Computational protocol: Development of Reference Transcriptomes for the Major Field Insect Pests of Cowpea: A Toolbox for Insect Pest Management Approaches in West Africa

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Protocol publication

[…] Insect samples were collected during the summer through fall of 2011 at 7, 11 and 9 locations in Benin, Burkina Faso and Niger respectively for A. craccivora, A. curvipes, C. tomentosicollis and M. sjostedti (). A total of 79, 1,920, 364 and 740 individual insect samples were collected respectively for A. craccivora, A. curvipes, C. tomentosicollis and M. sjostedti from these locations. Both the larval and adult life stages were sampled for all species and stored in RNAlater (Ambion, TX, USA) immediately after collection in the field. All samples from each species, from a single location, were pooled and total RNA was extracted from the insect samples at IITA Benin and INERA Burkina Faso using QIAGEN RNeasy RNA extraction kits (CA, USA) and following the manufacturer's instructions. The RNA was shipped to University of Illinois at Urbana-Champaign (UIUC), USA in 70% ethanol where it was resuspended in water and quantified by measuring the absorbance at 260 nm using a NanoDrop spectrophotometer (Thermo Scientific, DE, USA). The samples were then stored in an ultra-low temperature freezer (-80°C). Four normalized cDNA libraries were constructed and sequenced on a Roche 454 GS-FLX at the W.M. Keck Center for Comparative and Functional Genomics, Roy J. Carver Biotechnology Center, UIUC. Briefly, messenger RNA (mRNA) was isolated from 10μg of total RNA with the Oligotex kit (Qiagen, Valencia, CA). The mRNA-enriched fraction was converted to 454 barcoded cDNA libraries and normalized []. The barcoded libraries were pooled in equimolar concentration based on average fragment length and concentration. After library construction, the pooled libraries were quantified using a Qubit fluorometer (Invitrogen, CA, USA) and average fragment sizes were determined by analyzing 1µl of the samples on the Bioanalyzer (Agilent, CA, USA) using a DNA 7500 chip. The pooled library was diluted to 1 x 106 molecules/µl. Emulsion-based clonal amplification and sequencing on a full plate on the 454 Genome Sequencer FLX+ system was performed according to the manufacturer’s instructions (454 Life Sciences, CT, USA). Signal processing and base calling were performed using the bundled 454 Data Analysis Software v2.6. The raw sequence read data from the four insect pests were analyzed using the CLC Genomics Workbench 6.0.1 (Cambridge, MA, USA). Pre-processing of the raw reads from each of the four insect samples involved trimming each 454 read using a Phred quality score of 20 and also removing nucleotides < 50 bp from the ends. The adapter sequences were also trimmed from the raw reads. The processed read data from each of the four insect samples were assembled into contiguous sequences using parameters: mismatch cost = 2, insertion and deletion cost = 3, length fraction = 60% and similarity = 90%. After assembly, the vector contamination were removed using the UniVec database and also after assembly, human, bacterial, fish (Danio rerio), mouse (Mus musculus), Salmonella enterica, archeal and viral contamination were removed using a web-based version of DeConSeq [] using a coverage of 90% and a sequence identity threshold of 94%. The clean transcriptomes, with the contaminations removed, were then deposited at DDBJ/EMBL/GenBank for each of the four insect species. [...] The bacterial endosymbiont, Buchnera aphidicola, was identified from a BLASTn search against Buchnera (Taxid: 32199) in NCBI using the assembled A. craccivora contigs as queries. The A. craccivora contigs with relevant hits to Buchnera were extracted and further confirmatory analysis was performed using InterProScan on Blast2GO v2.6.5, and the associated KEGG pathways were investigated also using Blast2GO v2.6.5. […]

Pipeline specifications

Software tools BLASTN, InterProScan, Blast2GO
Databases KEGG
Application Metagenomic sequencing analysis
Organisms Vigna unguiculata, Homo sapiens, Aphis craccivora, Buchnera aphidicola