call-rna-transcript-vars

Call RNA variants in a long-read RNA-seq assembled transcriptome BAM file.

Usage

Template:

exacto call-rna-transcript-vars \
    --bam-file <bam_file> \
    --reference-genome-fasta-file <reference_genome_fasta_file> \
    --reference-gene-annotation-file <reference_gene_annotation_file> \
    --reference-gene-annotation-source <reference_gene_annotation_source> \
    --reference-gene-annotation-assembly <reference_gene_annotation_assembly> \
    --reference-gene-annotation-version <reference_gene_annotation_version> \
    --output-dir <output_dir> \
    --output-prefix <output_prefix> \
    [--num-threads NUM_THREADS] \
    [--chunk-size CHUNK_SIZE] \
    [--min-mapping-quality MIN_MAPPING_QUALITY] \
    [--min-terminal-softclip-length MIN_TERMINAL_SOFTCLIP_LENGTH] \
    [--temp-dir TEMP_DIR] \
    [--bkpt-rescue BKPT_RESCUE] \
    [--bkpt-rescue-min-ins-len BKPT_RESCUE_MIN_INS_LEN] \
    [--bkpt-rescue-realignment-gap-open-score BKPT_RESCUE_REALIGNMENT_GAP_OPEN_SCORE] \
    [--bkpt-rescue-realignment-gap-extend-score BKPT_RESCUE_REALIGNMENT_GAP_EXTEND_SCORE] \
    [--bkpt-rescue-realignment-k BKPT_RESCUE_REALIGNMENT_K] \
    [--bkpt-rescue-realignment-band-width BKPT_RESCUE_REALIGNMENT_BAND_WIDTH] \
    [--bkpt-rescue-realignment-min-score-fraction BKPT_RESCUE_REALIGNMENT_MIN_SCORE_FRACTION] \
    [--bkpt-rescue-realignment-min-query-coverage BKPT_RESCUE_REALIGNMENT_MIN_QUERY_COVERAGE] \
    [--bkpt-rescue-realignment-min-placed-fraction BKPT_RESCUE_REALIGNMENT_MIN_PLACED_FRACTION] \
    [--bkpt-rescue-realignment-max-pieces BKPT_RESCUE_REALIGNMENT_MAX_PIECES] \
    [--predict-nonsense-mediated-decay PREDICT_NONSENSE_MEDIATED_DECAY] \
    [--translation-strategy TRANSLATION_STRATEGY] \
    [--start-codons START_CODONS [START_CODONS ...]] \
    [--nmd-distance-threshold NMD_DISTANCE_THRESHOLD] \
    [--dna-variants-tsv-files DNA_VARIANTS_TSV_FILES [DNA_VARIANTS_TSV_FILES ...]] \
    [--dna-variant-match-buffer DNA_VARIANT_MATCH_BUFFER]

Example:

exacto call-rna-transcript-vars \
    --bam-file stitched/tumor_exacto_stitched_transcripts.sorted.bam \
    --reference-genome-fasta-file reference_genome.fasta \
    --reference-gene-annotation-file gencode.gtf.gz \
    --reference-gene-annotation-source gencode \
    --reference-gene-annotation-assembly hg38 \
    --reference-gene-annotation-version v45 \
    --output-dir rna_variants/ \
    --output-prefix tumor

Description

Call RNA variants in a long-read RNA-seq assembled transcriptome BAM file.

NoteAt a glance

Inputs: *.bam (assembled transcripts aligned with cs tags — in the exacto pipeline, the realigned stitched FASTA of stitch-reference-transcripts), *.fasta (reference genome), *.gtf.gz (gene annotation)

Outputs: {prefix}_exacto_assembled_transcripts.tsv and {prefix}_exacto_assembled_transcript_{model_alignments,variants,exons,splice_junctions,reference_transcript_matches,nmd_predictions,filter_status}.tsv under --output-dir

Typical next step: integrate-vars, translate-transcripts, quantify-rna-abundances

Required arguments

Flag Type Description
--bam-file str Input BAM file of assembled transcripts.
--reference-genome-fasta-file str Reference genome FASTA file.
--reference-gene-annotation-file str Reference gene annotation file.
--reference-gene-annotation-source str Reference gene annotation source.
--reference-gene-annotation-assembly str Reference gene annotation assembly (e.g. ‘hg38’).
--reference-gene-annotation-version str Reference gene annotation version (e.g. ‘v41’).
--output-dir str Output directory.
--output-prefix str Output prefix.

Optional arguments

Flag Type Default Description
--num-threads int 4 Number of threads.
--chunk-size int 10000 Number of reads processed per chunk.
--min-mapping-quality int 0 Minimum mapping quality.
--min-terminal-softclip-length int 4 Terminal soft-clip runs shorter than this many bases emit no variant record; 1-2 bp terminal clips are aligner anchor jitter, not junction or insertion evidence. Set 0 to disable.
--temp-dir str Temp directory.
--bkpt-rescue str2bool yes Whether to rescue breakpoints from insertions by re-aligning long insertion sequences.
--bkpt-rescue-min-ins-len int 100 Minimum insertion length to be considered for re-alignment (to rescue breakpoints).
--bkpt-rescue-realignment-gap-open-score int -5 Gap open score for insertion re-alignment (to rescue breakpoints).
--bkpt-rescue-realignment-gap-extend-score int -1 Gap extend score for insertion re-alignment (to rescue breakpoints).
--bkpt-rescue-realignment-k int 11 K-mer size for insertion re-alignment (to rescue breakpoints).
--bkpt-rescue-realignment-band-width int 50 Band width for insertion re-alignment (to rescue breakpoints).
--bkpt-rescue-realignment-min-score-fraction float 0.8 Minimum score fraction for insertion re-alignment (to rescue breakpoints).
--bkpt-rescue-realignment-min-query-coverage float 0.1 Minimum query coverage for insertion re-alignment (to rescue breakpoints).
--bkpt-rescue-realignment-min-placed-fraction float 0.9 Minimum placed fraction for insertion re-alignment (to rescue breakpoints).
--bkpt-rescue-realignment-max-pieces int 3 Maximum number of pieces for insertion re-alignment (to rescue breakpoints).
--predict-nonsense-mediated-decay str2bool yes Whether to predict nonsense-mediated decay for the open reading frames of each transcript.
--translation-strategy str (longest_orf|all_orfs) longest_orf Translation strategy: which complete open reading frames of each transcript to evaluate when predicting nonsense-mediated decay.
--start-codons str AUG One or more start codons. Pass multiple as: –start-codons AUG GUG CUG.
--nmd-distance-threshold int 50 Distance in nucleotides upstream of the last exon-exon junction beyond which a stop codon marks the transcript as NMD-sensitive.
--dna-variants-tsv-files str One or more DNA variant TSV files written by ‘call-germline-dna-vars’ or ‘call-somatic-dna-vars’ (column ‘origin’ required). An RNA variant with the same chromosomes, operations, variant type and sequence as a DNA variant, each position within –dna-variant-match-buffer bases (strand not compared), carries the DNA variant’s origin in the ‘origin’ column of the RNA variants TSV file.
--dna-variant-match-buffer int 1 Bases each position of a matching DNA variant may differ by.