CLI Workflow

Map reads to a reference genome

Map reads using pbmm2.

pbmm2 align --preset ISOSEQ --sort <input.bam> <ref.fa> <mapped.bam>

Collapse into unique isoforms

Collapse redundant transcripts into unique isoforms based on exonic structures using isoseq collapse.

isoseq3 collapse <mapped.bam> <collapsed.gff>

Sort input transcript GFF

Sort the transcript GFF file output from isoseq collapse.

pigeon sort <collapsed.gff> -o sorted.gff

Index the reference files

Index the genome annotation, (optional) CAGE peak, and (optional) intropolis files before classification.

pigeon index <gencode.annotation.gtf>
pigeon index <cage.bed>
pigeon index <intropolis.tsv>

Classify Isoforms

Classify isoforms into categories using the base required input.

pigeon classify <sorted.gff> <annotations.gtf> <reference.fa>

Alternatively, classify isoforms using supplemental reference information. Details in pigeon input.

pigeon classify <sorted.gff> <annotations.gtf> <reference.fa> --fl abundance.txt --cage-peak refTSS.bed --poly-a polyA.list

Filter isoforms

Filter isoforms from the classification output.

pigeon filter <classification.txt>

Report gene saturation

Gene saturation can be determined by subsampling the classification output and determining the number of unique genes at each subsample size.

pigeon report <classification.filtered_lite_classification.txt> <saturation.txt>

Make Seurat compatible input

Output files that are compatible with the downstream Seurat analysis package.

pigeon make-seurat --dedup molecules.fasta --group group.txt -d output_dir