GMT Tutorial Exercise 26

Converting "raw" Luminex data to genotypes with the SNPscorer macro

Background

• When a "FlexMAP microsphere"-based OLA or ASPE assay is carried out on the Luminex platform, each microsphere's internal dye (also referred to as its "bead address") and the associated reporter dye are detected.
• The Luminex-embedded analysis software reports the number of microspheres with a given bead address that were detected, and the "mean reporter dye intensity" associated with these microspheres.
• The SNPscorer macro (downloadable from GENica Marker Tools) converts this raw output into genotype scores through the following actions:
  1. "Bead addresses" are replaced with the name and allele of the assayed SNP
  2. "Mean reporter dye intensities" are corrected for background noise
  3. Thresholds for minimum microspher counts are applied
  4. Detection thresholds for minimum mean reporter dye intensity are applied
  5. "Allele ratios" are calculated from reporter dye intensities for pairs of SNP probes
  6. Allele ratio "cut off" thresholds are applied to make genotype calls
  7. AR values obtained for the set of samples that were analysed are graphically displayed, these scatter plots can be used e.g. to help decide whether AR values falling outside the AR value "cut off" points can be assigned a genotype or not.
• Please note: In our ASPE assays for SNP markers, extended probes are labelled with a fluorescent dye, therefore, "mean fluorescence intensity" is used alongside of "mean reporter dye intensity".

The current version of the "SNPscorer macro" was specifically created for users of the locally implemented SNP markers, 
but further developments to make the macro more generic are planned. For updated information on the status of this macro, 
please contact Matt Hayden.

The instructions below aim to give a basic overview of how to use the macro. 

For the tutorial exercise on importing SNP genotype data, please use the downloadable example file, see Exercise 3.

Workflow

1. Generate the raw Luminex data with the BioPlex Manager Software.
2. Download and "unzip" the SNPscorer macro from GENica Marker Tools.
3. Open the macro and enable the macro functions.
4. Open the "Work Flow" worksheet and follow its step-by-step instructions.
   
5. Save the table with allele ratio values and genotypes on the "GENica Export Table" worksheet as a "text file".
   If desired, this file can be imported into SNPDB, see tutorial exercise 27.