single cell amplification and sequencing of full-length RNAs by Nanopore platform
Nanopore rawdata basecalling use Guppy(v3.1.5) to generate the fastq data from electric signals.
guppy_basecaller -i /project/test/cell/ -s ./ -c /opt/ont/guppy/data/rna_r9.4.1_70bps_fast_prom.cfg -x auto -z -q 8000
barcode demultiplex use nanoplexer
nanoplexer -b barcode.fa -p /ouput/ input.fastq
filter low quality reads (qscore < 7) and short reads (length <100bp) use nanofilt
NanoFilt.py -l 100 -q 7 raw.fastq --logfile log > clean.fastq
NanoStat.py --fastq clean.fastq --outdir ./ --name clean.stat.xls
reads were identified, oriented and trimmed using Pychopper
python3 /export/pipeline/RNASeq/Software/Pychopper2/v2.0.3/bin/cdna_classifier.py -m edlib -u unclassified.fa -A aln_hits.bed -b primers.fa -c primer_config.txt -S statistics.txt -t 8 clean.fastq full_length_output.fq
genome alignments were performed with the arguments “-ax splice -uf -k14 --secondary=no”