SNP: Detection

Paul Berg

Single Nucleotide Polymorphisms (SNPs) are the most common type of sequence variation and probably account for more than 90% of sequence differences in most organisms. For most users, coding SNPs (cSNPs) are of particular interest. These SNPs have the potential to alter the coding sequence of genes and possibly also gene functionality.

In addition to pure contract genotyping, CIGENE can provide assistance with the identification and validation of SNPs.

For most species it is recommended that the SNPs in question have previously been identified and validated, but CIGENE will also assist in these procedures if necessary.

In connection with all SNP genotyping studies, we normally suggest that the users themselves perform the detection or selection of the SNPs to be used. However, CIGENE can help with SNP detection in specific candidate gene regions or in specific organisms where SNPs are not readily available.

The CIGENE SNP detection pipeline is designed to give users a complete solution from sequence information to validated SNPs. See here for prices.

Assay development and primer design

Experiments are set up in collaboration with the external users, based on their chosen sequence information. All sequences are run through RepeatMasker and primers are then designed to yield amplicons of approximately 1000 bp. The number of samples to be re-sequenced is carefully chosen together with the user.

PCR and Sequencing reactions

We set up all PCR reactions in 96 or 384 well plates, using our Beckman Biomek Fx pipetting robot. The resulting PCR products are purified and set up for all downstream processing using our post-PCR pipetting robot. All sequences are run in forward and reverse direction to ensure maximum reliability of the results.

Automated sequencing

Data is collected using fluorescent dye-terminator chemistry by our fully automated ABI 3730 sequencer. The traces are then compiled into contigs (when applicable) and transferred to our automated SNP detection software pipeline, consisting among other things of Phred/Phrap/Consed, polybayes etc.

Mutation/variation identification

We primarily use the Phred base call confidence scores to identify high probability heterozygotes. The variations are then mapped back to the original sequence and the variation together with the flanking sequence is used to design assays etc. for the SNP genotyping pipeline.

Please contact Sigbjørn Lien if you would like to make use of this service.


Updated: 21.04.06
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