Ambiguity Code Handling

josiahseaman commented 4 years ago

Problem: SARS-CoV2 genomes already downloaded have ambiguity codes in them. Our pipeline needs to handle this in some way.

Simon Heumos: Sequences like 'Y' or 'K' are encoding a nucleotide, 'C, T or U' and 'G, T or U' respectively. For more detailed information see the FASTA specification https://en.wikipedia.org/wiki/FASTA_format. I also ran a short Python script to validate each FASTA entry. Key function was https://biopython.org/DIST/docs/api/Bio.Alphabet-module.html#_verify_alphabet. All entries passed the test.

Add the Python script for completeness. sars_filter_proteins.py

from Bio import SeqIO
from Bio.Seq import Seq
from Bio.Alphabet import IUPAC
from Bio.Alphabet import _verify_alphabet
for record in SeqIO.parse("SARS-CoV-2.genbank.20200329.fasta", "fasta"):

If the script is run, no sequence identifier will be printed. Do you think this will be an issue for some downstream analysis like mapping or doing graph manipulation in vg? If so, we could mask all these ambivalent nucleotides with 'N'.

Josiah Seaman: My initial inclination is to leave the data the way it is. Those ambiguity codes are actual information after all. A far downstream feature would actually be unpacking those ambiguities into the matrix to see what is plausible based on extant variation in others, but that's low value at the moment. We also don't want to bloat the pangenome sequence with N's, almost better to leave those as a gap. ... With N's there's a new kind of coverage "sequence is known to be present, but we don't know what it is". Conclusion: preserve the ambiguity codes all the way up to FASTA chunks so that they can be visible in sequence download: https://github.com/graph-genome/Schematize/issues/36 In rendering, show ambiguity as gaps, no coverage.

Highest Priority: Pipeline doesn't crash or discard entire sample because of ambiguity codes.

6br commented 4 years ago

May I transfer this to the proper repository, e.g. component_segmentation or pipeline? I think this not affects the visualization itself.

josiahseaman commented 4 years ago

There's something important I've noticed about our dataset: Those "private insertion" nodes we've been hunting down contain ambiguity codes. We talked about these in April https://github.com/graph-genome/Schematize/issues/44 but currently they're being handled as completely novel sequence. That may be why ClustalW is giving different results; it would properly handle ambiguity codes as being a near trivial difference.

For example MT246470 starts with TTTATACCTTCSCCCC which separates it from others without ambiguities.

This requires some thought. We don't want to report a variant based only on an ambiguity code, which is what we're doing right now. But if we already had a variant nucleotide at that position, I'd be more inclinded to take an ambiguity seriously. One path for now would be to always make them N's, but that still causes the same problem of creating new nodes where they are not necessarily needed. The other option would be to set the sequence to match "consensus" where ambiguities are present. This would either need to be before seqwish, or in the alignment process itself. How does minimap handle ambiguities?

This is all I was able to find https://lh3.github.io/minimap2/minimap2.html --score-N INT Score of a mismatch involving ambiguous bases.

josiahseaman commented 4 years ago

Possible options:

[ ] Throw out all genomes containing ambiguities (stop gap solution)
[ ] Minimap can handle ambiguous bases
[ ] Seqwish can handle ambiguous bases
[ ] We could make a graph with a different tool that can handle ambiguous bases
[ ] Tool to make MSA, use consensus sequence to overwrite ambiguities
[ ] Write a script to transfer consensus sequence
[ ] Replace amiguities with N's or deletions? (vg does this)
- Doesn't really solve false diversity problem
[ ] Schematize handling -- doesn't address novel nodes problem
[ ] Check to see that remaining sequences don't have ambiguities

josiahseaman commented 4 years ago

Identified problematic sequences

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Last 6 Pathnames to be excluded:

['http://collections.lugli.arvadosapi.com/c=80946cc74d6f6ba751a8d7950d7b1c75+126/sequence.fasta',
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 'http://collections.lugli.arvadosapi.com/c=59172eea057dd24b550d969e1f4aac5f+126/sequence.fasta']

graph-genome / Schematize

Ambiguity Code Handling #44

Last 6 Pathnames to be excluded: