Howlett et al. BMC Genomics 2012, 13:211 http://www.biomedcentral.com/1471-2164/13/211 immersing its body in a water-filled glass vial, while its head protruded from a latex gasket. cDNA library preparation and sequencing Dissected chemosensory tissues were immersed immedi- ately in Trizol reagent (Invitrogen, 15596–026). Tissue was homogenized with a micro-pestle and stored at −80° C until RNA extraction. Total RNA purification was per- formed according to manufacturer’s instructions. Con- struction of two normalized cDNA libraries and 454 pyrosequencing were carried out at the W.M.Keck Cen- ter for Comparative and Functional Genomics, Roy J. Carver Biotechnology Center, University of Illinois at Urbana-Champaign. First, messenger RNA was isolated from 20 μg of total RNA using the Oligotex mRNA Mini kit (Qiagen, CA). cDNA was synthesized from 200 ng of mRNA using the SuperScriptW Double-Stranded cDNA Synthesis Kit (Invitrogen, CA) with 100 μM random hexamer primers (Fermentas, USA). Double-stranded DNAs were nebulized at 32 psi for one minute, concen- trated with a Qiaquick PCR minelute column (Qiagen, CA) and blunt-ended (25 μl water, 10 μl 10x T4 DNA Ligase buffer (NEB), 4 μl 10 mM dNTP mix, 5 μl T4 DNA polymerase (3 U/μl) (NEB), 1 μl Klenow polymer- ase (5 U/ μl) (NEB), and 5 μl Polynucleotide kinase (10 U/μl) (NEB). A dA-overhang was added at 3’ ends of cDNA by adding the following to the blunt-ended cDNA: 5 μl 10x buffer 2 (NEB), 10 μl 1 mM dATP and 3 μl Klenow exo-minus polymerase (5 U/μl) (NEB). The reaction was incubated at 37°C for 30 minutes and then cleaned up with a QIAquick MinElute column and eluted in 10 μl EB. The cDNA was adaptored with Titan- ium adaptors (454 Life Sciences, Branford, CT) by add- ing 9 μl water, 25 μl 2x Rapid Ligase buffer (Enzymatics, MA) 5 μl (50 μM) Titanium adapter A/B mix and 1 μl T4 DNA Ligase (600 U/μl (Enzymatics, MA) and incubated the ligation reaction at room temperature for 15 minutes. The reaction was cleaned up using a Qia- quick MiniElute column (Qiagen), eluting the cDNA in 20 μl EB. Adaptored cDNA was run on a E-GEL EX 2% agarose (Invitrogen, CA) following the manufacturer instructions and cDNAs in the size range of 400-800 bp were excised from the gel and purified with a Qiagen’s Gel Extraction kit and the cDNA was eluted in 30 μl EB. The gel- purified cDNA was used as template for ampli- fication in 50 μl PCR reactions containing 10 μl 5x Phu- sion Buffer HF (NEB), 25 μM Adapter A_For primer (5’CCATCTCATCCCTGCGTGTCTCCGACTCAGAC- GAGTGCGT3’), 25 μM Adapter B_For primer (5’C CATCCCCTGTGTGCCTTGGCAGTCTCAGT3’), 3% DMSO, 10 mM dNTPs and 1 U Phusion polymerase (Finnzymes/NEB, USA). The PCR conditions were as follows: 98°C for 30 seconds, followed by 10 cycles with 98°C for 10 seconds, 68°C for 30 seconds and Page 7 of 10 72°C for 30 seconds, with a final extension of 72°C for 5 minute and cleaned up with a Qiaquick minelute PCR column. The cDNA librares were normalized with the Trimmer Direct Kit (Evrogen, Russia). In brief, 300 ng of cDNA were denatured at 95°C for 5 minutes and allowed to reanneal at 68°C for 4 hours in the hybridization buffer included in the kit (50 mM Hepes, pH7.5 and 0.5 M NaCl). After the incubation, the reactions were treated with ¼ units of duplex spe- cific nuclease (DSN). The normalized cDNAs were then PCR amplified as described above. After library construction, the samples were quantified using a Qubit fluorometer (Invitrogen, CA) and average fragment sizes were determined by analyzing 1 μl of the samples on a Bioanalyzer (Agilent, CA) using a DNA 7500 chip. The libraries were mixed in equimolar con- centration and diluted to 1x106molecules/μl. Emulsion- based clonal amplification and sequencing on the 454 Genome Sequencer FLX Titanium system were per- formed according to the manufacturer’s instructions (454 Life Sciences, Branford, CT). Signal processing and base calling were performed using the bundled 454 Data Analysis Software version 2.0.01. Contig identification Primary cDNA sequences were assembled into approxi- mately 52,000 contigs. A standalone tblastx was run using default parameters for each of the OR, CSP, and OBP gene families, and a standalone tblastn was run using default parameters for GR gene families. For the ORs and GRs, all available sequences in the gene family from both Bombyx mori and Drosophila melanogaster were used as queries. In the other two cases, all protein sequences from Bombyx mori were used (see Additional file 2: Figure S2). All query sequences were downloaded from NCBI except for the Bombyx OBP sequences, which were obtained from the authors of [7]. All contigs with matches to at least one query sequence with an E value less than e-45 were retained and for each of these contigs the sequence within the range given in the Blast hit was used for further analyses. In some cases the Blast result gave multiple significant results for a contig in dif- ferent reading frames along the same strand. When these sequences were contiguous in both the query and in the contig, indicating that it was most likely the result of an indel sequencing error, the two protein sequences corresponding to the two reading frames in the contig were joined contiguously. Phylogenetic analysis For each gene family, amino acid query sequences and translated contig sequences were aligned using ClustalW under default parameters [38]. Phylogenetic analyses on these alignments were performed using Phylip 3.69