Allele QC for PomBase

A series of scripts for quality control of alleles. Currently used for PomBase, but could be adapted.

It checks that:

• Allele descriptions adhere to the allele nomenclature.
• Coordinates used in the allele descriptions are within the sequence length (E.g. we can't say a protein is missing aminoacids 120-150 if the protein length is 140).
• Aminoacids or nucleotides mentioned in allele descriptions occur at the indicated positions (E.g. if we say alanin 120 is mutated to glycine, check that the aminoacid at position 120 is indeed alanine).

TL;DR;

# Install dependencies
poetry install

# Activate python environment
poetry shell

# Set up the necessary transvar variables (you must have installed transvar, see next section)
. transvar_env_vars.sh
bash set_up_transvar.sh


# Run this script (See the comments in the subscripts)
bash run_analysis.sh

Installing

Python dependencies

To install the dependencies, we used poetry (see poetry installation instructions).

In the source directory run:

poetry install

This should create a folder .venv with the python virtual environment. To activate the virtual environment, then run:

poetry shell

Now when you call python, it will be the one from the .venv.

Other dependencies and setting up transvar

This project uses transvar. This requires to install some binaries.

# If you have linux and you want to install them globally
sudo apt install -y samtools tabix

# Installing globally in mac
brew install htslib samtools

# If you want to install them locally (see the content of the script)
# > basically downloads the libs and uses make to build the necessary bin files, then deletes all unnecesary source code
bash install_transvar_dependencies_locally.sh

Then, regardless of whether you are using local or global installation of samtools and tabix:

# Env vars (see script)
. transvar_env_vars.sh

# Build the transvar database, and test that it works
bash set_up_transvar.sh

What the pipeline does

The best thing is to look at the script run_analysis.sh, the subscripts are well documented.

Defining syntax rules in a grammar

These are used to interpret the allele descriptions, check that the sequence residues they refer to are correct, and to format the description correctly/

We define "syntax rules" representing the syntax of a type of mutation as dictionaries in a python list that we call a "grammar". The dictionaries are parsed into SyntaxRule objects (see models.py).

A full grammar can be found in grammar.py, and the best is to go through that example and the tests to understand how it works. Below an example of a rule to represent several single aminoacid mutations, in the form of VP120AA (Valine and Proline in position 120 and 121 replaced by Alanines).

aa = 'GPAVLIMCFYWHKRQNEDST'
aa = aa + aa.lower()
aa = f'[{aa}]'

{
        'type': 'amino_acid_mutation',
        'rule_name': 'single_aa',
        'regex': f'(?<=\\b)({aa})(\d+)({aa})(?=\\b)',
        'apply_syntax': lambda g: ''.join(g).upper(),
        'check_sequence': lambda g, gg: check_sequence_single_pos(g, gg, 'peptide'),
        'further_check': lambda g, gg: g[0] != g[2],
        'format_for_transvar': lambda g, gg: [f'p.{g[0]}{g[1]}{g[2]}']
    },

• type: the type of mutation (see below)
• rule_name: type + rule_name should be unique, it identifies the rule matching a particular mutation.
• regex: a regular expression with a pattern that represents a permisive syntax pattern for this type of mutation.
  • Note the use of {aa} inside the python f string to represent any aminoacid.
  • The pattern is a regex, so it can be permissive
  • The capture groups of the regex are passed to the functions below as a first argument.
• apply_syntax: a function that takes a tuple of re.Match[str], representing a match in a string to the pattern described in regex, and returns the correctly-formatted mutation.
  • In the example VP120AA, the groups are ('VP', '120', 'AA'), and the function returns VP120AA.
  • The function can be defined inline using lambda, or outside of the dictionary.
• check_sequence: a function that takes the two arguments below and checks whether the sequence position indicates exist (the index is within the boundaries of the sequence), and contain the indicated residue. If sequence is correct, should return empty string, otherwise, the residue or residues that are incorrect, see the examples in grammar.py and the tests.
  • A tuple of re.Match[str], representing a match in a string to the pattern described in regex
  • A "gene dictionary", see load_genome.py
• further_check: takes the same argument as above, and is used as an extra check on top of matching the regex pattern. In this example, it checks that the parts before and after the number are different (e.g. VP120VP will not be matched by this grammar rule, even if it matches the pattern).
• format_for_transvar: takes the same arguments as above, and returns the transvar formatted variant.

Defining allele categories

In PomBase we use categories for allele types, depending on the types of mutations they contain. These are described in a dictionary that uses frozenset objects as keys in grammar.py. For example:

allowed_types = {
    frozenset({'amino_acid_mutation'}): 'amino_acid_mutation',
    frozenset({'partial_amino_acid_deletion'}): 'partial_amino_acid_deletion',
    frozenset({'amino_acid_mutation','partial_amino_acid_deletion'}): 'partial_amino_acid_deletion'
    }

This is convenient because it allows to represent the fact that an allele that contains only the types of mutations indicated by a frozenset in the dictionary is of that type, e.g., if it has only amino_acid_mutation, it is of type amino_acid_mutation, if it contains amino_acid_mutation and partial_amino_acid_deletion, it is of type amino_acid_deletion_and_mutation.

Config file

See config.json, the variables included there are self-explaining. See also data/sgd/config.sgd.json for SGD.

New columns in allele file after analysis

• allele_parts: the substrings of the allele_description that match regex patterns in the grammar, separated by | characters. For example E325A G338D would result in E325A|G338D.
• needs_fixing: True or False depending on whether the allele needs fixing.
• change_description_to: if the correct nomenclature differs from allele_description, change_description_to contains the right syntax. E.g. for E325A G338D contains E325A,G338D.
• rules_applied: for each of the allele_parts, the syntax rule type and name as |-delimited type:name. E.g. for VP120AA,E325A contains amino_acid_mutation:multiple_aa|amino_acid_mutation:single_aa.
• pattern_error: contains the parts of the allele that are not picked up by any regular expression in the grammar.
• invalid_error: if the systematic_id or sequence of a gene product is missing.
• sequence_error: contains an error if the indicated position does not exist or if the aminoacid indicated at a certain position is not correct. Values to be homogenised in the future.
• change_type_to: if allele_type is not right, contains the right type. This comes from using the frozenset in grammar.py.
• auto_fix_comment: for alleles that can be auto-fixed, contains some info (e.g.):
  • syntax_error
  • type_error
  • syntax_and_type_error
  • multi_shift_fix: For a given publication with 4 or more alleles with errors, if shifting all coordinates by the same amount fixes the error, the fix is accepted. This fix has the lowest priority.
  • histone_fix: histone indexes have been often counted ignoring the first methionine, so if a shift by +1 in index fixes the sequence error in a histone, it is accepted. This type of fix takes second priority.
  • old_coords_fix, revision xxx: gene_coordinates: if using old gene coordinates the error is fixed, the fix is accepted. This type of fix takes max priority, as it is in principle more reliable.
• solution_index: Normally empty, but if more than one solution has been found to a sequence error, they have the index of the solution.

Optional - Using old coordinta changes for fixes

Some of the alleles for which sequence_errors are found might result from residue coordinates refering to previous gene structures. E.g. if the starting methionine has been changed, all residue coordinates are shifted. To fix this case, we use a genome change log produced with https://github.com/pombase/genome_changelog (for PomBase, see build_alignment_dict_from_genome.py). For DNA sequence, since the probability of getting the right nucleotide by chance is ~25%, we cannot be sure it is safe to switch coordinates even if that gives the right nucleotide.

For sgd data, we use a method that uses only the protein sequences: https://github.com/pombase/all_previous_sgd_peptide_sequences, see build_alignment_dict_from_peptides.py.

Running the API in Docker

docker build -t allele_qc_api .
docker run -d --name apicontainer -p 8000:80 allele_qc_api

Then if you go to http://localhost:8000/ you should be redirected to the API documentation and you can run a test request directly there.