type_variants

A python script to type predefined variants in a fasta-format alignment of SARS-CoV-2 sequences, which must be aligned to Wuhan-Hu-1 (discarding any insertions relative to it). Instructions for making an alignment are at the end of this README. See also the Genbank entry and the file MN908947.fa in this repository.

Frame-shifting mutations in coding sequence are ignored by this routine.

Usage

The variants to type must be defined by the user in a config file (see the example config.csv in this repository).

This file has one line per variant of interest, and each line must be in one of the following three formats:

snp:T6954C
del:11288:9
aa:orf1ab:T1001I

Any snp lines should consist of a :-separated string whose first part is "snp" and whose second part describes the reference allele, the 1-based position of the site, and the alternative allele.

Any del lines should consist of a :-separated string whose first part is "del", and whose second part is the 1-based first position of the deletion, and whose third part is the length of the deletion.

Any aa lines should consist of a :-separated string whose first part is "aa", and whose second part is the name of the coding sequence to query, and whose third part describes the reference amino acid, the 1-based number of the codon relative to the coding sequence it is in, and the alternative amino acid.

For snp and aa lines, if the reference allele does not match the allele at the specified coordinates in the reference fasta file, the program will write an error to stderr and exit.

To run the program:

python3 type_variants.py --fasta-in query.fasta --variants-config config.csv --reference MN908947.fa --variants-out out.csv

Which will produce the file out.csv:

❯ head out.csv
query,ref_count,alt_count,other_count,fraction_alt
seq1,0,9,0,1.0
seq2,0,7,2,0.7778
seq3,0,9,0,1.0
seq4,0,9,0,1.0
seq5,0,9,0,1.0
seq6,0,9,0,1.0
seq7,0,9,0,1.0
seq8,0,9,0,1.0
seq9,0,9,0,1.0
...

ref_count is the total number of reference alleles in this query

alt_count is the total number of predefined alternative alleles in this query

other_count is the total number of alleles in this query that are neither ref nor alt - this includes other valid alleles as well as missing data.

fraction_alt = alt_count / (ref_count + alt_count + other_count)

Appending genotypes to the output

You can also append the genotype of each variant in the config file to the output with the --append-genotypes flag:

python3 type_variants.py --fasta-in query.fasta --variants-config config.csv --reference MN908947.fa --variants-out out.withgenotypes.csv --append-genotypes

❯ head out.withgenotypes.csv
query,ref_count,alt_count,other_count,fraction_alt,snp:C3267T,del:11288:9,aa:orf1ab:T1001I,del:21765:6,snp:G24914C,aa:S:N501Y,snp:A28111G,aa:Orf8:Q27*,aa:N:S235F
seq1,0,9,0,1.0,T,del,I,del,C,Y,G,*,F
seq2,0,7,2,0.7778,T,X,I,X,C,Y,G,*,F
seq3,0,9,0,1.0,T,del,I,del,C,Y,G,*,F
seq4,0,9,0,1.0,T,del,I,del,C,Y,G,*,F
seq5,0,9,0,1.0,T,del,I,del,C,Y,G,*,F
seq6,0,9,0,1.0,T,del,I,del,C,Y,G,*,F
seq7,0,9,0,1.0,T,del,I,del,C,Y,G,*,F
seq8,0,9,0,1.0,T,del,I,del,C,Y,G,*,F
seq9,0,9,0,1.0,T,del,I,del,C,Y,G,*,F
...

Each genotype column contains the allele for that variant in each query sequence.

For amino acids, X denotes missing data/an untranslatable codon

For deletions, X denotes an allele that is neither the reference allele nor the deletion. Deletion variants are otherwise coded as ref (same nucleotide sequence as the reference) or del.

Making an alignment in Wuhan-Hu-1 coordinates

If you have a consensus fasta file containing sequences that haven't been aligned to Wuhan-Hu-1, you can make an alignment to feed to this python script using minimap2, the latest version of gofasta and the reference fasta file:

minimap2 -a -x asm5 MN908947.fa unaligned.fasta | gofasta sam toMultiAlign > aligned.fasta

Or potentially using MAFFT with the --keeplength option ("Keep alignment length" in the web app).