I have an issue with high FAM fluorescence that lis the likely cause of incorrect genotype calls.
I obtain twice as many FAM alleles relative to the reference genotypes of the control samples (64 loci in 6 individuals).
Reference genotypes come from Illumina RNA-seq with coverage >10 in all 6 individuals.
Allele counts in the reference genotypes:
Major allele (V): 523
Minor allele (F): 229
Allele counts in OpenArray genotypes:
Major allele (V): 263
Minor allele (F): 443
No Call: 23
FAM fluorescence intensity values are almost double those of VIC:
Mean ThroughHole.IntensityData.FAM.1: 5053.44
Mean ThroughHole.IntensityData.FAM.1: 3310.39
Fluorescence intensity in the throughholes is not uniform but shows a strong highlight on one edge of the throughholes that gives them a "3D" look (see attached OpenArray FAM image file from experiment) .
VIC and ROX images from the same imaging run appear normal; repeated imaging twice with same results.
Could the higher FAM fluorescence lead to genotype calls strongly biased towards F alleles?
Using TaqMan® OpenArray® 64 loci custom Genotyping Plate
All DNA samples were titrated to 12.5 ng/ul (genome size 0.8pg - Bluefin tuna)
gDNA was quantitated using PicoGreen assay fluorimetry, and qualitatively evaluated with agarose gel electrophoresis.
You need review the design of the TaqMan probes, if they are in the chain forward or reverse strand ("design strand", example "C" forward Fam or Vic, reverse "G" Fam or Vic, review the "AB Assay Information File") in C/G and T/A you can confuse the minor/major alleles in forward/reverse strand, is a common problem with different information (strand design) of companies Applied and illumina.
- Badges
- Science method