Single nucleotide polymorphism (SNP) development and typing
Semi-automated medium-scale SNP genotyping is supported at NEOF using the LGC KASP genotyping system. This powerful assay allows us to perform genotyping of non-model systems.
Various levels of support can be provided:
- SNP discovery: usually using new-generation sequencing
- DNA preparation and quantification
- SNP selection and array design
- SNP typing on the KASP system
- SNP data analysis
Our staff are highly experienced SNP genotypers, and have successfully completed numerous SNP-typing projects involving a wide range of species.
When planning the sequencing for SNP identification, it's very important to take great care when choosing the individuals to be sequenced. Careful consideration should be taken regarding the number of individuals sequenced and their population origins, to avoid causing any bias in the data produced.
For more information, please download the SNP selection checklist (Word, 77.5kB)
The DNA requirements for samples that are sequenced to identify SNPs are that a quantity of at least five micrograms of (pooled) high-quality cDNA is required from which the SNPs would be initially identified by sequencing.
Requirements concerning the sequences from which the primers to amplify the SNPs are designed
The amplicon should be approximately 80-90bp.
The assay chemistry requires a minimum of 100bp of polymorphism-free flanking sequence on either side of the target SNP to facilitate accurate and effective primer binding.
From experience, if another SNP appears within this flanking sequence, the hybridisation of the primer targeting that area will be affected, and consequently, the clustering of that SNP will be ambiguous. During the scoring process of model organism, for example, any sequences that contain an SNP other than the SNP of interest in the assay region will be flagged with a warning and affect the score of the assay.
In summary, we recommend you make sure that SNPs are flanked by sufficiently long sequences. A minimum of 70 bp of sequence on either side of the SNP variant is required; however, 100 bp (or longer) sequences are preferred.
These flanks should be free of other SNPs, repeat regions and runs of mononucleotide bases. The SNP-array primers should not be designed from sequences with more than three repeat units (eg CACACA) or 3 consecutive mononucleotides (eg AAA).
Numbers of SNPs and samples
The KASP system is intended for small to medium-scale SNP projects. We support projects to obtain up to 250K data points. The combination of numbers of SNPs x samples can vary to suit the project - for example, 250K data points could be obtained by typing 10 SNPs x 25,000 samples or 250 SNPs x 1000 samples.
Please note that up to 30% of SNPs may fail (usually 10-20%) in a non-model species and this should be taken into account when selecting how many SNPs you require.
Normally we only offer projects up to a maximum of 250K data points.
(For projects requiring more than 768 SNPs, we recommend running Illumina Goldengate on an iScan, for typing 1536 SNPs we recommend a different platform (eg BeadStation), and finally, we suggest using Infinium chips for projects above 3000 SNPs).
Numbers of samples
Within the KASP framework, reagents are typically sold in batches of 50,000 reactions (1 kit). However, these could be used in various SNP-sample number combinations - for example, 10 SNPs x 5000 samples or 200 SNPs x 250 samples.
The plates that we use here at Sheffield for the SNP typing have 384 wells.
Examples of the SNP-Sample combinations:
|1 kit||2 kits||3 kits||4 kits||5 kits||6 kits||7 kits|
The largest project we can support is a maximum of 5 kits (250K data points, or possibly more following discussion).
Please bear in mind that 20,000 data points will take approximately 1 week of full-time work. Obtaining 50,000 data points would take 2.5 weeks. To complete the maximum sized project (250K data points) would require 12.5 weeks of full-time work (the labour involves sample preparation, processing and analysis.)
In summary: The smallest project we can offer is unlimited and can be any size (NB one kit contains 50,000 reactions). The largest project we normally offer is five kits (250,000 reactions).
If you would like more information, or to discuss your proposed project informally, please contact Dr Deborah Dawson.
DNA requirements of samples for SNP typing
For the SNP genotyping of each individual, the amount of DNA required depends on the number of SNPs to be typed and the genome size of the study species. For example, to genotype 384 mouse SNPs we would need a total of 2.5-3 micrograms of DNA per individual.
For full details of how to calculate the amount of DNA required, download this PDF document (PDF, 212kB).
DNA should be buffered in lowTE (10 mM Tris-HCl pH 8.0, 0.1mM EDTA).
Genomic DNA should be provided at the calculated concentration and quantified using a spectrophometer / fluorometer. Please provided details of the concentration and gel photos to show the quality of the DNA before shipping the DNA.
For each SNP reaction the amount of DNA required (calculated based on the genome size) needs to be provided in a volume of 1.5 microlitres. For example, if a project was using 96 SNPs we would need a minimum of 144 microlitres of DNA, plus 20% extra for repeats and pipetting error.
Individuals are genotyped in set numbers of multiples, so experiments should be designed with this in mind.
The DNA needs to be supplied in semi skirt plates at the calculated concentration based on genome size and number of SNPs. The absolute minimum concentration required is 5 ng/microlitre and the minimum volume of DNA required is 5 microlitres. Any samples with less than the amount of DNA required (based on genome size) are likely to fail, so the use of whole genome amplification (WGA) prior to SNP typing should be considered, or these samples should only be included if you have no choice.
We advise to send samples to Sheffield using the same method as used by the LGC company, see the LGC SNPline brochure (PDF, 788kB).
Samples should be sent on dry ice by courier (same-day or next-day delivery) to Dr Gavin Horsburgh at the address below. Please send samples as early in the week as possible to avoid their delivery getting delayed over the weekend (ie please do not post later than Wednesday).
When posting samples, please label the parcel with "Freeze on arrival" and address to:
Dr Gavin Horsburgh
Molecular Ecology Lab (B71)
Department of Animal & Plant Sciences
c/o Biodistribution Centre
Alfred Denny Building
University of Sheffield
- SNP typing checklist (Word, 77.5kB)
- LGC Genomics KASP Assay Design Factsheet v3 (PDF, 148kB)
- DNA requirements for your KASP SNP genotyping project (PDF, 212kB)
- LGC SNPline brochure (PDF, 788kB)
- KASP quick start guide (PDF, 336kB)
- KASP Brochure (PDF, 400kB)
- KASP-genotyping chemistry user guide (PDF, 452kB)
- Species tested with KASP technology (PDF, 156kB)
The NERC Environmental Omics Facility (NEOF)
Making state-of-the-art molecular genetics facilities and training available to the UK science community.