Supplemental Methods
General procedure for normalization of pooling data
The Illumina BeadStation software is designed to call genotypes, but it also
reports the raw intensity data for each of two alleles of a SNP assay. These data can be
adapted to the measurement of pooled DNA. Corrections need to be made since the ratio
of the two dye intensities can vary widely across assays. We derived correction factors
for each SNP from raw intensity data from individuals assayed on the genotyping
platform. We expected that the precision of the Illumina platform, where each SNP is
interrogated with approximately 30 identical bead probes, would make it possible to
correct signals with high accuracy. Since DNA from a heterozygous individual is
equivalent to DNA from a pool with a 50% allele frequency, we calculated the correction
factor k for each SNP (1), based on the average ratio of dye intensities (A:B) across all
known heterozygotes. The mean k value was 0.84, indicating that such unequal
fluorescence is the norm and correction is therefore necessary.
We applied k to the relative allele signal (RAS = A / (A + B)) for each SNP to
obtain a raw allele frequency [RAFk = A / (A + k*B)], a value which is aligned to the
true 50:50 allele position for that SNP assay. For each SNP, extreme values were
normalized to data obtained from the RAS values of known homozygotes, who are
equivalent to pools of with 0% or 100% allele frequencies, respectively (2). The
homozygote RAS values for each SNP were averaged and used to normalize the RAFk
value to a 0-to-1 scale [(RAFk - mean (BB) / mean (AA)]. The average RAS for A in true
BB homozygotes was 0.039, and the average RAS for B in true AA homozygotes was
0.035, indicating that normalization results in a more precise allele frequency estimate.
Testing of normalization procedure
We tested our normalization procedure by pooling equimolar amounts of DNA
from 88 neurologically normal individuals (plate NDPT008, Coriell Institute of Medical
Research, Camden, NJ) who had been individually genotyped with the Infinium I
technology (data provided courtesy of J.H.). Three pools were made to test the variance
introduced by the pooling process itself. Variance attributable to the genotyping chip was
estimated by measuring aliquots of each pool with two InfiniumI chips and one or two
InfiniumII chips (the two platforms share 23,907 SNPs in common). The 3 correction
factors (k, average RAS(AA) , and average RAS(BB)) were calculated from 240 subjects
individually genotyped on the same machine using the InfiniumI chip (unpublished data)
and from the 270 HapMap individuals genotyped by Illumina, Inc. using the InfiniumII
chip.
As expected, the uncorrected RAS values correlated less well with allele
frequencies estimated from the individual genotype data (r=0.86, p < 10-16) than did
RAFk and the fully normalized values (r=0.95, p<10-16). Pools systematically
overestimated allele frequencies even after correction, especially alleles who true
frequency was <5% (median difference between true frequency and normalized
frequency = -0.016).
Normalization of pooled data from the NIMH and German samples
We measured each pool on at least two chips. In pools derived from the NIMH
sample, the mean within-pool measurement standard deviation of transformed SNP
measurements was 0.017. Only 1.27% of SNP/pool combinations had a standard
deviation greater than 0.05. In pools derived from the German sample, the standard
deviation was larger: 0.042, and 23% of the SNP/pool combinations had a standard
deviation greater than 0.05. This probably reflects lower quality for some of the DNA
specimens in the German sample.
1. Hoogendoorn B, Norton N, Kirov G, Williams N, Hamshere ML, Spurlock G et al.
Cheap, accurate and rapid allele frequency estimation of single nucleotide
polymorphisms by primer extension and DHPLC in DNA pools. Hum Genet 2000;
107:488-493.
2. Craig DW, Huentelman MJ, Hu-Lince D, Zismann VL, Kruer MC, Lee AM et al.
Identification of disease causing loci using an array-based genotyping approach on
pooled DNA. BMC Genomics 2005; 6:138.
Supplemental References
Bipolar disorder linkages
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