IsoSeqAnalysis

Steps of my analysis used in IsoSeq

HPC settings

#SBATCH --time 48:00:00 #SBATCH --nodes=1 #SBATCH --ntasks=1 #SBATCH --cpus-per-task=12 #SBATCH --mem=500GB

WT steps for the CPR1 gene:

Step 1: Lima for primer removal

lima m64146_230816_190133.hifi_reads.bam PacBioPrimers.fa movieX.fl.bam --isoseq --peek-guess

Step 2: Isoseq Refine for Poly A Tail removal

isoseq refine movieX.fl.3prime_3p--5prime_5p.bam PacBioPrimers.fa movieX.flnc.bam --require-polya

Step 3: IsoSeq Cluster2 for clustering reads

isoseq cluster2 movieX.flnc.bam WT_clusters.bam

Step 4: Pbmm2 Allign for alligning reads to a reference

pbmm2 align --preset ISOSEQ --sort WT_clusters.bam CPR1_Genomic_flank_genes_rev_comp.fa WT_CPR1_mapped.bam

Step 5: Isoseq Collapse for collasping reads into unique isoforms

isoseq collapse WT_CPR1_mapped.bam WT_CPR1_collapsed.gff