HEAD OFFICE: MERTON HOUSE, CROESCADARN CLOSE, CARDIFF CF23 8HF UK ALSO OFFICES AT LONDON, HOUSTON, SINGAPORE, CARMARTHEN TEL:+44 (0) 29 20540000 FAX:+44 (0) 29 20540111 E MAIL: [email protected]WEB: www.minton.co.uk REGISTRATION NUMBER: ENGLAND 435262 SNP Validation Dr Sarah Parker and Mr John Robinson August 2014
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H E A D O F F I C E : M E R T O N H O U S E , C R O E S C A D A R N C L O S E , C A R D I F F C F 2 3 8 H F U K A L S O O F F I C E S A T L O N D O N , H O U S T O N , S I N G A P O R E , C A R M A R T H E N
T E L : + 4 4 ( 0 ) 2 9 2 0 5 4 0 0 0 0 F A X : + 4 4 ( 0 ) 2 9 2 0 5 4 0 1 1 1 E M A I L : m t d @ m i n t o n . c o . u k W E B : w w w . m i n t o n . c o . u k R E G I S T R A T I O N N U M B E R : E N G L A N D 4 3 5 2 6 2
Appendix I Porcine Genotype Control Samples .................................................................................................... 15
Appendix II Bovine Genotype Control Samples ..................................................................................................... 16
Appendix III Porcine - Individual SNP plots ............................................................................................................. 17
Appendix IV Bovine - Individual SNP plots .............................................................................................................. 21
Appendix V Porcine Sensitivity plots ..................................................................................................................... 25
Appendix VI Bovine Sensitivity plots ...................................................................................................................... 27
Appendix VII Porcine breed panels ...................................................................................................................... 29
Appendix VIII Bovine breed panels ....................................................................................................................... 33
Appendix IX TaqMan results .................................................................................................................................. 37
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Appendix V
2 Introduction
This report describes the methods and results of an external validation study to examine the performance of the
genetic breed identification assays developed during Defra research project FA0125, for two porcine breeds (Berkshire
and Large Black) and two bovine breeds (Welsh Black and Traditional Hereford), using the SOP provided. Please refer
to the Final Project Report for full details of this project.
The aims of the external validation study were:
i) to demonstrate performance of the assays in an external laboratory.
ii) to examine the performance of individual genotype control samples.
iii) to examine how the assays would perform in identifying the correct breed of origin for commercial samples
2.1 Proposed Validation Plan
To satisfy the aims of this study, a validation plan was agreed between MTD and TRACE Wildlife Forensics Network.
The following were assessed for each of the four breed panels:
i) Consistency / reproducibility of control sample SNP genotypes.
ii) Sensitivity evaluation of the assays across a range of DNA concentrations
iii) Sample type evaluation using DNA extracted from previously frozen samples.
iv) Sample assignment analysis for breed control individuals and commercial samples
In addition to validating the performance of each breed assay, a comparison was made between genotypes generated
using KASP chemistry, as detailed in the SOP and the alternative Taqman chemistry. The latter is less economical but
also, potentially, more robust during inter-laboratory transfer. Therefore, although not part of the SOP validation, a
direct comparison between the two genotyping chemistries was included.
3 Methods
The breed-specific assays contain between 8 and 14 individual, breed-informative SNP markers. Kasp chemistry was
used as an economical means of genotyping individual SNPs and assignment analysis was conducted on the combined
SNP panel, to either verify or refute the claimed breed of the samples tested. SNP assays were trialled using both
individuals of known genotype and breed (control samples), as well as commercial/market test samples. Due to time
limitations, a sub-set of SNP assays and individuals were selected for sensitivity analysis to assess the consistency of
genotype assignment across a range of DNA concentrations. Details of the control and test samples, and breed-panel
SNPs are given below.
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Appendix V
3.1 Genotype and breed control samples
The control samples provided by TRACE are shown in Table 1and Table 2. Expected SNP genotype data were also
All SNPs were genotyped using the StepOne plus (Applied Biosystems) system. Kasp assays used for each breed panel are shown in Table 5 (porcine) and Table 6 (bovine).
Table 5 SNP panel details for each of the porcine assays tested.
TaqMan assays were ordered for four porcine SNP markers (SNP 07, 23, 43 and 47), to compare genotyping
efficiency and ease of use with that of the KASPar assays. The four porcine SNPs selected had previously shown
variable results using Kasp assays. However, due to technical limitations, design of a Taqman assay for SNP 23 was
not possible and thus was substituted with SNP 24. Taqman assays were tested on Berkshire and Large Black breed-
control samples.
4 Results
4.1 Validation of SNP genotypes
Expected control sample genotypes for each assay can be seen in Appendix I (porcine SNPs) and Appendix II (bovine
SNPs). In some instances, when the assay was first run, discrepancies were observed between expected and observed
genotypes for some of the porcine and bovine assays. However, when the assays were re-run using additional
genotype positive control samples, the majority of assays performed as expected. This reasons for the initial
discrepancy are unclear, but may be due to variability in the quality of original control DNA. Allelic discrimination plots
for each of the Kasp assays trialled can be seen in Appendix III (porcine SNPs) and Appendix IV (bovine SNPs). These
results are summarised in Table 7 and 8.
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Appendix V
Table 7 Summary of porcine Kasp assay genotype results
Assay Comments
SNP 04 The assay performed well. Control samples produce the expected genotypes.
SNP 07 The assay performed well. Control samples produce the expected genotypes.
SNP 08 The assay performed well. However, homozygote genotypes opposite to those expected.
SNP 23 The assay performed well. Control samples produce the expected genotypes.
SNP 24 The assay performed well. Control samples produce the expected genotypes.
SNP 27 The assay is not performing well. No clear genotype clusters.
SNP 31 The assay has mixed success. Ambiguity in assigning genotypes is reduced when more samples are included on the allelic discrimination plot.
SNP 32 The assay performed well. Control samples produce the expected genotypes.
SNP 36 The assay performed well. Control samples produce the expected genotypes.
SNP 43 The assay performed well. Control samples produce the expected genotypes.
SNP 47 The assay performed well. Control samples produce the expected genotypes.
SNP 48 The assay performed well. Control samples produce the expected genotypes.
SNP 57 The assay performed well. Control samples produce the expected genotypes.
SNP 60 The assay performed well. Control samples produce the expected genotypes.
SNP 73 The assay performed well. Control samples produce the expected genotypes.
SNP 80 The assay performed well. Control samples produce the expected genotypes.
SNP 93 The assay performed well. Control samples produce the expected genotypes.
SNP 96 The assay performed well. Control samples produce the expected genotypes.
Table 8 Summary of bovine Kasp assay genotyping results
Assay Comments
snp 00717 Initial results place heterozygotes and and homozygote2 individuals in the same position on the allelic discrimination plot. No homozygote1 controls available. Genotype clusters were better resolved when the sample number was increased.
snp 02704 The assay performed well. Control samples produce the expected genotypes.
snp 03887 The assay performed well. Control samples produce the expected genotypes.
snp 06722 The assay performed well. Control samples produce the expected genotypes.
snp 07095 The assay performed well. Control samples produce the expected genotypes.
snp 12182 The assay performed well. Control samples produce the expected genotypes.
snp 24444 The assay performed well. Control samples produce the expected genotypes.
snp 24495 The assay performed well. Control samples produce the expected genotypes.
snp 24725 The assay performed well. However, homozygote genotypes appear opposite to those expected. Possible ambiguity due to lack of one homozygote control.
snp 32639 The assay performed well. Control samples produce the expected genotypes.
snp 33112 The assay performed well. Control samples produce the expected genotypes.
snp 33168 The assay performed well. Control samples produce the expected genotypes.
snp 36999 The assay performed well. Control samples produce the expected genotypes.
snp 38045 The assay performed well. Control samples produce the expected genotypes.
snp 42261 The assay performed well. Control samples produce the expected genotypes.
snp 43303 The assay performed well. Control samples produce the expected genotypes.
snp 45370 The assay performed well. Control samples produce the expected genotypes.
snp 49510 The assay performed well. Control samples produce the expected genotypes.
snp 51421 The assay performed well. Control samples produce the expected genotypes.
snp 53248 The assay performed well. Control samples produce the expected genotypes.
Page 7 of 37
Appendix V
4.2 Sensitivity testing
4.2.1 Porcine sensitivity testing
Porcine SNP Assays snp.07, snp.32 and snp.96 performed as expected with DNA concentrations of 4, 2, 1 and 0.5ng/µL
concentrations. The allelic discrimination plots for each of the assays are given in Appendix V. It is noted that the more
dilute the sample, the closer the resultant points are to zero. This is understandable as a reduction in the amount of
amplification product, and therefore fluorescence, is expected when lower quantities of start material (i.e. template)
are used. When fluorescent signals are reduced, each of the three expected genotype clusters (homozygote 1,
heterozygote and homozygote 2) may appear closer together on the allelic discrimination plot reducing genotype
clusters resolution.
4.2.2 Bovine sensitivity testing
Bovine SNP Assays snp.12182, snp.33112 and snp.43510 also performed well at initial DNA-template concentrations
of 20, 10, 5 and 2.5ng/µL concentrations. The allelic discrimination plots for each of the assays are given in Appendix
VI. Unlike the porcine sample sensitivity results, the bovine samples did not show performance reducing with DNA
concentration. However, as the overall concentrations of the bovine DNA were greater than for the porcine samples
further dilution of the bovine samples would be needed to directly compare the results. Overall the results
demonstrate the performance of the Kasp assays across the range of DNA concentrations given in the SOP.
4.3 Breed-panel validation
Breed control and test samples were genotyped using the breed-specific panels. Allelic discrimination plots for each
of the porcine assays are given in Appendix VII where BK- and LB- prefixes identify Berkshire and Large Black samples,
respectively. Allelic discrimination plots for all bovine assays are given in Appendix VII where WB- denotes samples of
Welsh Black and THF signifies Traditional Hereford.
4.3.1 Porcine SNP-panel validation
For the Berkshire panel, SNP 27 did not perform well and it was not possible to reliably assign genotypes. All other SNP
assays performed well and genotypes were assigned unambiguously. For the Large Black panel, SNP 57 did not perform
well initially and no genotypes were assigned. However, repetition of the assay allowed genotypes to be assigned for
all samples. All other SNP assays performed well and genotypes were assigned unambiguously.
Genotype results from the StepOne output were translated into four-digit genotype scores using the genotype key
provided in the porcine breed identification SOP (see Final Project Report for details). Translated genotype values
are provided in Tables 9 and 10.
Page 8 of 37
Appendix V
Table 9 Four-digit genotype codes generated for Berkshire breed-control and test samples. SNP markers that did not produce unambiguous genotypes are highlighted in yellow.
SNP 73 SNP 04 SNP 23 SNP 60 SNP 27 SNP 43 SNP 24 SNP 08 SNP 31 SNP 47