Computational protocol: Sequencing and De Novo Assembly of the Western Tarnished Plant Bug (Lygus hesperus) Transcriptome

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Protocol publication

[…] Prior to analyses, all adapter nucleotides were trimmed and the 454 sequences were de novo assembled using the newbler 2.6 software package (454 Life Sciences/Roche). The resulting isotigs were analyzed using BLASTx against the GenBank non-redundant database with an e-value cut-off of 1e−5. A tBLASTn analysis was performed using the most current (Sept, 2012) L. hesperus protein sequences. BLASTx-based comparative analyses of the L. hesperus transcriptome protein coding sequences were performed using putative protein sequences from D. melanogaster, A. pisum, and P. humanus humanus. Protein sequences shared across the species in question were determined using the online algorithm Venny (http://bioinfogp.cnb.csic.es/tools/venny/index.html). Homologous protein domains from translated L. hesperus transcriptomic sequences were identified by searching against the Pfam database using HMMER3 . Blast2GO , was used to assign putative functionalities, GO terms, and KEGG (Kyoto Encyclopedia of Genes and Genomes) based metabolic pathways , . Final GO assignments were defined based on a 10% filter for all three processes profiled at level 2. All other settings for the analysis were maintained at their defaults. For phylogenetic analysis, putative L. hesperus Hsp sequences and those of other insect species were aligned using MAFFT v6.814b , as implemented in Geneious 5.6.5, and analyzed in MEGA 5 using default maximum parsimony settings with 1000 bootstrap replications. […]

Pipeline specifications

Software tools Newbler, BLASTX, TBLASTN, VENNY, HMMER, Blast2GO, MAFFT, Geneious, MEGA
Databases Pfam KEGG
Applications Phylogenetics, Amino acid sequence alignment
Organisms Lygus lineolaris, Lygus hesperus, Gossypium hirsutum