Microphaser

Microphaser is a tool for phasing small tumor DNA sequences - e.g. coding for small peptides - in linear time.
It can be used in tumor neoantigen prediction to generate the neo-peptidome.

Installation

Microphaser is available for installation via conda. Use conda install -c biconda microphaser to easily install the current version.

Usage

Input

To use microphaser, you need the following input files:

• a sorted and indexed bam file containing mapped tumor reads
• a reference genome in fasta format
• a matching gene and transcript annotation in gtf format
• a bcf/vcf file containing germline and somatic variants, where somatic variants should be flagged with a SOMATIC INFO tag
• optional: a bcf/vcf file containing only germline variants

Output

Microphaser returns three important files:

• two filtered fasta files containing all neopeptides and their wildtype counterparts for further use with MHC-binding prediction tools
• an info file in tsv format containing meta-information about every neopeptide

The info table consist of the following fields:

• id: peptide identifier as found in the fasta files
• transcript: Ensembl transcript name
• gene_id: Ensembl gene name
• gene_name: Gene symbol
• chrom: Chromosome
• offset: Position of the neopeptide on the chromosome
• freq: Frequency of the neopeptide occurring in all reads overlapping the peptide position
• depth: Read depth at the peptide position
• nvar: number of variants in the neopeptide
• nsomatic: number of somatic variants in the neopeptide
• nvariant_sites: number of variant sites in the range of the neopeptide
• nsomvariant_sites: number of somatic variant sites in the range of the neopeptide
• strand: Strand orientation of the transcript (forward or reverse)
• somatic_positions: Positions of the somatic variants in the neopeptide
• somatic_aa_change: Somatic Amino Acid changes occuring in the neopeptide
• germline_positions: Positions of germline variants in the neopeptide
• germline_aa_change: Germline Amino Acid changes occuring in the neopeptide
• normal_sequence: Nucleotide sequence of the wildtype peptide
• mutant_sequence: Nucleotide sequence of the neopeptide

Run

Currently, microphaser consists of four different submodules:

• somatic (returns neopeptides and their corresponding normal peptides)
• normal (returns all normal peptides of the patient)
• build_reference (returns a binary file representing the patients normal peptidome)
• filter (compares neopeptides against the normal peptidome and removes self-similar candidates)

You can run microphaser like this:

Phasing of the tumor reads and variants:

microphaser somatic tumor.bam -r reference.fasta -b all_variants.bcf -t neopeptides.info.tsv -n peptides.wt.fasta < annotation.gtf > peptides.mt.fasta

Generation of the patients healthy peptidome:

microphaser normal normal.bam -r reference.fasta -b germline_variants.bcf < annotation.gtf > healthy_peptides.fasta

Building the reference binary file of the healthy peptidome:

microphaser build_reference -r healthy_peptides.fasta -o peptides.bin > peptides.translated.fasta

Filtering the neopeptide candidates from subcommand microphaser somatic:

microphaser filter -r peptides.bin -t neopeptides.info.tsv -o neopeptides.filtered.info.tsv -n normal_peptides.filtered.fasta > neopeptides.filtered.fasta

Authors

• Jan Forster (https://github.com/jafors)
• Johannes Köster (https://koesterlab.github.io)