1. Quality control

2. Classification

QC and Classification, protocol version="2.3.0" id="RS_IsoSeq.1", default parameters.

	smrtpipe.py --distribute  --params=settings.xml --output=outputdir xml:input.xml 2> smrtpipe.stderr 1> smrtpipe.stdout

3. Clustering

Mapping and phasing

	perl phase_allotetraploid_pipeline.pl –flnc flnc.fastq --gmap_genome_directory database/ --gmap_genome_database databasename –outdir ./result --reference_fasta ref.fasta

Doing isoform-level-cluster according to alignments.

	python collapse_isoforms_by_sam.py -c 0.90 -i 0.90 --input flnc.fastq --fq -s flnc.sort.sam -o all

Consensus, each cluster generate one consensus sequence.

	perl analysis_cluster.pl all.collapsed.group.txt flnc.sort.sam flnc.fastq  > flnc.best.sort.sam

Doing isoform-level-cluster again.

	python collapse_isoforms_by_sam.py -c 0.90 -i 0.90 --input chose.fq --fq -s flnc.best.sort.sam -o all.consensus

Convert bam format to gff format.

	samtools view -bS all.consensus.collapsed.rep.fq.sam > all.consensus.bam
	bedtools bamtobed  -split -i all.consensus.bam > all.consensus.bed
	perl bed2cDNA_match.pl all.consensus.collapsed.rep.fq all.consensus.collapsed.rep.fq.sam > all.consensus.cDNA_match.gff

4. Transcriptome analysis

Alternative splicing analysis.

	python alternative_splice.py -i all.consensus.cDNA_match.gff -g ref.gtf -f ref.fasta -o ./ -os -as -ats T -op

Alternative polyadenylation analysis.

	perl polyA_position.pl all.consensus.collapsed.gff all.consensus.collapsed.rep.fq flnc.sort.sam > transcript_polyA.result

Finding fusion gene.

	python fusion_finder.py --input flnc.fastq --fq -s flnc.sort.sam -o ./fusion

Finding non-coding RNA.

	python PLEKModelling.py -lncRNA high_quality_lncRNA.fa -prefix species -mRNA mRNA.fasta
	python PLEK.py  -fasta flnc.fasta -out lncRNA.predicted -thread 10 -range species.range -model species.model -k 4

5. program list

changed to https://github.com/fancy1124/pipeline-for-isoseq

• smrtanalysis (http://www.pacb.com/products-and-services/analytical-software/smrt-analysis/)
• gmap (http://research-pub.gene.com/gmap/)
• phase_allotetraploid_pipeline.pl (attached)
• collapse_isoforms_by_sam.py from pbtranscript-tofu (https://github.com/PacificBiosciences/cDNA_primer)
• analysis_cluster.pl in-house Perl script
• bed2cDNA_match.pl in-house Perl script
• samtools (http://www.htslib.org/)
• bedtools (https://github.com/arq5x/bedtools2/)
• alternative_splice.py developed by ourselves (attached)
• polyA_position.pl
• fusion_finder.py (https://github.com/PacificBiosciences/cDNA_primer/blob/master/pbtranscript-tofu/pbtranscript/pbtools/pbtranscript/fusion_finder.py)
• PLEKModelling.py from plek (https://sourceforge.net/projects/plek/files/)
• PLEK.py from plek (https://sourceforge.net/projects/plek/files/)