nextpolish安装_Iso-seq3安装与使用（全长转录组分析）

使用conda安装Iso-seq3

conda create -n anaCogent5.2 python=2.7 anaconda

source activate anaCogent5.2

conda install -n anaCogent5.2 biopython

conda install -n anaCogent5.2 -c http://conda.anaconda.org/cgat bx-python

conda install -n anaCogent5.2 -c bioconda isoseq3

conda install -n anaCogent5.2 -c bioconda pbccs

conda install -n anaCogent5.2 -c bioconda lima

#The packages below are optional:

conda install -n anaCogent5.2 -c bioconda pbcoretools # for manipulating PacBio datasets

conda install -n anaCogent5.2 -c bioconda bamtools # for converting BAM to fasta

conda install -n anaCogent5.2 -c bioconda pysam # for making CSV reports

Running IsoSeq

Typical workflow:

1. Generate consensus sequences from your raw subread data

$ ccs movie.subreads.bam movie.ccs.bam –noPolish –minPasses 1

2. Generate full-length reads by primer removal and demultiplexing

$ cat primers.fasta

>primer_5p

AAGCAGTGGTATCAACGCAGAGTACATGGGG

>primer_3p

AAGCAGTGGTATCAACGCAGAGTAC

$ lima movie.ccs.bam primers.fasta movie.fl.bam –isoseq –no-pbi

3. Remove noise from FL reads

$ isoseq3 refine movie.fl.P5–P3.bam primers.fasta movie.flnc.bam

4. Cluster consensus sequences to generate unpolished transcripts

$ isoseq3 cluster movie.flnc.bam unpolished.bam –verbose

5. Optionally, polish transcripts using subreads

$ isoseq3 polish unpolished.bam movie.subreads.bam polished.bam

6. Map unpolished or polished transcripts to genome and collapse transcripts based on genomic mapping

$ pbmm2 align unpolished.bam reference.fasta aligned.sorted.bam –preset ISOSEQ –sort

$ isoseq3 collapse aligned.sorted.bam out.gff

or $ isoseq3 collapse aligned.sorted.bam movie.ccs.bam out.gff