Functional Annotation of Cotesia congregata Bracovirus: Identification of Viral Genes Expressed in Parasitized Host Immune Tissues
The cDNA libraries were prepared by using SMARTer™ PCR cDNA Synthesis Kit (Clontech). First-strand cDNA synthesis was performed with 3’ SMART CDS Primer II A as described in the supplier protocol and using 995 and 70 ng of total RNA from fat body and hemocytes respectively. Double-strand cDNAs were prepared by long-distance PCR (LD-PCR) from 1 and 10 µl of first-strand cDNA from fat body and hemocytes respectively. A pool of three independent LD-PCRs was used for deep sequencing of each cDNA library.   Reads generated by 454 sequencing were trimmed for poly(A), 454  adaptors (A and B) and PCR primers. The origin of reads was established by mapping on two reference genomes, M. sexta  (Manduca Base: www.agripestbase.org) and CcBV (chromosomal form) (Bézier et al. 2013). Mapping was performed using GS Reference Mapper from Newbler software package (version 2.5.6, Roche) with the following parameters: -cdna -ss 1 –sl 16 –ml 80% -mi 95 –d 10. To distinguish and annotate genes belonging to gene families and sharing high sequence similarity, CcBV reads were aligned to CcBV genome using Exonerate with the following parameters: -m cdna2genome –geneseed 100 –codonwordlimit 1 –refin region. For each cluster of reads aligned with a given genomic region, a consensus sequence was generated by de novo assembling reads using GS De Novo Assembler from Newbler software package with the following parameters: -cdna -it 1 –ig 1 –icc 1 –ss 1 –sl 16 –ml 90% -mi 80.