Can´T Find The Snps With Samtools (Only Get Indels)

Question

[Pilar E.]

Hello everybody,

Could anyone tell me how to get SNPs using SAMtools???. I am learning how to do it but can´t get the SNPs, I only get the Indels. I am working with a mutant I created from an E coli genome (just for practice). The mutant was created with 5 mismatches, 5 insertions and 5 deletions (using Biopieces).

The lenght and coverage is here:

read_fasta -i  <fasta ref genome>.fna | shred_seq -s 100 -c 50 | add_ident -k SEQ_NAME | write_fasta -x -o  <rrreads>.fna

The alignment was made with BWA . Then I used SAMtools protocol (as described in some of the posts) like this :

samtools mpileup -uf <refgenome>.fasta result.sort.bam > results.bcf

and then ,

bcftools view -vcg results.bcf - > test.vcf

If anyone can help me .. Thanks!!! :)

[Michael]

might be the alignment step, too. did you enable mismatches for the alingments? Because, if just by chance only indels were allowed but 0 mismatches, then you cannot call SNPs. Can you provide the bwa parameters?

[Pilar E.]

Thanks for answering !Here is the BWA :

read_fasta -i reference.fasta | mutate_seq -n 5 | indel_seq -i 5 -d 5 | write_fasta -xo mutant.fna read_fasta -i mutant.fna | shred_seq -s 100 -c 50 | add_ident -k SEQ_NAME | write_fasta -xo reads.fna mkdir BWA (for alignment files) bwa index -p BWA/Ecoli reference.fasta bwa aln BWA/Ecoli reads.fna > reads.sai bwa samse -f result.sam BWA/Ecoli reads.sai reads.fna

Do you see anthing wrong??? Thanks for helping Jeremy! :)

[jimkozubek]

Pilar,

I am having trouble with this same situation.

I have a VCF file with only indels. Did you figure out why this occured?

Jim

Answer 1

[Swbarnes2]

First simple thing to try: samtools mpileup -Buf. Sometimes, the BAQ calculations will wrongly believe real SNPs to be undiagnosed indels, and will drop their quality in the pileup step such that they can't be called as SNPs

Since you know where your missing SNPs are, look at them in the pileup text file. If the SNP is detected, but the quality score is terrible, despite being fine in the .bams, that's what happened.