Entering edit mode
10.1 years ago
Max Ivon
▴
130
Hello everybody,
Could anybody tell me why when I'm using samtools it doesn't find Heterozygous Indels (homozygous are found correctly as SNPs)?? Here is the command lines used:
bwa index -p ref gla.fasta
bwa mem -t 4 ref reads.fastq > proj.sam
samtools view -bS proj.sam > proj.bam
samtools sort proj.bam sorted_proj
samtools index sorted_proj.bam
samtools mpileup -C50 -L30000 -ugf gla.fasta sorted_proj.bam | bcftools view -bvcg - > proj.raw.bcf
bcftools view proj.raw.bcf > pr.flt.vcf
In how large indels are you interested in? samtools will identify only very small indels, you may consider other tools for larger indels and/or rearrangements.
I'm interested in small indels. I've got an alignment where i can see heterozygous insertion of one G but samtools dont see it.
Are reads confirming that insertion near the reference ends? Do they have sufficient quality score? How many there are?
And I'm back to the problem (got problems with health) :) Mutation occurs approximately in the middle of the amplicon. If i remove all reads with quality lower then 30, then SamTools finds it properly. More interesting, I;ve found that mutation occurs only in reads sequenced from left to right (12,000 out of 13,000 reads got altered nucleotide according to SamTools) when in reads, sequenced from right to left there is no it at all. So may be it is the problem of Sequencing (was performed on Ion Torrent PGM). I found even more such deletion/insertions and all are G or C. So is there mutation in heterozygote or no?????
P.S. I found that in SamTools code is written to work only with the first 8,000 reads or less. And this maximum value can not be changed with input parameters. Strange.
Sounds interesting, can you show us a screenshot of alignments, please?
If the mutation only turns up in one direction, something is wrong. I don't think you can believe that's a real indel.