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 Iso-Seq:

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

Bulk Iso-Seq:

isoseq collapse --do-not-collapse-extra-5exons <mapped.bam> <flnc.bam> <collapsed.gff>

Note: The optional <flnc.bam> input is required to get the correct FLNC counts for bulk Iso-Seq in the flnc_count.txt supplemental file.

Prepare reference files for pigeon

As of version 1.1.0 pigeon prepare replaces the pigeon sort and pigeon index tools. Use pigeon prepare to sort and index the genome annotation, (optional) CAGE peak, and (optional) intropolis files before classification. This step ensures that all records for a given chromosome/scaffold are contiguous within the file. Additionally, if a reference fasta is provided, the fai index will be generated.

pigeon prepare <gencode.annotation.gtf> <reference.fa> <cage.bed> <intropolis.tsv>

or input a file of file names

pigeon prepare files.fofn

More information about pigeon reference input can be found here.

Prepare input transcript GFF

Use prepare to sort the transcript GFF file output from isoseq collapse.

pigeon prepare <collapsed.gff>

Classify Isoforms

Transcript classification

Classify isoforms into categories using the base required input.

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

Adding supplemental reference information to classification output

Additionally, supplemental reference information can be added to the classification.txt output. Additional reference details can be found in pigeon input.

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

Alternatively use provided reference sets here.

pigeon classify <sorted.gff> --ref Human_hg38_Gencode_v39.referenceset.xml

Adding FLNC counts to classification output

FLNC counts can be added to the classification.txt output. Pigeon uses supplemental files from isoseq collapse to to add counts.

For single-cell Iso-Seq, use the abundance.txt output from isoseq collapse. This file contains the deduped FLNC counts and cell barcodes.

pigeon classify <collapsed.sorted.gff> <annotations.gtf> <reference.fa> --fl abundance.txt

For bulk Iso-Seq, use the flnc_count.txt output from isoseq collapse. This file contains the FLNC counts after isoseq refine separated by sample if applicable.

pigeon classify <collapsed.sorted.gff> <annotations.gtf> <reference.fa> --fl flnc_count.txt

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 <collapsed.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 and isoform- level saturation can be determined by subsampling the classification output and determining the number of unique genes / isoforms at each subsample size.

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

For bulk Iso-Seq that has been clustered using cluster2, use the --exclude-singletons option.

pigeon report --exclude-singletons <classification.filtered_lite_classification.txt> <saturation.txt>

Make Seurat-compatible gene- and isoform- count matrix for single-cell Iso-Seq

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 isoseq groupdedup. The collapse.group.txt file is obtained after running isoseq 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

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