Supplemental Digital Content 3 - Appendix 3. Genotyping Methods
The Affymetrix GeneChip MegAllele System (Affymetrix, Inc. in conjunction
with ParAllele BioScience), incorporates Molecular Inversion Probe (MIP) technology.
The basic principles of MIP have been previously described in Hardenbol et al. 2003.(1)
For our custom SNP panel, single oligonucleotide probes are simultaneously annealed to
150ng of high quality genomic DNA to form a circularized probe surrounding each SNP.
Each probe consists of a recognition sequence flanking each SNP to be genotyped, a
universal PCR primer sequence, and a unique tag sequence complementary to a specific
site on the MegAllele TrueTag Array. After hybridization, each hybridized probe/DNA
mixture is then split into 4 samples and undergoes dNTP addition (1 different dNTP for
each sample) and ligation to fill in the single base gap, producing covalently sealed
probes for the appropriate nucleotides for each SNP. Each probe is then cleaved from the
genomic DNA, undergoes a unimolecular rearrangement, and lineralizes into an
amplifiable product. PCR with universal primers amplifies the probes. Only those probes
that were ligated can be amplified. Allele specific primers are added to the amplicons,
and a second PCR reaction provides an allele specific label to each probe. The probes are
then hybridized to the MegAllele TrueTag Array using the chip hybridization system.
Each hybridized chip is washed and stained using the GeneChip Fluidics Station, and
scanned with the Affymetrix GeneChip Scanner 3000. The array data is collected and
analyzed by the TrueCall Analyzer Software (Affymetrix, Inc.)
Genotyping using the sequenom perform was done using the iPLEX Gold
method. iPLEX reagents and protocols were used for multiplex PCR single base primer
extension (SBE) reactions. Multiplexed assays typically contain between
10-36 SNPs. Reactions were desalted and SBE products measured using the
MassARRAY system, and mass spectra analyzed using TYPER software (Sequenom,
San Diego), in order to generate genotype calls and allele frequencies (for complete
details see iPLEX Application Note, Sequenom, San Diego).
The SNPlexTM Genotyping System uses the Applied Biosystems oligonucleotide
ligation assay (OLA) combined with multiplex PCR technology to achieve allelic
discrimination and target amplification. The basic principles of SNPlex chemistry are
made possible through the use of a set of universal PCR primer sequences, a set of SNPspecific ligation probes and multi-color ZipChuteTM Probes that target unique allele
sequences of single nucleotide polymorphisms (SNPs) in genomic DNA (gDNA) samples
as described in Tobler et al, 2005.(2) During the OLA procedure, allele-specific probes
and linkers as well as universal linkers ligate to and phosphorylate 80ng of high quality
gDNA. The probes contain a unique ZipCode TM sequence for the ZipChuteTM probe that
binds to hybridized PCR product described later in this section. After the OLA reaction,
exonucleases purify the OLA product by digesting portions of the ligated product,
unligated, partially ligated oligonucleotides and gDNA. The purified product is
amplified with universal forward and reverse primers resulting in a double-stranded
amplicon with one biotinylated strand. The PCR product and positive hybridization
control is bound to a streptavidin-coated hybridization plate. The double stranded
amplicon is separated with the biotinylated strand remaining bound to the plate.
Fluorescently labeled ZipChuteTM probes hybridize to the ZipCode TM sequence. To
establish a sizing calibration curves, a fluorescently labeled size standard is added. The
sample with the ZipChuteTM probes are eluted from the hybridization plate and
transferred into a 96-well reaction plate, Allelic Ladder is dispensed into the reaction
plate and the plate is loaded into the Applied Biosystems 3130xl DNA Analyzer. The
Applied Biosystem 3130xl enables the separation and detection of SNP genotypes using
standard capillary arrays. The array data is collected and analyzed by GeneMapper
Software (Applied Biosystems).
1. Hardenbol, P, Baner, J, Jain, M, et al. Multiplexed genotyping with sequence-tagged
molecular inversion probes. Nat Biotechnol 2003; 21:673.
2. Tobler, A R, Short, S, Andersen, M R, et al. The SNPlex genotyping system: a
flexible and scalable platform for SNP genotyping. J Biomol Tech 2005; 16:398.