Hi Rasmus,
mpileup outputs the candidate indel
MyRef 120 . G GGAAT
which is then not called by bcftools (*). The -v option is not present,
therefore all input records are printed on output. The -A option is not
present, therefore unused ALT alleles are trimmed. The INDEL tag is a
reminiscence of the site being a candidate indel.
I hope this helps
Petr
(*) that is, with the default options, it would be called with -mP 0.01
On Fri, 2014-10-31 at 15:56 +0100, Rasmus Borup Hansen wrote:
> Hi! I'm getting VCF files with some strange INDELS from bcftools. The
> shell script below contains everything needed to reproduce it (I'm
> using bwa 0.7.10-r789, samtools 1.1, and bcftools 1.1) and it outputs
> the following:
>
>
> Lines for position 120 when the VCF is generated by "samtools
> mpileup":
>
>
> MyRef 120 . G <X> 0 .
> DP=3;I16=0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0;QS=0,0;MQ0F=0 PL 0,0,0
> MyRef 120 . G GGAAT 0 .
> INDEL;IDV=2;IMF=0.666667;DP=3;I16=0,0,1,0,0,0,30,900,0,0,60,3600,0,0,25,625;QS=0,1;SGB=-0.379885;MQ0F=0
> PL 30,3,0
>
>
> Lines for position 120 when the VCF is generated by "bcftools call":
>
>
> MyRef 120 . G . 0 .
> DP=3;MQ0F=0;AN=0;DP4=0,0,0,0;MQ=. GT .
> MyRef 120 . G . 2.1484 .
> INDEL;IDV=2;IMF=0.666667;DP=3;SGB=-0.379885;MQ0F=0;AN=1;DP4=0,0,1,0;MQ=60
> GT 0
>
>
> Output from "samtools mpileup" around position 120:
>
>
> MyRef 110 G 2 ., mD
> MyRef 111 T 2 ., bD
> MyRef 112 G 2 ., eD
> MyRef 113 T 2 ., jD
> MyRef 114 T 2 C, kD
> MyRef 115 G 1 . m
> MyRef 116 G 1 . e
> MyRef 117 G 1 . j
> MyRef 118 G 1 . i
> MyRef 119 A 1 . k
> MyRef 120 G 0
> MyRef 121 A 0
> MyRef 122 C 1 . I
> MyRef 123 T 1 . k
> MyRef 124 C 2 ., gA
> MyRef 125 A 2 Gg dA
> MyRef 126 G 2 ., _A
> MyRef 127 T 2 ., iF
> MyRef 128 G 2 ., fH
> MyRef 129 C 2 ., jJ
> MyRef 130 C 2 ., iI
>
>
> It's the output from "bcftools call" that has ALT="." while the
> "INDEL" flag is present that worries me. Is this a bug, or just
> something I don't understand (yet)?
>
>
> The shell script to reproduce the above output:
>
>
> #!/bin/bash
>
>
> # Make directory for data
> mkdir -p tmp-data
> cd tmp-data
>
>
> # Redirect stderr.
> exec 2>stderr
>
>
> # Use this short refence.
> cat >ref <<EOF
> >MyRef
> GGTGGCGAAGGCGGCTTGCTGGACAAATACTGACGCTGAGGTGCGAAAGCGTGGGGAGCA
> AACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTGGGGAG
> ACTCAGTGCCGCAGCTAACGCAATAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGA
> EOF
>
>
> # Use these reads.
> cat >reads.fastq <<EOF
> @Instrument:1:FlowCell:1:1:1:1/1
> ACCTTGCGGTCGTACTCCCCAGGTGGAGTGCTTATTGCGTTTGCTGCGGCACCGACCATCTCTGGCCAACACCTAGCACTCATCGTTTACGGCGTGGA
> +
> CCCFFFFFHFHHGJJJJ3EHGIJ?FHDHFHJJIJJJJJJHIJJJJJJIJHFFDDDDDDDDDDEDCDDDDDDDDDDCCCCDDDDDDBDDDDDDDDDDDD
> @Instrument:1:FlowCell:1:1:1:1/2
> GGTGGCGAAGGCGGCTTACTGGACTGTTACTGACGCTGAGTCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACG
> +
> CCBFFFFFHHHHHJJJJJJIJJIIJJJIJJJJJIJJJIJJHHHHHFFFDE9=BDDDDDDDDDDDDDDDDDDDDEDDDDDDADDDEDDDBDDDDDDDD<
> @Instrument:1:FlowCell:1:2:2:2/1
> TCAACCTTGCGGTCGTACTCCCCAGGTGGAGTGCTTATTGCGTTAGCTGCGGCACCGAGGATTCCTCCCCGACACCTAGCACTCATCGTTTACGGCG
> +
> BCCFFFFFHHHGFHIJJGHIIIIHDH?GDFI*BGIHIIJGEGAEEHIHJIGHFFB?@DDBBCDCDCCDBDDDBBDB9CCDDD@?CDDDDDD?@@DBB
> @Instrument:1:FlowCell:1:2:2:2/2
> GGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTCGGGGAGGAATCCTCGGTGCCGCAGCTAACGCAATAAGCACTCCACCTGGGGAGTACGACCGC
> +
> BBBDFDF?FHHHFHHHIJIJJJIHIHGJIGIJADGHJDGGIGGGFHHHHHF>DACCDBDBDDDDDBDD@DDCDDDDDDDCDDDCBBB@DDCDDDBDBB
> EOF
>
>
> # Prepare indices of the reference.
> samtools faidx ref
> bwa index ref
>
>
> # Align the reads to the reference.
> bwa mem -p -t 4 -R '@RG\tID:MySample\tSM:MySample' ref reads.fastq >
> alignment.sam
>
>
> # bwa-mem needs to infer the insert size distribution from data. You
> # have to mix the read pair with at least tens of other pairs. For
> # this reason bwa doesn't think the reads are properly paired, so we
> # set the flags manually.
> perl -pe '@_=split /\t/; if ($_[0] !~ /^@/) { @_[1] |= 2;
> $_=join("\t", @_) }' alignment.sam > fixed_alignment.sam
>
>
> # Sort the alignment.
> samtools sort -T samtools.sort fixed_alignment.sam -O bam >
> sorted_alignment.bam
>
>
> # Index the sorted alignment.
> samtools index sorted_alignment.bam
>
>
> # Make a file containing the ploidy of the sample (for "bcftools call
> -m").
> echo -e >sample.tab "MySample\t1"
>
>
> # Generate vcf using samtools.
> samtools mpileup -r MyRef:110-130 -vuf ref sorted_alignment.bam >
> pos110-130.samtools.vcf
>
>
> # Generate vcf using bcftools.
> samtools mpileup -r MyRef:110-130 -uf ref sorted_alignment.bam |
> bcftools call -m -S sample.tab -O v > pos110-130.bcftools.vcf
>
>
> # Generate mpileup data.
> samtools mpileup -r MyRef:110-130 -f ref sorted_alignment.bam >
> mpileup.tab
>
>
> # Output results.
> echo -e "\nLines for position 120 when the VCF is generated by
> \"samtools mpileup\":\n"
> grep -P '^MyRef\t120' pos110-130.samtools.vcf
> echo -e "\nLines for position 120 when the VCF is generated by
> \"bcftools call\":\n"
> grep -P '^MyRef\t120' pos110-130.bcftools.vcf
> echo -e "\nOutput from \"samtools mpileup\" around position 120:\n"
> cat mpileup.tab
> echo
>
>
> ### END OF SCRIPT
>
>
>
>
> Best,
>
>
> Rasmus Borup Hansen
>
> Intomics is a contract research organization specialized in deriving
> core biological insight from large scale data. We help our clients in
> the pharmaceutical industry develop tomorrow's medicines better,
> faster, and cheaper through optimized use of biomedical data.
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> System Administrator Diplomvej 377
> Scientific Programmer DK-2800 Kgs. Lyngby
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