Flo min 106d r9 flow cell

Three biological replicates of the seedling and callus samples from the same tissue samples used for Illumina RNA-Seq were sequenced by the Nanopore direct cDNA sequencing protocol. Briefly, mRNA was first isolated from 10 ug total RNA with Poly(A) RNA Selection Kit (Lexogen), followed by direct cDNA library preparation with SQK-DCS109 kit (Oxford Nanopore). The protocol version for library preparation was DCS_9090_v109_revB_04Feb2019. The cDNA library was loaded onto a FLO-MIN106D R9 flowcell and sequenced on MinION (Oxford Nanopore).
FAST5 raw data was converted to FASTQ data using the basecaller Guppy version 3.4.5 (Oxford Nanopore) with default parameters. Two steps of trimming were employed. Adapter sequence was first trimmed by porechop (version 0.2.4) (https://github.com/rrwick/Porechop) with parameters "--check_reads 10000 --adapter_threshold 100 --end_size 100 --min_trim_size 5 --end_threshold 80 --extra_end_trim 1 --middle_threshold 100 --extra_middle_trim_good_side 5 --extra_middle_trim_bad_side 50", and then poly A was trimmed by the software cutadapt (version 2.6) 70 with the options of " -g T{12} -e 0.1 -a A{12} -n 100". Trimmed reads were aligned to A188Ref1 as unstranded spliced long reads using MiniMap2 (version 2.14) 71 with the parameter "ax splice". Merged alignments from three replicates were input to StingTie2 for generating assembled transcripts.