SNP’s: Which Method is Best for Your Study?

SNP Detection Methods available in the DNA Analysis Facility Sequence SNaPshot Allelic Discrimination Assay DHLPC SNPlex

Joint RG Study Unfortunately, little information is available regarding the specific advantages and limitations of even the most routinely used mutation detection techniques. The primary goal is to compare operational parameters for the most popular mutation/polymorphism screening methods. In the first year of this joint proposal, we will conduct a pilot study engaging only members of the participating research groups: DSRG, FARG and NARG. This pilot study will allow us to refine the survey design and prepare a broader based study for year 2 directed at the greater scientific community.

Sequencing Methodology Your Lab: gDNA extraction PCR amplification of region of interest DNA Analysis Facility Cycle Sequence Reaction Sequence Run

Sequencing: Data Analysis TYMS SNP: AAGTTTTTACACTTT(C/T)ATTTCTCTGTGGCT

Sequencing: Data Analysis Observed Mixed Ratios of Heterozygous peaks

Sequence Data Analysis Confirmed Mixed Ratios of Heterozygous peaks

Joint RG Data: Sequencing TNF GAGGGGCATG(A/G)GGACGG Sample # Forward Reverse 1 G C 2 3 4 5 G/A C/T 6 7 8 9 10 11 12

Joint RG Data: Sequencing AR SNP: Forward sequence slips on CAG repeat

Joint RG Study Sequence Data Comparison TYMS, MTHFR, and TNF SNP’s 32/36 calls matched the true sample identity. Of the four calls missed: 3 calls missed the 2nd allele when it was at 5%. 1 call missed the 2nd allele when it was at 12.5%. AR SNP 12/12 calls matched the true sample identity in Reverse direction only.

Sequence Summary Sequence Multiplex Limited Throughput Medium Advantages: well established technology, easy sample preparation, and can easily get confirmation from 2nd strand. Disadvantages: Some problems with sequence context, only limited ability to multiplex SNP’s. Sequence Multiplex Limited Throughput Medium

SNaPshot Methodology Your Lab: DNA Analysis Facility gDNA extraction PCR amplification Purify PCR product SNaPshot Reaction SAP treatment DNA Analysis Facility Add size standard and perform Genescan Run

SNaPshot Analysis Liz 120 Size Standard

Joint RG Data: SNapShot

Joint RG Study SNaPshot Data Comparison TYMS, MTHFR, TNF and AR SNP’s 44/48 calls matched the true sample identity. Of the 4 calls missed: 3 calls missed the 2nd allele when it was at 5%. 1 call missed the 2nd allele when it was at 12.5%.

SNaPshot Summary Sequence SNaPshot Multiplex Limited Yes, 7-10 SNP’s Advantages: this system is designed for multiplexing and you have two options: multiplex the SNaPshot reaction or pooled separate reactions. Disadvantages: primers need to be specifically designed, primers >30bp need to be HPLC purified, optimization of the SNaPshot reaction can take time. Sequence SNaPshot Multiplex Limited Yes, 7-10 SNP’s Throughput Medium

Real-time qPCR Method

Allelic Discrimination Assay Methodology Your Lab: gDNA extraction PCR amplification with 2 dual-labelled probes DNA Analysis Facility Perform End point plate read and Analysis

Allelic Discrimination Assay: Data Analysis

Joint RG Data

Joint RG Data

Joint RG Data

Joint RG Data

Joint RG Data

Joint RG Data

Joint RG Study ADA Data Comparison TYMS and MTHFR (both alleles represented) 19/24 calls matched the true sample identity. Of the 5 calls that were missed: 2 calls missed the 2nd allele when it was at 5%. 2 calls missed the 2nd allele when it was at 12.5%. 1 call missed the 2nd allele when it was at 25%. TNF and AR SNP’s (no 2nd allele present) 16/24 calls matched the true sample identity. 8 heterozygous samples were miscalled as homozygous.

Allelic Discrimination Assay: Summary Advantages: ABI has a huge collection of pre-designed SNP assays so little optimization is needed. Disadvantages: can’t multiplex. Sequence SNaPshot ADA Multiplex Limited Yes, 7-10 SNP’s No Throughput Medium High

DHPLC Methodolgy DNA Analysis Facility Run sample on Transgenomic Wave Your Lab gDNA extraction Design specific primers PCR amplification DNA Analysis Facility Run sample on Transgenomic Wave

DHPLC Data Analysis

Joint RG Data MTHFR SNP

AR SNP was unable to be interpreted. Joint RG Data AR SNP was unable to be interpreted.

Joint RG Study DHPLC Data Comparison TYMS and MTHFR SNP’s 19/24 calls matched the true sample identity. Of the 5 missed calls: 2 calls missed the 2nd allele when it was at 5%. 2 calls missed the 2nd allele when it was at 12.5% 1 sample was a low signal. AR and TNF SNP’s AR: all calls undetermined TNF: couldn’t design suitable primers

DHPLC Summary Advantages: high throughput screening method Disadvantages: can only distinguish heterozygous from homozygous with no ability to make actual base calls, some sequence parameters can make primer design difficult. Sequence SNaPshot ADA DHPLC Muliplex Limited Yes, 7-10 SNP’s No Throughput Medium High

SNPlex Methodology

SNP’s: Which Method is Best for Your Study? How many SNP’s? How many samples? Sequence SNaPshot ADA DHPLC SNPlex Multiplex Limited Yes, 7-10 SNP’s No 48 SNP’s Throughput Medium High

DNA Analysis Facility The facility provides: Consultations on assay selection. Comprehensive support for assay design. Fast and inexpensive sample processing. Comprehensive support for data analysis.