Building a De Novo Transcriptome

The transcriptome step involves constructing a de novo based transcriptome from the full length transcripts.

Minimap2 is first used to construct an initial mapping to the reference genome. Then, Pinfish is used to polish the genome-based transcript to obtain the transcriptome. This is done by first clustering reads with similar intron and exon structure,

This part of the pipeline is primarly based on the ont_tutorial_pinfish released by Nanopore Technologies.

Usage

Input -

/path/to/genome/fasta/X.fa
Results/Pychopper/X.pychop.fastq

Output - Results/Gffcompare/nanopore.combined.gtf

Configuration

Below are changes that can be configured in the config.yaml file or explicitly specified in the command line.

Minimap2

minimap2_opts_junction: TRUE - Use annotation to improve splice junction mapping.

minimap2_opts: -uf - Required for stranded data.

minimum_mapping_quality: 5 - Minimum mapping quality.

minimap2_opts_polished: -s - Required for stranded data.

Pinfish

spliced_bam2gff: 0.25 - Stringency of porechop heuristic.

minimum_cluster_size: 3 - Minimum size to cluster transcripts.

exon_boundary_tolerance: 10 - Exon boundary tolerance.

terminal_exon_boundary_tolerance: 50 - Terminal exon boundary tolerance.

minimum_isoform_percent: 1 - Minimum percentage of reads necessary to call an isoform.

collapse_internal_tol: 5 - Internal exon boundary tolerance.

collapse_five_tol: 500 - 5' boundary tolerance.

collapse_three_tol: 50 - 3' boundary tolerance.

spliced_bam2gff_opts_pol: -s - Required for stranded data.

Output Folder Structure

| -- ReferenceData/
    | -- junctions.bed
| -- Results/
    | -- Minimap2/
        | -- ReferenceFasta.mmi
        | -- merged.mapping.bam
    | -- Pinfish/
        | -- raw_transcripts.gff
        | -- clustered_pol_transcripts.pol_gff
        | -- cluster_memberships.tsv
        | -- clustered_transcripts_collapsed.gff
        | -- polished_transcripts.fas
        | -- polished_reads_aln_sorted.bam
        | -- polished_transcripts.gff
        | -- polished_transcripts_collapsed.gff
        ...