absence of significant homology, an ORF was labeled as a hypothetical protein. BLAST searches also were performed against InterPro and PFAM. RNAs were identified by running BLAST searches against RFAM and tRNA-scan.