4.3. PacBio Library Preparation, Sequencing and Preprocessing

Total RNA for each of the four tissues was constructed library separately according to the PacBio Isoform Sequencing (Iso-Seq) experimental protocol. PacBio libraries were sequenced on the PacBio Sequel platform (Pacific Biosciences, Menlo Park, CA, USA). To obtain Polymerase reads, raw reads were used to remove data containing 0 or 2 DNA molecules as templates. Subreads were obtained by removing the connector and the data with a length less than 50 bp. The subreads.bAM file is processed by the Circular Consensus Sequence algorithm to obtain the CCS Sequence. The above data processing is carried out in SMRTlink v. 6.0 software (http://www.pacb.com/products-and-services/analytical-sofware/smrt-analysis/), and the parameters are set as: -minLength 50, -maxLength 15000, -minPasses 2, -min_seq_len 200, -minPredictedAccuracy 0.8, -minZScore -9999, -maxDropFraction 0.8, -min_seq_len 200, -minReadScore 0.65, -no_polish TRUE.

Arrow was used to calibrate the consensus sequence using the nFL sequences [64]. And the parameters were set as: Hq_quiver_min_accuracy 0.99, bin_BY_Primer false, bin_size_KB 1, Qv_TRIM_5p 100, Qv_TRIM_3p 30. To further improving the sequencing accuracy, validate the polished consensus sequence with second-generation data using LoRDEC V0.7 software and the parameters are set as follows: -K 23, -S 3 [65].

Using CD-HIT v. 4.6.8 to cluster the corrected transcript sequences according to the 95% similarity between the sequence, and the parameters are set as: -c 0.95, -T 6, -G 0, -aL 0.00, -aS 0.99, -AS 30 [66]. Select the core conserved gene set of terrestrial plants, namely embryophyta_odb9 (Creation date: 13 February 2016, number of species: 30, number of BUSCOs: 1440), and use BUSCO v. 3.0.2 to evaluate the completeness of the full-length transcriptome sequences [67].

The PacBio Iso-Seq FL transcriptome data were deposited in the Sequence Read Archive (SRA) of NCBI as follows: root: SRR12058216; stem: SRR12058215; leaf: SRR12058214; male cone: SRR12058213.