Entering edit mode
8.4 years ago
Aiah
▴
10
Hi I have tried to generate a VCF file using this command
samtools mpileup -ugf A1163.fa.fai aP1.bam 2>stderr.out | bcftools view -bvcg - > aP1.raw.bcf
bcftools view aP1.raw.bcf | perl vcfutils.pl varFilter -D2 > aP1.flt.vcf
and it generated a small file without SNPs I tiered changing the depth to 2 but still nothing any suggestions ?
here is the generated VCF file
##fileformat=VCFv4.1
##INFO=<ID=DP,Number=1,Type=Integer,Description="Raw read depth">
##INFO=<ID=DP4,Number=4,Type=Integer,Description="# high-quality ref-forward bases, ref-reverse, alt-forward and alt-reverse bases">
##INFO=<ID=MQ,Number=1,Type=Integer,Description="Root-mean-square mapping quality of covering reads">
##INFO=<ID=FQ,Number=1,Type=Float,Description="Phred probability of all samples being the same">
##INFO=<ID=AF1,Number=1,Type=Float,Description="Max-likelihood estimate of the first ALT allele frequency (assuming HWE)">
##INFO=<ID=AC1,Number=1,Type=Float,Description="Max-likelihood estimate of the first ALT allele count (no HWE assumption)">
##INFO=<ID=AN,Number=1,Type=Integer,Description="Total number of alleles in called genotypes">
##INFO=<ID=IS,Number=2,Type=Float,Description="Maximum number of reads supporting an indel and fraction of indel reads">
##INFO=<ID=AC,Number=A,Type=Integer,Description="Allele count in genotypes for each ALT allele, in the same order as listed">
##INFO=<ID=G3,Number=3,Type=Float,Description="ML estimate of genotype frequencies">
##INFO=<ID=HWE,Number=1,Type=Float,Description="Chi^2 based HWE test P-value based on G3">
##INFO=<ID=CLR,Number=1,Type=Integer,Description="Log ratio of genotype likelihoods with and without the constraint">
##INFO=<ID=UGT,Number=1,Type=String,Description="The most probable unconstrained genotype configuration in the trio">
##INFO=<ID=CGT,Number=1,Type=String,Description="The most probable constrained genotype configuration in the trio">
##INFO=<ID=PV4,Number=4,Type=Float,Description="P-values for strand bias, baseQ bias, mapQ bias and tail distance bias">
##INFO=<ID=INDEL,Number=0,Type=Flag,Description="Indicates that the variant is an INDEL.">
##INFO=<ID=PC2,Number=2,Type=Integer,Description="Phred probability of the nonRef allele frequency in group1 samples being larger (,smaller) than in group2.">
##INFO=<ID=PCHI2,Number=1,Type=Float,Description="Posterior weighted chi^2 P-value for testing the association between group1 and group2 samples.">
##INFO=<ID=QCHI2,Number=1,Type=Integer,Description="Phred scaled PCHI2.">
##INFO=<ID=PR,Number=1,Type=Integer,Description="# permutations yielding a smaller PCHI2.">
##INFO=<ID=QBD,Number=1,Type=Float,Description="Quality by Depth: QUAL/#reads">
##INFO=<ID=RPB,Number=1,Type=Float,Description="Read Position Bias">
##INFO=<ID=MDV,Number=1,Type=Integer,Description="Maximum number of high-quality nonRef reads in samples">
##INFO=<ID=VDB,Number=1,Type=Float,Description="Variant Distance Bias (v2) for filtering splice-site artefacts in RNA-seq data. Note: this version may be broken.">
##FORMAT=<ID=GT,Number=1,Type=String,Description="Genotype">
##FORMAT=<ID=GQ,Number=1,Type=Integer,Description="Genotype Quality">
##FORMAT=<ID=GL,Number=3,Type=Float,Description="Likelihoods for RR,RA,AA genotypes (R=ref,A=alt)">
##FORMAT=<ID=DP,Number=1,Type=Integer,Description="# high-quality bases">
##FORMAT=<ID=DV,Number=1,Type=Integer,Description="# high-quality non-reference bases">
##FORMAT=<ID=SP,Number=1,Type=Integer,Description="Phred-scaled strand bias P-value">
##FORMAT=<ID=PL,Number=G,Type=Integer,Description="List of Phred-scaled genotype likelihoods">
##INFO=<ID=PR,Number=1,Type=Integer,Description="# permutations yielding a smaller PCHI2.">
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT
Can you first try to save the output of each step (samtools mpileup, bcftools view, bcftools view...>vcf)? Does the problem solely occur in the final step?
I am not very expert, but why did you provide A1163.fa.fai instead of A1163.fa? It is true that in the manual it says "indexed", but this means that you have to provide the FASTA and also have it indexed, but NOT provide the index name. Also, posting the stderr.out error file would be useful.
Thank you for your reply -I used the A1163.fa.fai file for the reason you mentioned. Are you saying that I should give the file a different name after indexing ?
-If I ran the command with A1163.fa I get the fallowing message: [bcf_sync] incorrect number of fields (0 != 5) at 0:0
I have tried sorteing my bam file but still no SNPs.
I think you need to address this first error in order to get output. First, can you check your BAM file isn't truncated using SAMtools view? Second, can you make sure that the FASTA file you're using for variant calling is exactly the same you used for alignment?
Thank you all I have checked BAM file isn't truncated and Yes I am using the same ref fasta file after indexing and tried it without .
Is your problem solved?