Assembly, expression analysis and annotation

Raw reads obtained after sequencing were filtered by removing Illumina adapter using “cut-adapt”⁴⁰ tool and in house “Perl scripts” was used to remove low quality bases (Q < 20) and to obtain processed reads. The trimmed reads were then de novo assembled using Velvet (Version 1.2.09) & Oases (Version 0.2.8) with kmer size of 41^41,42. To calculate FPKM values, trimmed reads were first aligned to the assembled transcriptome using Bowtie2 program. Up to 1-mismatch was allowed in the seed region (length = 31 bp). Assembled contigs with >150 bp and expression value of >1 FPKM were annotated using BLASTX programme against various public databases including NCBI non-redundant (nr) protein database with set parameters of E-value >10⁻⁵ and similarity score of >40%, UniProt database for assigning Gene Ontology (GO) terms⁴³, and KEGG using KEGG Automatic Annotation Server (KAAS; http://www.genome.jp/kaas-bin/kaas_main?mode=partial)⁴⁴ for pathway assignment.