Nes that share BLASTX similarity with respective databases. (B) Number of all annotated Odontotermes formosanus unigenes is figured out based on the summation of both unique sequence-based annotations and unique domain-based annotations. The circle “a” and “b” indicate the two subsets of O. formosanus unigenes with sequence-based annotations and domain-based annotations, respectively. doi:10.1371/journal.pone.0050383.gTranscriptome and Gene Expression in TermiteTable 2. Frequency of EST-SSRs in the head transcriptome of Odontotermes formosanus.Motif length Repeat numbers 4 Di Tri Tetra Penta Hexa Toatl ??935 280 137 1,352 11.59 5 ?1,642 679 43 17 2,381 20.42 6 1,140 1,126 171 14 14 2,465 21.14 7 905 1,115 29 7 3 2,059 17.66 8 801 275 29 5 6 1,116 9.57 9 707 74 22 6 4 813 6.97 10 593 60 8 2 0 663 5.69 .10 479 242 59 28 4 812 6.Total4,625 4,534 1,932 38539.66 38.88 16.57 3.3 1.doi:10.1371/journal.pone.0050383.tto Solexa sequencing 35013-72-0 web analysis at the Beijing Genomics Institute (BGI; Shenzhen, China). RNA quality and quantity were verified using a NanoDrop 1000 spectrophotometer and an Agilent 2100 Bioanalyzer prior to further processing at BGI, and 25331948 RNA integrity was confirmed with a number value of 8.6. The samples for transcriptome analysis were prepared using Illumina’s kit following manufacturer’s recommendations. Briefly, mRNA was purified from 44.4mg of total RNA using oligo (dT) magnetic beads. Fragmentation buffer was added for generation of short mRNA fragments. Taking these short fragments as templates, random hexamer-primer was used to synthesize the first-strand cDNA. The second-strand cDNA is synthesized using buffer, dNTPs, RNaseH and DNA polymerase I, respectively. Short fragments are purified with QiaQuick PCR extraction kit and resolved with EB buffer for end reparation and adding poly (A). After that, the short fragments were connected with sequencing adapters. And, after the agarose gel electrophoresis, the suitable fragments were selected for the PCR amplification as templates. At last, the library could be sequenced using Illumina HiSeqTM 2000.called contigs. Then the reads were mapped back to contigs; with paired-end reads it was able to detect contigs from the same transcript as well as the distances between these contigs. Trinity connected the contigs, and gets sequences that cannot be extended on either end. Such sequences were defined as unigenes. When multiple samples from a same species were sequenced, unigenes from each sample’s assembly could be taken into further process of sequence splicing and redundancy removing with sequence clustering software to acquire non-redundant unigenes as long as possible.Analysis of Illumina Sequencing ResultsUnigene sequences were firstly aligned by BLASTX to databases like nr, Swiss-Prot, KEGG and COG (E-value ,0.00001), retrieving proteins with the highest sequence similarity with the given unigenes along with their protein functional annotations, the results about this were included in the folder annotation. With nr annotation, we used Blast2GO program to get GO annotation of unigenes. After Methionine enkephalin getting GO annotation for every unigene [24], we used WEGO software to do GO functional classification for all unigenes and to understand the distribution of gene functions of the species from the macro level [25]. With the help of KEGG database, we could further study genes’ biological complex behaviors, and by KEGG annotation we could get pathway annotation for unigenes. When predict.Nes that share BLASTX similarity with respective databases. (B) Number of all annotated Odontotermes formosanus unigenes is figured out based on the summation of both unique sequence-based annotations and unique domain-based annotations. The circle “a” and “b” indicate the two subsets of O. formosanus unigenes with sequence-based annotations and domain-based annotations, respectively. doi:10.1371/journal.pone.0050383.gTranscriptome and Gene Expression in TermiteTable 2. Frequency of EST-SSRs in the head transcriptome of Odontotermes formosanus.Motif length Repeat numbers 4 Di Tri Tetra Penta Hexa Toatl ??935 280 137 1,352 11.59 5 ?1,642 679 43 17 2,381 20.42 6 1,140 1,126 171 14 14 2,465 21.14 7 905 1,115 29 7 3 2,059 17.66 8 801 275 29 5 6 1,116 9.57 9 707 74 22 6 4 813 6.97 10 593 60 8 2 0 663 5.69 .10 479 242 59 28 4 812 6.Total4,625 4,534 1,932 38539.66 38.88 16.57 3.3 1.doi:10.1371/journal.pone.0050383.tto Solexa sequencing analysis at the Beijing Genomics Institute (BGI; Shenzhen, China). RNA quality and quantity were verified using a NanoDrop 1000 spectrophotometer and an Agilent 2100 Bioanalyzer prior to further processing at BGI, and 25331948 RNA integrity was confirmed with a number value of 8.6. The samples for transcriptome analysis were prepared using Illumina’s kit following manufacturer’s recommendations. Briefly, mRNA was purified from 44.4mg of total RNA using oligo (dT) magnetic beads. Fragmentation buffer was added for generation of short mRNA fragments. Taking these short fragments as templates, random hexamer-primer was used to synthesize the first-strand cDNA. The second-strand cDNA is synthesized using buffer, dNTPs, RNaseH and DNA polymerase I, respectively. Short fragments are purified with QiaQuick PCR extraction kit and resolved with EB buffer for end reparation and adding poly (A). After that, the short fragments were connected with sequencing adapters. And, after the agarose gel electrophoresis, the suitable fragments were selected for the PCR amplification as templates. At last, the library could be sequenced using Illumina HiSeqTM 2000.called contigs. Then the reads were mapped back to contigs; with paired-end reads it was able to detect contigs from the same transcript as well as the distances between these contigs. Trinity connected the contigs, and gets sequences that cannot be extended on either end. Such sequences were defined as unigenes. When multiple samples from a same species were sequenced, unigenes from each sample’s assembly could be taken into further process of sequence splicing and redundancy removing with sequence clustering software to acquire non-redundant unigenes as long as possible.Analysis of Illumina Sequencing ResultsUnigene sequences were firstly aligned by BLASTX to databases like nr, Swiss-Prot, KEGG and COG (E-value ,0.00001), retrieving proteins with the highest sequence similarity with the given unigenes along with their protein functional annotations, the results about this were included in the folder annotation. With nr annotation, we used Blast2GO program to get GO annotation of unigenes. After getting GO annotation for every unigene [24], we used WEGO software to do GO functional classification for all unigenes and to understand the distribution of gene functions of the species from the macro level [25]. With the help of KEGG database, we could further study genes’ biological complex behaviors, and by KEGG annotation we could get pathway annotation for unigenes. When predict.