Question: Hi all,

I've received a set of BAM files , the variant were called with bcftools

${bcftools_exe} mpileup -Ou -f "${REF}" \
--bam-list "${bam_list}" \
--regions-file "${bedfile}" \
--annotate 'FORMAT/AD,FORMAT/ADF,FORMAT/ADR,FORMAT/DP,FORMAT/SP,INFO/AD,INFO/ADF,INFO/ADR' \
--redo-BAQ --adjust-MQ 50 --min-MQ 30 |\
${bcftools_exe} call \
--ploidy GRCh37 \
--multiallelic-caller \
--variants-only -O z -o	"output.vcf.gz"

but I suspect there is a cross-contamination between the sample, because many of the HOM_REF genotypes contain a few ALT allele.

The variants were called with samtools, but some genotypes called as HOM_REF contain a few ALT

+---------+---------+--------+-------+-------+-----+-----+-----------+----+
| Sample | Type | AD | ADF | ADR | DP | GT | PL | SP |
+---------+---------+--------+-------+-------+-----+-----+-----------+----+
| 28D0609 | HOM_REF | 206,15 | 97,9 | 109,6 | 221 | 0/0 | 0,255,255 | 4 |
| 37D1676 | HOM_REF | 154,10 | 89,5 | 65,5 | 164 | 0/0 | 0,229,255 | 1 |
| 13D0720 | HET | 170,59 | 92,27 | 78,32 | 229 | 0/1 | 134,0,255 | 5 |
| 37D1631 | HOM_REF | 155,16 | 73,8 | 82,8 | 171 | 0/0 | 0,76,255 | 0 |
| 57D1188 | HOM_REF | 85,0 | 39,0 | 46,0 | 85 | 0/0 | 0,255,255 | 0 |
| 14D2313 | HOM_REF | 101,0 | 50,0 | 51,0 | 101 | 0/0 | 0,255,255 | 0 |
| 24D2314 | HOM_REF | 48,0 | 18,0 | 30,0 | 48 | 0/0 | 0,144,255 | 0 |
| 24D0430 | HOM_REF | 64,0 | 31,0 | 33,0 | 64 | 0/0 | 0,193,255 | 0 |
| 18D0610 | HOM_REF | 55,0 | 29,0 | 26,0 | 55 | 0/0 | 0,166,255 | 0 |
+---------+---------+--------+-------+-------+-----+-----+-----------+----+

Some samples were sequenced in the same flowcell/lane.

How can I validate the hypothesis of a cross contamination ?

I was suggested to use **verifyBamID** but as far as I understand, It need another VCF called with another method (?)

I also tried to use Gatk **ContEst** but I've no idea of what I'm doing...

java -ja GenomeAnalysisTK.jar -T ContEst -I bam.list -R human_g1k_v37.fasta -o out.metrics --genotypes my.vcf.gz -pf 1000G_phase1.snps.high_confidence.b37.vcf --min_genotype_depth 20 -L 22

INFO 10:17:00,850 ContEst - Total sites: 31803838
INFO 10:17:00,860 ContEst - Population informed sites: 310728
INFO 10:17:00,861 ContEst - Non homozygous variant sites: 310728
INFO 10:17:00,861 ContEst - Homozygous variant sites: 0
INFO 10:17:00,861 ContEst - Passed coverage: 0
INFO 10:17:00,861 ContEst - Results: 0

any suggestion ?

Answer: A really nice method is [GATK CalculateContamination][1] and gives you an exact contamination estimate. It works if you have WGS/WES data (to provide sufficient coverage for enough SNPs). They provide a reference VCF for human genome. It needs to be in a specific format, so can be tricky to generate for other species, especially since population frequencies may not be known.

I've been using [Bamkin][2], which is fairly simple and crude, but seems to work sufficiently well to detect sample mixups. I processed hundreds of samples and it's always been clear when some of them are problematic, at least when you have multiple samples from supposedly the same individual. The nice thing is it will work with smaller targeted panels or ChIP-seq or RNA-seq. You can also tell if the contamination is coming from other samples in the same batch of samples.

[1]: https://software.broadinstitute.org/gatk/documentation/tooldocs/current/org_broadinstitute_hellbender_tools_walkers_contamination_CalculateContamination.php
[2]: https://github.com/igordot/bamkin