## 2 Results and Discussion  
### Transcriptome Sequencing, de novo Assembly and General Metabolism  
Sequencing was performed on RNA extracted from the roots of _T. laciniata_, utilizing two lanes of an Illumina GAIIx platform. A total of 66,776,746 high quality (HQ) reads with an average sequence length of 104.5 bp were obtained from 76,028,196 raw reads after the initial quality-filtering step. The HQ reads were assembled into 76,565 contigs with the use of the Trinity RNA-Seq assembly package [29], with a minimum length cut off set at 300 bp. 64.58 million of the 66.78 million reads were used in the assembly, with the rest not being assembled to larger contigs. The contigs had an N50 of 1261 bp, with a range of 300-16,683 bp and a total GC content of 41%.  
The assembly for our sequencing reads was initially carried out with both the Trinity and Velvet assemblers. In general, multi-kmer assemblies are slightly poorer in terms of N50 values [30], but we found that the Trinity assembler was significantly better at resolving splice alternates in the dataset than Velvet (data not shown). The Trinity assembly also produced less duplicates or assembly chimeras, which are often introduced in a Velvet/Oases multi-run merging stage, hence generating a lower total number of long reads than the Velvet output. In addition, the ratio of reads recruited for assembly was greater in the Trinity assembly suggesting better coverage of the transcriptome. Since Trinity has this resolving power, its results were chosen for analysis.  
The 76,565 assembled contigs obtained from the Trinity assembly were successively annotated with MAGPIE [31] through comparison with the sequences in major public protein databases (KEGG, the non-redundant NCBI Protein collection, the plant subset of Refseq, Interpro, and the NCBI Conserved Domain Database). A total of 47,408 contigs, accounting for 61.9% of the total contigs, were annotated. 52,101 were associated with Gene Ontology terms of which 43,816 contigs received high level hits (\(E<10^{-35}\)) (Tables S1 and S2: S1 is the summary of the annotation obtained from NCBI and S2 is the summary of the annotations obtained from InterPro including GO annotation).  
The Illumina dataset can be used to substantially aid the discovery of novel genes involved in the general metabolism as well as specialized metabolism. When using _de novo_ assembled transcriptomes for the identification of novel transcripts, it is first important to determine whether the depth of the sequencing is sufficient to enable the accurate and complete assembly of the majority of expressed transcripts. Using protein sequences of the well-described and highly conserved tricarboxylic acid (TCA) cycle as BLAST queries against our assembled contigs, we determined that transcripts encoding all of these enzymes could be identified (Table 1). Full length transcripts encoding all TCA cycle components enzymes were present in our assembly and were represented by between 7805 (in the case of Dihydrolipoyl dehydrogenase) and 194838 (in the case of isocitrate dehydrogenase) sequencing reads. This indicated that the depth of sequencing obtained from our Illumina sequencing of the _T. laciniata_ transcriptome was sufficient for the assembly of complete transcripts, and also that the Trinity algorithm used for assembly was accurate. It is also important to note that little or no degradation of RNA had occurred prior to sequencing; otherwise, assembled contigs would not be full length.  
Based on the search and annotation results, several definite sequence features could be used to identify the specific terpene synthases, cytochromes P450, aldehyde and possibly alcohol dehydrogenase involved in the modification of terpene skeletons. Candidate genes related to sesquiterpene backbone biosynthesis and similar to sesquiterpene synthases have been identified and will be discussed in detail, similarly for downstream enzymes involved in terpenoid biosynthesis such as cytochromes P450.