## 2 Results
### RNA Sequencing and Transcriptomic Assembly  
Full-length transcriptome of _C. porrectum_ was generated from normalized full-length cDNAs from pooled poly(A) RNA of one sample (leaf) and subjected to single molecule real-time (SMRT) sequencing via the PacBio Sequel system. In total, two SMRT cells generated 9,624,949 subreads (13.3 Gb); the average read length was 1389.96 bp and an N50 were 2147 bp. A total of 683,767 reads of insert (ROIs) were generated and 104,062 those sequences containing two primers and a poly-A tail were identified as full-length reads of inserts(ROIs), which were subsequently classified into chimeric and nonchimeric reads. Of these ROIs, 289,102 were identified as full-length non-chimeric (FLNC) reads with an average read length 2819.5 bp (Figure 1a). Full-length nonchimeric sequences were clustered, and with the Quiver algorithm, each cluster was corrected and integrated into a consensus sequence. The consensus sequence output by Quiver was divided into low-quality sequences (low QV, LQ) and high-quality sequences (high QV, HQ). A total of 213586 full-length consensus transcripts, including 133,966 HQ (\(>99\%\) accuracy) transcripts and 79620 LQ transcripts, were generated. Information on each library of clustered reads is shown in Table S1, and the sequence length and sequence quality of the low-QV and high-QV sequences were compared (Figure S1). Subsequent analyses only used high-QV consensus sequences. Iso-Seq produced more unigenes with lengths \(>2000\) bp than Illumina sequencing.  
These results indicated that PacBio Iso-Seq provides a practical method for producing full-length transcripts without assembly steps and is an important improvement for transcriptome studies of species without reference genomes.  
\begin{table}
\begin{tabular}{c c c c c c c c c c c} \hline
**Rla** & **Rlb** & **Component** & **Y\_11 (\%)** & **Y\_12 (\%)** & **Y\_13 (\%)** & **F\_11 (\%)** & **F\_12 (\%)** &  
Library clusters and high-quality sequences obtained after error correction were finally merged to eliminate redundancy. After this process, the transcriptome of _C. porrectum_ had a total of 104,062 transcripts with a total of 306,337,921 bp; the average sequence length was 2944 bp, and the N50 length was 5449 bp (Table 2). For the length distribution of subreads, see Figure 1b, and the length distribution of nonredundant transcripts is shown in Figure 1c.  
After clustering and error correction, the TransDecoder software was used to identify the candidate coding regions in each transcript. First, the longest open reading frame was extracted. Then, using BLAST, the SwissProt database was compared with Hnmscan to search for homology to Pfam domains to predict the coding regions. A total of 89,103 complete transcripts were obtained. The details of the prediction results are listed in Table 3, and the coding sequence(CDS) length distribution is shown in Figure 1d.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Sample** & **Total Isoforms** & **Total Base (bp)** & **Mean Length (bp)** & **N50 (bp)** \\ \hline Total & 104,062 & 306,337,921 & 2944 & 5449 \\ \hline \hline \end{tabular}
\end{table}
Table 2: Summary of the final transcript sequences obtained after the elimination of redundancy.  
Figure 1: PacBio single-molecule long-read sequencing of _C. porrectum_. (**a**) Reads classification from PacBio Iso-Seq. (**b**) distribution of subreads length. (**c**) Read length distribution. (**d**) Length distribution of all isoforms coding sequence(CDS).  
\begin{table}
\begin{tabular}{c c} \hline \hline
**Software** & **Total Number** & **Total Length** & **Mean Length** & **N50** & **N70** & **N90** & **GC (\%)** \\ \hline Blast & 88763 & 66645204 & 750 & 1041 & 687 & 363 & 44.66 \\ ESTScan & 340 & 205098 & 603 & 729 & 501 & 336 & 43.75 \\ Overall & 89103 & 66850302 & 750 & 1038 & 687 & 363 & 44.66 \\ \hline \hline \end{tabular}
\end{table}
Table 3: Statistics of CDS prediction results.