Computational protocol: SNP Miniplexes for Individual Identification of Random‐Bred Domestic Cats†

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Protocol publication

[…] A previous study used 148 intergenic SNPs to examine domestic cat population structuring . The genotypic data from this previous study were analyzed to select a subset of BGA SNPs for the forensic applications in this study. The statistical program FSTAT V.2.9.3 was used to determine G ST and heterozygosity data, based on Nei, Weir, and Cockerham estimators , , . The software program POPGENE V.1.32 was used to perform the pair‐wise linkage disequilibrium (D') tests based on Ohta's method , . Total variance of linkage disequilibrium was measured in di‐loci (DIT)², within population (DIS)² and between populations (DST)². A subset of the 148 SNPs (n = 49) were chosen based upon the following criteria: (i) high heterozygosity (>0.35), (ii) low G st (≤0.06), and (iii) low linkage disequilibrium (LD) across all random‐bred populations used in the previous study.SNPs from 13 genes that are causal for 29 EVCs in cats, including sex and also blood type, were included to complement the BGA SNPs , , (Table ). Combining 49 BGA and 29 EVC SNPs, 78 SNPs were analyzed to develop the SNP panels. Excluding the familial cats, the same statistical analyses, and Shannon's information index (H') , , were re‐calculated on the selected SNPs for the cat populations analyzed in this study. [...] The sequences for each SNP locus were obtained from either GenBank or prior studies , and primers for PCR amplification were designed using online software Primer3Plus and NetPrimer (www.premierbiosoft.com/netprimer/index.html). Each primer pair was tested for potential primer dimers and secondary hairpin structures using the Auto Dimer software (www.cstl.nist.gov/strbase/AutoDimerHomepage/AutoDimerProgramHomepage.htm). All SNP and single base extension (SBE) primers (see below) were verified with the sequence databases at the National Center for Biotechnology Information (NCBI) via BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Seven PCR amplicons amplifying the genes ASIP, CMAH, FGF5, SHH, TYR, TYRP1, and ZFXY were designed to evaluate multiple EVC variants within each amplicon. Primers for PCR amplification were desalted (Integrated DNA Technology (IDT) Coralville, IA) (Table S1).A singleton PCR and direct Sanger‐sequencing was performed to test all primer pairs for amplification of the correct product and to verify correct genotype calls for each locus (data not shown). To optimize the multiplex PCRs, a temperature gradient of 50–70°C was tested, MgCl2 concentration was varied from 2.0–8.0 mM, and the PCR primer concentrations were adjusted to balance product amplification at each locus. The final PCR multiplex reactions were conducted in a 15 μL reaction volume, containing 1 U Taq polymerase (Denville Scientific, South Plainfield, NJ), 800 mM of each dNTP (ThermoFisher Scientific, Waltham, MA), 2 μL of the PCR primers, premixed with 0.2–1.0 μM of each primer (IDT), 1× PCR Buffer with 0.5% BSA, 5.0 mM MgCl2, and 5 μM of betaine. The thermal cycling conditions were 94°C for 4 min, 35 cycles of 94°C for 30 sec, 63°C for 30 sec, 72°C for 10 sec, and final extension of 72°C for 10 min. All amplifications were performed using the GeneAmp PCR Thermal Cycler 9700 System (Applied Biosystems). To verify all loci amplified their expected amplicon size, amplification products were examined on a 4% agarose gel with 0.05% ethidium bromide for 120 min at 90 V. Unincorporated dNTPs and excess primers were removed by adding 2 μL of ExoSAP‐IT (Affymetrix, Santa Clara, CA) to the post‐PCR product, which was incubated at 37°C for 20 min followed by 15 min at 75°C for enzyme inactivation. [...] SBE primers were designed using online software, BatchPrimer3 (v.1.0) . SNPplexes containing >10 SNP loci per panel were developed following criteria per the ABI SNaPshot® Multiplex Kit protocol (Applied Biosystems) and the Primer Focus kit . The melting temperature of the primers ranged from 60 ± 3°C. The mobility ranges were from 24 to 90 bp (Table S2). Opposite allele combinations such as A/T and C/G were used for overlapping SNP loci when necessary. All SBE primers were HPLC purified (IDT). […]

Pipeline specifications

Software tools POPGENE, Primer3, NetPrimer, BatchPrimer3
Databases STRBase
Applications Population genetic analysis, qPCR
Organisms Felis catus