Genotyping

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Genotyping

• PAAR, member of PGRN
• Ed Cook, Soma Das University of Chicago
• Peter Imle St. Judes

2
Many SNP typing methods

• PCR-direct sequencing with Polyphred v 4 allele
  calling (shorter sequencing pyrosequencing)
• PCR-RFLP
• Allele-specific PCR (/- locked nucleic acids)
• Oligonucleotide ligation (OLA Nickerson)
• Includes bead array by Illumina for multiplexing
• Hybridization/quencher-TaqMan (SNPs on demand and
  by design), Molecular Beacons
• Primer extension (based on sequence, extension of
  different length by ddNTP of specific
  base)(Sequenom)
• SBE (detection methods-FP,dHPLC,CE,tag array)

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Single base extension (SBE)

• Detection methods
• Fluorescent polarization (FP-TDI)(? Cheapest SBE
  method, but relatively low assay conversion rate)
• dHPLC (? highest SBE conversion rate, can be
  duplexed or more)
• CE (SNaPshot)(can be pooled)
• Tag array (Orchid, Affymetrix, homegrown)(allows
  high rate of multiplexing PCR and pooling SBE
  products)

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SNP Genotyping Advantages and Disadvantages

• Low cost per genotype and high throughput, but
  only when running thousands of subjects per SNP
  for a few SNPs
• Availability of equipment (location-specific)
• Equipment without separation or scanning (e.g.
  FP, TaqMan)(either may be run by LjL fluorescent
  96-384 microplate reader)
• Requirement (or not) of labeled primers or ddNTPs
  (SBE-dHPLC)
• Requirement of relatively expensive
  reporter/quencher probes (TaqMan, Molecular
  Beacons)(cost depends on numbers of samples to
  genotype)
• High conversion rate (e.g. OLA, SBE-dHPLC, ?
  TaqMan)
• Ease of typing (TaqMan, one reaction, one plate)

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(No Transcript)
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SBE-DHPLC

• Sample is amplified by PCR
• PCR product is purified by SAP and
• exoI to eliminate excess primers and
• dNTPs
• An extension reaction is done using a
• primer directly adjacent to the base in
• question and ddNTPs
• Only one base is added to the 3-end of
• the primer resulting in an extension
• product containing the SNP
• The samples are denatured and run
• on the DHPLC
• Extension products separate based on
• base content and hydrophobicity of

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GENES ANALYSED BY SBE-DHPLC in Das lab
GENE POLYMORPHISM UGT2B7 -161T/C (promoter
SNP) VDR Ex8283G/A (intron 8
SNP) PGP/MDR1 2677G ? T (exon
21) PGP/MDR1 3435C ? T (exon 26) RFC 80G ?
A MTHFR Glu429Ala GSTP1 Ile105Val
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RFC 80 G?A
C
9
RFC 80 G?A (R27H)
u.p.
C
T
A/A
G/A
A/A
G/G
G/G
Allele frequencies A 43.7 G 56.3
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5
PCR reaction, when Taq extends over a hybridized
probe, 5 nuclease activity cleaves 5 base and
reporter dye is no longer quenched (U substituted
for T, so that UNGase will destroy any
contaminants with products from a previous assay)
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Assay on Demand (TaqMan)
384 well microplate, 5 ul reaction volume, 6 ng
DNA, amplify 9600 mode on 9700, read same plate
as LJL HE384A
1 per genotype for 300 samples, 0.40 for 750
subjects core charge e.g. 22 assays for
EGFR, 17 of 21 intervals lt 11 kb (mean 8.8 kb)
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TPH2 (new gene - ? assays)

• Walther DJ, et al. (2003) Synthesis of serotonin
  by a second tryptophan hydroxylase isoform.
  Science 29976
• AK094614
• Homo sapiens cDNA FLJ37295 fis, clone
  BRAMY2015311, moderately similar to TRYPTOPHAN
  5-MONOOXYGENASE (EC 1.14.16.4)
• chr1272284699-72311212
• 12q21.1
• cM 83.19 markers flank it (Marshfield
  sex-averaged)
• Between D12S80 and D12S1040
• Celera Gene Name hCG25174

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Search assays on demand (TPH2)

• https//abassays.celera.com/cdsEntry/Marketing/snp
  _overview.jsp

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SNPs that have to be typed Algorithm (subject to
change)

• A) Genotyping
• 1) FP-TDI (lt 200 samples or no TaqMan assay)
• 2) TaqMan (gt 200 samples and available)
• 2) SBE-dHPLC
• 3) Whatever works, but cost probably increased
  (molecular beacon, OLA,PCR-RFLP,)

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SNP typing approach (continued)

• B) QC (Mendelian, double-recombinant, HWE
  checks)
• 1) should 2 or more methods with non-overlapping
  primers be used to be sure no errors? SNPs in
  primersif association negative if positive?
• 2) should allele calling images (sequence traces)
  be part of PAAR (and PGRN) network data
• 3) is ability to reduce errors of SBE-dHPLC a
  significant advantage for triallelic SNPs?

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Dense SNP coverage of a region

• More efficient when every SNP doesnt have to be
  typed (to allow choosing one or two methods and
  automating them)
• Assays on demand (TaqMan) focused on genes
• Other approaches to selecting and typing other
  SNPs (e.g. FP-TDI)

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Other Adventures in SNP Typing

• Outsourcing (Adventures in purchasing?)
• Illumina pricing based on large number of SNPs
  (e.g. 1152)
• Sequenom (better for a large number of SNPs)
• DNAprint (Orchid)(some concerns about one of
  homozygote clusters overlapping negatives)

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Pooling?

• Pooling (sequenom, SNaPshot, dHPLC,
  pyrosequencing, direct sequencing)(probably not
  Orchid arrays or Affy flex arrays)

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Length Variants

• Microsatellites, small insertion/deletions
• Relatively straightforward with end-labeled
  primers and capillary electrophoresis instruments
  (e.g. 3100,3700, 3730)
• Can be relatively easily multiplexed (for PCR)
  and pooled (for injection) with up to 20 markers
  (with up to 5 fluorescent dyes)
• Genome scans available locally (e.g. U Chicago)
  or through CIDR, NHLBI, DeCODE
• SNP genome scans
• Affy (now gt10K, 100K planned for 2004)

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Whole Genome Analysis

• Affymetrix Human Mapping 10K XbaI
• 11,560 SNPs spanning the genome
• Average heterozygosity 33
• Preliminary Results
• 90-95 call rate
• Straightforward Assay
• Automated allele calling (in contrast to
  microsatellite allele calling)