S in NCBIof best hitsratio of best hit / Acetovanillone sequences in NCBIrank 4 2 3 1 5 6 7 9Gallus gallusChickenAnolis carolinensisCarolina Anole lizard1 2 23,Taeniopygia guttataZebra finchMeleagris gallopavoWild turkeyMonodelphis domesticaGray short-tailed opossumOrnithorhynchus anatinusDuckbill PlatypusXenopus laevisAfrican Clawed FrogHomo sapiensHumanMus musculusMousetotalsAnolis carolinensisCarolina Anole lizardAll birdsChicken, Zebra finch, and Wild 17,607 TurkeytotalsThe number of top BLAST hits and the number of sequences in the NCBI protein database per species were used to calculate the enrichment in top hits per species relative to the number of sequences in the database. The top half of the table lists the top nine species, the bottom half compares the combined hits for all birds with the hits to the Carolina Anole Lizard. doi:10.1371/journal.pone.0066357.tRed-Eared Slider Turtle Embryonic TranscriptomeRed-Eared Slider Turtle Embryonic TranscriptomeTable 4. TCA cycle enzymes present in the T. scripta developmental transcriptome.EC number 1.1.1.37 1.1.1.41 1.1.1.42 1.2.4.1 1.2.4.2 1.3.5.1 1.3.99.1 1.8.1.4 2.3.1.12 2.3.1.61 2.3.3.1 2.3.3.8 4.1.1.32 4.1.3.6 4.2.1.2 4.2.1.3 6.2.1.4 6.2.1.5 6.4.1.Enzyme name Malate dehydrogenase Isocitrate dehydrogenase (NAD+) Isocitrate dehydrogenase (NADP+) Pyruvate dehydrogenase (acetyl-transferring) Oxoglutarate dehydrogenase (succinyl-transferring) Succinate dehydrogenase (ubiquinone) Succinate dehydrogenase Dihydrolipoyl dehydrogenase Dihydrolipoyllysine-residue acetyltransferase Dihydrolipoyllysine-residue succinyltransferase Citrate (Si)-synthase ATP citrate synthase Phosphoenolpyruvate carboxykinase (GTP) Citrate (pro-3S)-lyase Fumarate hydratase Aconitate hydratase Succinate oA ligase (GDP-forming) Succinate oA ligase (ADP-forming) Pyruvate carboxylaseGenbank accession numbers JW457473, JW460952 JW313702, JW460801, JW464649 JW315818, JW459818, JW460815 JW458559, JW463459 JW425409, JW443178, JW460829, JW489499, JW460830, JW460831 JW460432, JW463499 JW317082, JW461916 JW459096 JW313827 JW464483 JW458401, JW459037 JW305869, JW460741, JW460742 JW288259, JW288260, JW461270, JW461271 JW402608, JW460741, JW460742 JW319039 JW321248, JW461661 JW305869, JW310451 JW305869, JW460741, BI 78D3 web JW463477, JW460742 JWPredicted proteins in the transcriptome were 23148522 mapped to the TCA KEGG metabolic pathway using Blast2Go. doi:10.1371/journal.pone.0066357.tdehyde in PBS, rehydrated in a MeOH/PBT series, treated with proteinase K, and then washed again in PBT. Fixed embryos were probed with a digoxygenin-labeled RNA probe for BMP5 which was detected with an anti-digoxygenin alkaline phosphatase conjugated antibody.ResultsTotal RNA from stage 14 and stage 17 [7] Trachemys scripta embryos was prepared separately, pooled and used to generate 188,674,651 single 100 bp sequences using an Illumina HiSeq 2000. These sequences were assembled without a reference genome using the Trinity package [23] which is capable of assembling and reporting allelic variation and alternatively spliced transcripts. Trinity produced 465,923 contigs with lengths over 150 bp. In these sequences 50 of the total sequence length was contained in the 61,333 sequences longer than 757 bp. Over 1676428 half of the contigs were shorter than 250 bp and most of these short sequences did not code for proteins. We decided to remove all contigs smaller than 250 bp to simplify our analysis. This left 231,876 sequences with 50 of the total sequence length contained in 37,485 sequ.S in NCBIof best hitsratio of best hit / sequences in NCBIrank 4 2 3 1 5 6 7 9Gallus gallusChickenAnolis carolinensisCarolina Anole lizard1 2 23,Taeniopygia guttataZebra finchMeleagris gallopavoWild turkeyMonodelphis domesticaGray short-tailed opossumOrnithorhynchus anatinusDuckbill PlatypusXenopus laevisAfrican Clawed FrogHomo sapiensHumanMus musculusMousetotalsAnolis carolinensisCarolina Anole lizardAll birdsChicken, Zebra finch, and Wild 17,607 TurkeytotalsThe number of top BLAST hits and the number of sequences in the NCBI protein database per species were used to calculate the enrichment in top hits per species relative to the number of sequences in the database. The top half of the table lists the top nine species, the bottom half compares the combined hits for all birds with the hits to the Carolina Anole Lizard. doi:10.1371/journal.pone.0066357.tRed-Eared Slider Turtle Embryonic TranscriptomeRed-Eared Slider Turtle Embryonic TranscriptomeTable 4. TCA cycle enzymes present in the T. scripta developmental transcriptome.EC number 1.1.1.37 1.1.1.41 1.1.1.42 1.2.4.1 1.2.4.2 1.3.5.1 1.3.99.1 1.8.1.4 2.3.1.12 2.3.1.61 2.3.3.1 2.3.3.8 4.1.1.32 4.1.3.6 4.2.1.2 4.2.1.3 6.2.1.4 6.2.1.5 6.4.1.Enzyme name Malate dehydrogenase Isocitrate dehydrogenase (NAD+) Isocitrate dehydrogenase (NADP+) Pyruvate dehydrogenase (acetyl-transferring) Oxoglutarate dehydrogenase (succinyl-transferring) Succinate dehydrogenase (ubiquinone) Succinate dehydrogenase Dihydrolipoyl dehydrogenase Dihydrolipoyllysine-residue acetyltransferase Dihydrolipoyllysine-residue succinyltransferase Citrate (Si)-synthase ATP citrate synthase Phosphoenolpyruvate carboxykinase (GTP) Citrate (pro-3S)-lyase Fumarate hydratase Aconitate hydratase Succinate oA ligase (GDP-forming) Succinate oA ligase (ADP-forming) Pyruvate carboxylaseGenbank accession numbers JW457473, JW460952 JW313702, JW460801, JW464649 JW315818, JW459818, JW460815 JW458559, JW463459 JW425409, JW443178, JW460829, JW489499, JW460830, JW460831 JW460432, JW463499 JW317082, JW461916 JW459096 JW313827 JW464483 JW458401, JW459037 JW305869, JW460741, JW460742 JW288259, JW288260, JW461270, JW461271 JW402608, JW460741, JW460742 JW319039 JW321248, JW461661 JW305869, JW310451 JW305869, JW460741, JW463477, JW460742 JWPredicted proteins in the transcriptome were 23148522 mapped to the TCA KEGG metabolic pathway using Blast2Go. doi:10.1371/journal.pone.0066357.tdehyde in PBS, rehydrated in a MeOH/PBT series, treated with proteinase K, and then washed again in PBT. Fixed embryos were probed with a digoxygenin-labeled RNA probe for BMP5 which was detected with an anti-digoxygenin alkaline phosphatase conjugated antibody.ResultsTotal RNA from stage 14 and stage 17 [7] Trachemys scripta embryos was prepared separately, pooled and used to generate 188,674,651 single 100 bp sequences using an Illumina HiSeq 2000. These sequences were assembled without a reference genome using the Trinity package [23] which is capable of assembling and reporting allelic variation and alternatively spliced transcripts. Trinity produced 465,923 contigs with lengths over 150 bp. In these sequences 50 of the total sequence length was contained in the 61,333 sequences longer than 757 bp. Over 1676428 half of the contigs were shorter than 250 bp and most of these short sequences did not code for proteins. We decided to remove all contigs smaller than 250 bp to simplify our analysis. This left 231,876 sequences with 50 of the total sequence length contained in 37,485 sequ.
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