MORFAL (Molecular Open Reading Frame AnaLyzer)

MORFAL is a pipeline build for the prediction of TIS and annotation of uORF (upstream open reading frame).

Requirements

• Samtools (tested on version 1.14) with hg19 fasta file
• Netstart (tested on version 1.0c) in singularity container
• VEP (tested on release-109.3)
• UTRannotator (https://github.com/ImperialCardioGenetics/UTRannotator) in singularity conntainer

Usage

Usage: python3 /path/to/MORFAL.py [-gtf gtf_file] [-vcf vcf_file] [-O outdir]

Option:
-gtf,    --gtf  TEXT      /Path/to/gtf file
-vcf,    --vcf  TEXT      /Path/to/vcf files
-O,      --outdir  TEXT    /Path/to/output directory
-hg,     --hg19fasta  TEXT      /Path/to/hg19 fasta file
-net,    --netstart_container  TEXT      /Path/to/netstart singularity container
-UTR,    --UTRannotator  TEXT      /Path/to/UTRannotator singularity container

Annotation Output

The output annotation is named output_annotation.txt . The other files are outputs from tools or from the concatenation and filtering stage. The annotations can be read in 4 sections: VCF and GTF information, Netstart prediction and delta scores, UTRannotator annotations and GnomAD population frequencies.

VCF and GTF information :

• identifiant : unique identifier created for each variant/transcript pair
• chr : chromosome carrying of the variant
• position : genomic position of the variant
• c. : position of the variant relative to the start of the CDS
• ref : affected nucleotide of wild-type sequence
• alt : mutated nucleotide of the mutated sequence
• name_gene : name of the gene affected by the variant
• gene_id : Ensembl identifier of the gene
• transcript_id : Ensembl identifier of the transcript
• strand : strand direction -> + if strand is sense, - if strand is antisense

Netstart prediction and delta scores

• score_ATG_CDS(pos_ns) : score predicted by Netstart for TIS and their position in the transcript
• delta_ATG_CDS : delta score for TIS
• delta_min : smallest delta score for transcript ATGs
• score_delta_min(pos) : score predicted by Netstart for the smallest delta score and their position in the transcript
• delta_max : highest delta score for transcript ATGs
• score_delta_max(pos) : score predicted by Netstart for the highest delta score and their position in the transcript
• abolition(pos) : score and position of lost ATG if available
• new(pos) : score and position of created ATG if available
• diff_phy_new : difference between CDS score and new ATG score

UTRannotator annotations

• Distance_CDS : distance between start of uORF and CDS
• Distance_stop : distance between start and stop of uORF
• Distance_Cap_start : distance between CAP and start of uORF
• Kozak_context : kozak sequence
• Kozak_strength : strength of sequence kozak
• type : type of uORF: uORF, OutOfFrame_oORF, inFrame_oORF
• type_ref : uORF reference type if this change
• Distance_stop_ref : distance between start and stop of uORF reference if it change
• evidence : whether the uORF disrupted has any translation evidence
• UTR_consequence : output the variant consequences of a given 5 prime UTR variant: uAUG_gained, uAUG_lost, uSTOP_gained, uSTOP_lost, uFrameshift
• nb oORF_inframe : number of existing inframe overlapping ORFs (inFrame_oORF) already within the 5 prime UTR
• nb oORF_outframe : number of existing out-of-frame overlapping ORFs (inFrame_oORF) already within the 5 prime UTR
• nb uORF : number of existing uORFs with a stop codon within the 5 prime UTR
• consequence : variant consequence on the transcript
• impact : strength of impact of variant on the transcript

Frequencies GnomAD

• Clin_sig : ClinVar classification
• gnomad_combi : frequency of existing variant in gnomAD exomes combined population
• gnomad_afr : frequency of existing variant in gnomAD exomes African/American population
• gnomad_ame : frequency of existing variant in gnomAD exomes American population
• gnomad_jew : frequency of existing variant in gnomAD exomes Ashkenazi Jewish population
• gnomad_east_asi : frequency of existing variant in gnomAD exomes East Asian population
• gnomad_fin : frequency of existing variant in gnomAD exomes Finnish population
• gnomad_eur : frequency of existing variant in gnomAD exomes Non-Finnish European population
• gnomad_other_combi : frequency of existing variant in gnomAD exomes other combined population
• gnomad_south_asi : frequency of existing variant in gnomAD exomes South Asian population