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.
Single-cell IsoSeq:
isoseq3 collapse <mapped.bam> <collapsed.gff>
Bulk IsoSeq:
isoseq3 collapse --do-not-collapse-extra-5exons <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>
If you want to generate a filtered GFF, you need to also provide the GFF that was used as input to pigeon classify
pigeon filter <classification.txt> --isoforms <sorted.gff>
The expected output consists of:
*.filtered_lite_classification.txt
*.filtered_lite_junctions.txt
*.filtered_lite_reasons.txt
*.sorted.filtered_lite.gff (only if --isoforms is used)
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 <dedup.fasta> --group <collapse.group.txt> -d <output_dir> <classification.filtered_lite_classification.txt>
The dedup.fasta file is obtained after running isoseq3 groupdedup or isoseq3 dedup. The collapse.group.txt file is obtained after running isoseq3 collapse.
The output will consist of:
Make-seurat output:
<output_dir>/annotated.info.csv
<output_dir>/info.csv
<output_dir>/genes_seurat/barcodes.tsv
<output_dir>/genes_seurat/genes.tsv
<output_dir>/genes_seurat/matrix.mtx
<output_dir>/isoforms_seurat/barcodes.tsv
<output_dir>/isoforms_seurat/genes.tsv
<output_dir>/isoforms_seurat/matrix.mtx