SNP Validation. Dr Sarah Parker and Mr John Robinson August 2014

Transcription

1 SNP Validation Dr Sarah Parker and Mr John Robinson August 2014 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 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 : ( 0 ) F A X : ( 0 ) E M A I L : m t 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

2 1 Contents 1 Contents Introduction Proposed Validation Plan Methods Genotype and breed control samples Commercial test samples Sensitivity analysis Breed panel SNP details TaqMan comparison Results Discussion/Conclusion Appendix I Porcine Genotype Control Samples Appendix II Bovine Genotype Control Samples Appendix III Porcine - Individual SNP plots Appendix IV Bovine - Individual SNP plots Porcine Sensitivity plots I Bovine Sensitivity plots II Porcine breed panels III Bovine breed panels Appendix IX TaqMan results Page 1 of 37

3 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. Page 2 of 37

4 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 provided for each of the control samples. Table 1 Porcine Genotype and Breed controls Genotype controls MTD shorthand Breed controls Breed MTD shorthand PIGW1 w1 BK1 Berkshire BK1 PIGW2 w2 BK2 BK2 PIGW3 w3 BK3 BK3 PIGW4 w4 LB1 Large Black LB1 PIGW5 w5 LB2 LB2 PIGW6 w6 LB3 LB3 PIGW7 w7 PIGW8 w8 PIGW9 w9 PIGO4 o4 PIGO5 o5 PIGO8 o8 PIGO9 o9 PIGH13 h13 PIGH75 h75 Table 2 Bovine Genotype and Breed controls Genotype controls MTD shorthand Breed controls Breed MTD shorthand COW003 cow 003 WB1 Welsh Black WB1 COW009 cow 009 WB2 WB2 COW010 cow 010 WB3 WB3 COW011 cow 011 THF1 Traditional THF1 COW035 cow 035 THF2 Hereford THF2 COW038 cow 038 THF3 THF3 COW040 cow 040 COW49W cow 49W COW55W cow 55W COW56W cow 56W COW57W cow 57W COW71W cow 71W COW76W cow 76W COW81W cow 81W 3.2 Commercial test samples Commercial samples were supplied by MTD and labelled Market to distinguish them from the breed-control sample supplied. DNA was extracted by MTD as per the SOP, using an automated Qiacube system (Qiagen) and quantified using a Nanodrop (Thermoscientific). Page 3 of 37

5 3.3 Sensitivity analysis To evaluate performance at reducing DNA amounts, the control samples were subject to serial dilution and then tested using the SNP assays specified for porcine (Table 3) and bovine (Table 4) samples. Higher concentrations of DNA were used for the bovine assay as the concentration range provided by Kasp manufacturers was previously found to be insufficient for this application. Table 3 Porcine sensitivity testing matrix (Berkshire {BK} and Large Blank {LB}) Samples tested Porcine SNP Sample Concentrations MTD shorthand BK1 SNP , 2.0, 1.0 & 0. 5ng/µL BK1 4, BK1 2, BK1 1, BK1 0.5 BK2 SNP , 2.0, 1.0 & 0. 5ng/µL BK2 4, BK2 2, BK2 1, BK2 0.5 BK3 SNP , 2.0, 1.0 & 0. 5ng/µL BK3 4, BK3 2, BK3 1, BK3 0.5 LB1 SNP , 2.0, 1.0 & 0. 5ng/µL LB1 4, LB1 2, LB1 1, LB1 0.5 LB2 SNP , 2.0, 1.0 & 0. 5ng/µL LB2 4, LB2 2, LB2 1, LB2 0.5 LB3 SNP , 2.0, 1.0 & 0. 5ng/µL LB3 4, LB3 2, LB3 1, LB3 0.5 Table 4 Bovine sensitivity testing matrix (Welsh Black {WB}) Samples tested Bovine SNP Sample Concentrations MTD shorthand WB1 SNP , 10.0, 5.0 & 2. 5ng/µL WB1 (20), WB1 (10), WB1 (5), WB1 (2.5) WB2 SNP , 10.0, 5.0 & 2. 5ng/µL WB2 (20), WB2 (10), WB2 (5), WB2 (2.5) WB3 SNP , 10.0, 5.0 & 2. 5ng/µL WB3 (20), WB3 (10), WB3 (5), WB3 (2.5) 3.4 Breed panel SNP details 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. Berkshire Large Black KASPar Assays MTD shorthand KASPar Assays MTD shorthand 004 SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP SNP 96 Page 4 of 37

6 Table 6 SNP panel details for each of the bovine assays tested Welsh Black Traditional Hereford KASPar Assays MTD shorthand KASPar Assays MTD shorthand B_tauSNP00717 snp B_tauSNP12182 snp B_tauSNP03887 snp B_tauSNP49510 snp B_tauSNP07095 snp B_tauSNP45370 snp B_tauSNP12182 snp B_tauSNP06722 snp B_tauSNP33168 snp B_tauSNP38045 snp B_tauSNP42261 snp B_tauSNP24495 snp B_tauSNP43303 snp B_tauSNP36999 snp B_tauSNP53248 snp B_tauSNP24444 snp TaqMan comparison 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 breedcontrol 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. Page 5 of 37

7 Table 7 Summary of porcine Kasp assay genotype results Assay SNP 04 SNP 07 SNP 08 SNP 23 SNP 24 SNP 27 SNP 31 SNP 32 SNP 36 SNP 43 SNP 47 SNP 48 SNP 57 SNP 60 SNP 73 SNP 80 SNP 93 SNP 96 Comments The assay performed well. However, homozygote genotypes opposite to those expected. The assay is not performing well. No clear genotype clusters. The assay has mixed success. Ambiguity in assigning genotypes is reduced when more samples are included on the allelic discrimination plot. Table 8 Summary of bovine Kasp assay genotyping results Assay snp snp snp snp snp snp snp snp snp snp snp snp snp snp snp snp snp snp snp snp Comments 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. The assay performed well. However, homozygote genotypes appear opposite to those expected. Possible ambiguity due to lack of one homozygote control. Page 6 of 37

8 4.2 Sensitivity testing 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. 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 Bovine sensitivity testing Bovine SNP Assays snp.12182, snp and snp 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 II where BK- and LB- prefixes identify Berkshire and Large Black samples, respectively. Allelic discrimination plots for all bovine assays are given in II where WB- denotes samples of Welsh Black and THF signifies Traditional Hereford 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 7 of 37

9 Snp49510 Snp33112 Snp12182 Snp00717 Snp51421 Snp45370 Snp38045 Snp36999 Snp32639 Snp24725 Snp24495 Snp24444 Snp06722 Snp02704 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 bk bk bk Bk-MTD Table 10 Four-digit genotype codes generated for Large Black breed-control and test samples SNP 23 SNP 80 SNP 96 SNP 48 SNP 07 SNP 57 SNP 93 SNP 32 SNP 47 SNP 36 LB LB LB LB-MTD Bovine SNP-panel validation For the Welsh Black panel, all breed-control samples were genotyped unambiguously at all panel-snps. Conversely, market sample genotypes could not be resolved for SNP assays snp.24725, snp and snp All sample genotypes were unambiguously resolved for Traditional Hereford samples. Genotype results from the StepOne output were again translated into four-digit genotype scores using the genotype key provided in the bovine breed identification SOP (see Final Project Report for further detail). Translated genotype values are provided in Tables 11 and 12. Table 11 Four-digit genotype codes assigned to Welsh Black samples. SNP-genotypes that failed to be unambiguously resolved are highlighted in yellow. WB1, WB2, WB3, WB-MTD, Page 8 of 37

10 Snp12182 Snp00717 Snp53248 Snp43303 Snp42261 Snp33168 Snp07095 Snp03887 Table 12 Four-digit genotype codes assigned to Traditional Hereford samples THF1, THF2, THF3, THF-MTD, Assignment Analysis Following the relevant SOP, Geneclass software was loaded onto the PC and a worked example, provided as an Appendix to the SOP, was used to gain familiarisation with the software. Assignment analysis was then performed on the re-coded genotype data (previously presented in Tables 9-12) Porcine Assignment Analysis The control and test sample genotype profiles were compared to the relevant breed reference dataset. The Geneclass output results are given in Figure 1 for Berkshire samples and Figure 2 for Large Black. All test samples were assigned to their correct breed of origin (all >99.7%), and therefore no further analysis was performed on these samples. It should be noted however, that all Berkshire samples were missing data for snp.027. Owing to a limited availability of sample DNA and the problematic amplification of snp.027 it was not possible to genotype samples at this marker. Under the SOP guidelines, assignment analysis would not normally be performed on samples that were missing data, due to a lack of confidence in the final result. Figure 1 GeneClass output for Berkshire sample assignment Page 9 of 37

11 Figure 2 GeneClass output for Large Black sample assignment Bovine Assignment Analysis The control and test sample genotype profiles were compared to the relevant breed reference dataset. The Geneclass output results are given in Figure 3 for Traditional Hereford samples and Figure 4 for Welsh Black. All Traditional Hereford samples were assigned to their correct breed of origin (all >99.0 %), and therefore no further analysis was performed on these samples. Two of the Welsh black samples, WB1 and WB-MTD, were correctly assigned with scores of 79.4 % and 95.7% respectively. It should be noted however, WB-MTD was missing data, thus reducing confidence in the final result. Figure 3 GeneClass output for Traditional Hereford sample assignment. Figure 4 GeneClass output for Welsh Black sample assignment. Page 10 of 37

12 Despite all SNP data being present, Welsh Black samples WB2 and WB3 were ranked as non-welsh black (87.6% and 95.4% respectively). Subsequent analysis was performed to calculate exclusion probabilities for the misassigned samples and the results from GeneClass are provided in Figure 5. As the probability for both of the misassigned samples is >0.05, the samples cannot be fully excluded as being from the Welsh Black breed. However, probability values generated for each of these samples (see SOP for details on the Analysis Sheet calculations) suggests that both WB2 and WB3 are likely to be non-welsh Black in origin (see Figure 6 and Figure 7). Figure 5 Welsh Black exclusion probability analysis Welsh Black ANALYSIS SHEET WB2 Table A Breed identified in GeneClass2 (Rank 1) Code -Log(L) Claimed breed of origin Code -Log(L) Welsh Black WB Welsh Black WB Non-Welsh Black Non-WB Non-Welsh Black Non-WB Likelihood ratio (LR) Table B Claimed Breed Probability table Breed identified in Geneclass2 (Rank 1) Welsh Black Non-Welsh Black Welsh Black x Non-Welsh Black x Result P(belonging to non-target breed, given the claimed origin) = Figure 6 Analysis sheet for sample WB2 Page 11 of 37

13 Welsh Black ANALYSIS SHEET WB3 Table A Breed identified in GeneClass2 (Rank 1) Code -Log(L) Claimed breed of origin Code -Log(L) Welsh Black WB Welsh Black WB Non-Welsh Black Non-WB Non-Welsh Black Non-WB Likelihood ratio (LR) Table B Claimed Breed Probability table Breed identified in Geneclass2 (Rank 1) Welsh Black Non-Welsh Black Welsh Black x Non-Welsh Black x Result P(belonging to non-target breed, given the claimed origin) = Figure 7 Sample WB3 4.5 TaqMan comparison All the samples were unambiguously genotyped for each of the four porcine SNP assays selected for the TaqMan/KASP comparison. The plots obtained from these four assays are given in Appendix IX. The majority of the samples and positive control samples demonstrate tight clustering. Although the genotype data generated was the same for both Taqman and Kasp assays, the former required no optimisation and performed consistently well under the manufacturers recommended conditions. Page 12 of 37

14 5 Discussion/Conclusion The SOP provided was very comprehensive and gave detailed information about how to proceed from sample acquisition through to assignment of an unknown sample. DNA extracted was sufficient in both quality and quality for downstream usage and the DNeasy chemistry detailed in the SOP can be automated to allow high throughput of samples. Initially there was some discrepancy in the performance of genotype control samples and some individuals did not genotype as expected. Advice from Trace and supplementary information enabled alternative positive control samples to be used which appears to have resolved the original problem.. On the whole, the StepOne software was able to assign the majority of the bovine genotypes, with only a small amount of manual assignment required. The porcine StepOne results needed far more user input to assign individual genotypes to either a homozygote or heterozygote cluster The difference in resolution observed between the porcine and bovine samples may be due to the concentration of DNA used in the PCR reaction. The SOP has taken into account the wide range of DNA concentrations over which the assay can be applied but the need for repeat runs to obtain unambiguous genotypes may be greater when lower concentrations of template are used. The results of the sensitivity analysis also demonstrate an increase in ambiguity in resolving genotype clusters among low DNA concentrations, suggesting that assays are likely to perform more consistently using higher concentrations of the DNA template in each Kasp reaction. Assay snp.027 did not show consistent clustering of the samples/positives and thus this assay needs to be revisited. The original assay components may have degraded and thus, a route forward would be to re-order the KASPer SNP assay 27 and re run the samples. The MTD provided sample of Welsh Black was not assigned a genotype for SNP assays SNP 24725, SNP & SNP All other samples were able to be assigned a genotype. For the porcine samples all Berkshire and Large Black samples were assigned to their correct breed of origin using the GeneClass2 software. For the bovine samples, all Traditional Hereford samples were assigned correctly to breed with a score greater than 99.9%. Whilst two of the Welsh Black samples were correctly assigned to the Welsh Black breed (despite missing data), two (WB2 and WB3) were ranked as non-welsh black (87.6% and 95.4% respectively). Subsequent exclusion analysis indicated that whilst these samples are likely to be non-welsh Black in origin, they could not confidently be excluded from the Welsh Black breed. Further discussion with TRACE revealed that the misassignment was due to a reversal of expected genotypes for both of the samples, likely caused by the lack of a control sample for one of the homozygote genotypes and therefore ambiguity in assigning genotype. TaqMan assays produced clear genotype data without any issues and using the manufacturers recommended conditions. When TaqMan allelic discrimination plots were compared to their KASP equivalent, the TaqMan plots showed tighter clustering and genotype resolution for all but one SNP assay. Page 13 of 37

15 Future directions Once the positive control for each SNP assay are verified to give consistent and reliable results, then routine use of this analysis, should be no more complicated than any other routine DNA test. However, initially the set up will be time-consuming, requiring meticulous and detailed implementation. Another consideration is the large volume of data that is generated, and data manipulation/transfer should be kept to a minimum to reduce transcription errors. The cost of the assays is a consideration, KASP assays are cheap in terms of DNA analysis techniques but, as shown here, the TaqMan assays provide more reliability and better quality data. However, this statement is based on a relatively small sample size and further validation would be needed to confirm this. Whilst this increased quality comes as a more expensive option, the nature of the assays and their intended future use suggests that data quality and confidence in the genotypes produced, is of paramount importance. However, with either chemistry, two to three 96 well plates are required per sample to cover all SNPs (with no replicates) from a single panel. This approach therefore is not a one plate, quick turnaround option. With the ensuing time and cost implications this brings, if large numbers of samples were to be analysed, it would be worth investigating the microarray route, as all the SNP s could be placed on one chip. Further validation work is needed to include a greater number of breed samples and potentially assess the performance of each panel on cross breeds. Furthermore, the Welsh Black samples were not tested against the Traditional Hereford panel and vice versa, so further cross-over work may substantiate the breed-specificity of the panels designed. Page 14 of 37

16 Appendix I Porcine Genotype Control Samples Expected genotypes for porcine control samples. Assays highlighted in green required additional control samples to unambiguously resolve genotype clusters. Genotype control tube Hom 1 Control tube ID sorted by genotyp e PIGW5 PIGW4 PIGW8 PIGO9 PIGW2 PIGW1 PIGW1 PIGW7 PIGW7 PIGW2 PIGW2 PIGW8 PIGW9 PIGW9 PIGW9 PIGW3 PIGO9 PIGO4 PIGW9 PIGO9 PIGW3 PIGW3 Hets PIGW2 PIGW2 PIGW5 PIGW5 PIGO4 PIGO4 PIGH13 PIGW1 PIGW2 PIGW4 PIGO5 Hom 2 PIGW3 PIGW7 PIGW5 PIGO5 PIGO5 PIGH13 PIGW2 PIGW2 PIGW5 PIGW5 PIGW1 PIGW1 PIGW4 PIGW4 PIGO8 PIGW2 PIGW3 PIGW4 PIGW4 PIGW8 PIGW8 PIGW6 PIGW6 PIGW4 PIGO9 PIGO8 PIGO8 PIGW5 PIGW5 PIGW9 PIGO8 Genotype control tube Hom 1 Control tube ID sorted by genotyp e PIGW1 PIGW1 PIGW3 PIGW3 PIGW3 PIGW1 PIGW1 PIGW7 PIGW7 PIGW6 PIGW6 PIGW7 PIGW7 PIGW7 PIGW1 PIGW8 PIGW8 Hets PIGW6 PIGW6 PIGW7 PIGW7 PIGO5 PIGO5 PIGH75 PIGW6 PIGW6 PIGW4 PIGO5 Hom 2 PIGW7 PIGW7 PIGW7 PIGO5 PIGH75 PIGW7 PIGW7 PIGW5 PIGW5 PIGW4 PIGW4 PIGW2 PIGW2 PIGO4 PIGO4 PIGW4 PIGO5 PIGO5 PIGW9 PIGW9 PIGW3 PIGW3 PIGO9 PIGO9 PIGW5 PIGO8 PIGO8 Genotype control tube Hom 1 Control tube ID sorted by genotyp e PIGW1 PIGW1 PIGW4 PIGO5 PIGW3 PIGW3 PIGW3 PIGW3 PIGW6 PIGW6 PIGW5 PIGW5 PIGW6 PIGW6 PIGW6 PIGW6 Hets PIGW7 PIGW7 PIGW2 PIGW2 PIGW4 PIGO8 PIGW4 PIGO8 Hom 2 PIGW7 PIGW9 PIGW2 PIGW8 PIGW8 PIGW7 PIGW7 PIGO8 PIGO8 PIGW5 PIGW5 PIGW5 PIGW5 PIGO9 PIGO9 PIGW7 PIGW7 PIGW9 PIGO5 Page 15 of 37

17 B_tauSNP24444 B_tauSNP12182 B_tauSNP12182 B_tauSNP07095 B_tauSNP06722 B_tauSNP03887 B_tauSNP03887 B_tauSNP02704 B_tauSNP02704 B_tauSNP00717 B_tauSNP38045 B_tauSNP36999 B_tauSNP33168 B_tauSNP33112 B_tauSNP33112 B_tauSNP32639 B_tauSNP24725 B_tauSNP24725 B_tauSNP24495 B_tauSNP24495 B_tauSNP53248 B_tauSNP51421 B_tauSNP49510 B_tauSNP49510 B_tauSNP45370 B_tauSNP45370 B_tauSNP43303 B_tauSNP42261 Appendix II Bovine Genotype Control Samples Expected genotypes for bovine control samples. Assays highlighted in green required additional control samples to unambiguously resolve genotype clusters. Hom1 (FAM) Hets Hom2 (HEX) cow 009 cow 009 cow 040 cow 040 cow 011 cow 009 cow 035 cow 035 cow 009 cow 81W cow 81W cow 76W cow 76W cow 71W cow 56W cow 040 cow 040 cow 55W cow 009 cow 003 cow 003 cow 009 cow 035 cow 55W cow 57W cow 009 cow 009 cow 003 cow 55W cow 56W cow 56W cow 035 cow 035 cow 76W cow 81W cow 56W cow 56W cow 011 cow 038 cow 57W cow 57W cow 56W cow 81W cow 49W cow 003 cow 003 cow 003 cow 038 cow 76W cow 040 cow 57W cow 81W cow 81W cow 038 cow 040 cow 57W cow 57W cow 76W Hom1 (FAM) Hets Hom2 (HEX) cow 035 cow 035 cow 038 cow 038 cow 81W cow 009 cow 55W cow 81W cow 011 cow 040 cow 035 cow 76W cow 038 cow 040 cow 038 cow 038 cow 040 cow 76W cow 011 cow 011 cow 035 cow 035 cow 040 cow 035 cow 035 cow 010 cow 011 cow 010 cow 81W cow 81W cow 71W cow 71W cow 76W cow 56W cow 56W cow 71W cow 57W cow 71W cow 003 cow 56W -ve cow 038 cow 003 cow 003 cow 49W cow 003 cow 009 cow 56W cow 56W -ve cow 71W cow 49W cow 003 cow 011 cow 55W cow 55W Hom1 (FAM) Hets Hom2 (HEX) cow 003 cow 55W cow 009 cow 009 cow 035 cow 035 cow 040 cow 49W cow 81W cow 038 cow 49W cow 009 cow 76W cow 038 cow 81W cow 76W cow 57W cow 010 cow 035 cow 035 cow 011 cow 011 cow 035 cow 55W cow 009 cow 71W cow 71W cow 71W cow 009 cow 009 cow 71W cow 010 cow 035 cow 49W cow 010 cow 010 cow 003 cow 003 cow 71W cow 040 cow 011 cow 57W cow 57W cow 56W cow 56W cow 038 Page 16 of 37

18 Appendix III Porcine - Individual SNP plots Allelic discrimination plots of all genotype controls for each porcine SNP assay. Individual sample details are provided in Table 1 of this validation report. Blue and red dots represent the two homozygote genotypes and green signifies a heterozygote. Page 17 of 37

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22 Appendix IV Bovine - Individual SNP plots Allelic discrimination plots of bovine genotype control samples for each of the bovine SNP assays tested. Individual sample details are provided in Table 2 of this report. Blue and red dots represent the expected homozygote genotypes and green signifies a heterozygote. Page 21 of 37

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26 Porcine Sensitivity plots Allelic discrimination plots of all samples tested under the sensitivity analysis. Samples are labelled BK for Berkshire samples, LB for Large Black samples and genotype controls are indicated by a W prefix. Blue, red and green circles represent the genotype control samples for each SNP, as previously described. Page 25 of 37

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28 I Bovine Sensitivity plots Allelic discrimination plots of all samples tested under the sensitivity analysis. Samples are labelled WB for Welsh Black samples, THF for Traditional Hereford samples and genotype controls are indicated by a W prefix. Blue, red and green circles represent the genotype control samples for each SNP, as previously described. Page 27 of 37

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30 II Porcine breed panels Allelic discrimination plots of all samples tested with the relevant breed panel SNPs. Samples are labelled BK for Berkshire samples, LB for Large Black samples and details can be found in Table 2 of this report. Genotype control samples are indicated by a W prefix and blue, red and green circles represent the expected homozygote and heterozygote control genotypes, as previously described. Berkshire Page 29 of 37

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32 Large Black Page 31 of 37

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34 III Bovine breed panels Allelic discrimination plots of all samples tested with the relevant breed panel SNPs. Samples are labelled WB for Welsh Black samples, THF for Traditional Hereford samples, and details can be found in Table 2 of this report. Genotype control samples are indicated by a W prefix and blue, red and green circles represent the expected homozygote and heterozygote control genotypes, as previously described. Welsh Black Page 33 of 37

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36 Traditional Hereford Page 35 of 37

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38 Appendix IX TaqMan results Allelic discrimination plots of all samples tested with Taqman SNP assays. Samples are labelled BK for Berkshire samples, LB for Large Black samples and details can be found in Table 2 of this report. Genotype control samples are indicated by a W prefix and blue, red and green circles represent the expected control homozygote and heterozygote genotypes, as previously described. Page 37 of 37