SNP Validation - Cotmore · WB2 SNP 49510 20.0, 10.0, 5.0 & 2. 5ng/µL WB2 (20), WB2 (10), WB2 (5),...

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

SNP Validation

Dr Sarah Parker and Mr John Robinson

August 2014

mailto:[email protected]

http://www.minton.co.uk/

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Appendix V

1 Contents

1 Contents ......................................................................................................................................................... 1

2 Introduction ................................................................................................................................................... 2

2.1 Proposed Validation Plan ................................................................................................................................ 2

3 Methods ......................................................................................................................................................... 2

3.1 Genotype and breed control samples ............................................................................................................. 3

3.2 Commercial test samples ................................................................................................................................ 3

3.3 Sensitivity analysis .......................................................................................................................................... 4

3.4 Breed panel SNP details .................................................................................................................................. 4

3.5 TaqMan comparison ....................................................................................................................................... 5

4 Results ............................................................................................................................................................ 5

5 Discussion/Conclusion .................................................................................................................................. 13

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

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 Hereford

THF1

COW035 cow 035 THF2 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).

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Appendix V

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 07 4.0, 2.0, 1.0 & 0. 5ng/µL BK1 4, BK1 2, BK1 1, BK1 0.5



LB1 SNP 07 4.0, 2.0, 1.0 & 0. 5ng/µL LB1 4, LB1 2, LB1 1, LB1 0.5



Table 4 Bovine sensitivity testing matrix (Welsh Black {WB})

Samples tested Bovine SNP Sample Concentrations MTD shorthand

WB1 SNP 12182 20.0, 10.0, 5.0 & 2. 5ng/µL WB1 (20), WB1 (10), WB1 (5), WB1 (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

S.scrSNP004 SNP 04 S.scrSNP007 SNP 07










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Appendix V

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 00717 B_tauSNP12182 snp 12182








3.5 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 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 08 The assay performed well. However, homozygote genotypes opposite to those expected.



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.












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 24725 The assay performed well. However, homozygote genotypes appear opposite to those expected. Possible ambiguity due to lack of one homozygote control.












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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.

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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

bk1 0101 0202 0202 0101 0000 0101 0101 0101 0101 0101

bk2 0101 0202 0202 0101 0000 0101 0101 0101 0101 0101

bk3 0101 0202 0202 0101 0000 0101 0101 0101 0102 0102

Bk-MTD 0101 0202 0202 0101 0000 0101 0101 0101 0101 0102

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

LB1 0101 0101 0101 0101 0202 0101 0101 0101 0202 0202

LB2 0101 0101 0101 0101 0202 0101 0101 0101 0202 0202

LB3 0101 0101 0101 0101 0202 0101 0101 0101 0202 0202

LB-MTD 0101 0101 0101 0101 0202 0101 0101 0101 0202 0202

4.3.2 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.32639 and snp.38045. 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.

Snp

0270

4

Snp

0672

2

Snp

2444

4

Snp

2449

5

Snp

2472

5

Snp

3263

9

Snp

3699

9

Snp

3804

5

Snp

4537

0

Snp

5142

1

Snp

0071

7

Snp

1218

2

Snp

33112

Snp

4951

0

WB1, 0101 0202 0202 0102 0202 0202 0101 0101 0202 0101 0202 0102 0101 0101

WB2, 0202 0101 0102 0102 0202 0102 0101 0102 0102 0101 0102 0101 0101 0102

WB3, 0202 0101 0102 0102 0202 0202 0102 0101 0202 0101 0102 0101 0101 0102

WB-MTD, 0202 0101 0102 0101 0000 0000 0101 0000 0101 0101 0202 0102 0102 0102

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Appendix V

Table 12 Four-digit genotype codes assigned to Traditional Hereford samples

Snp

03

88

7

Snp

07

09

5

Snp

33

16

8

Snp

42

26

1

Snp

43

30

3

Snp

53

24

8

Snp

00

71

7

Snp

12

18

2

THF1, 0102 0202 0101 0101 0101 0202 0101 0102

THF2, 0202 0202 0101 0101 0101 0202 0101 0101

THF3, 0102 0202 0101 0101 0101 0202 0101 0101

THF-MTD, 0102 0202 0101 0101 0101 0102 0101 0101

4.4 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)

4.4.1 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

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Appendix V

Figure 2 GeneClass output for Large Black sample assignment

4.4.2 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.

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Appendix V

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

Figure 6 Analysis sheet for sample WB2

Welsh Black ANALYSIS SHEET WB2

Table A

Breed identified in GeneClass2 (Rank 1) Code -Log(L) Code -Log(L)

Welsh Black WB WB 7.529

Non-Welsh Black Non-WB 6.678 Non-WB

Likelihood ratio (LR) 0.851

Table B

Welsh Black Non-Welsh Black

Welsh Black x 0.985289303

Non-Welsh Black 0.994059729 x

Result P(belonging to non-target breed, given the claimed origin) = 0.985

Cla

ime

d

Bre

ed

Breed identified in Geneclass2 (Rank 1)

Claimed breed of origin

Welsh Black

Non-Welsh Black

Probability table

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Appendix V

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.

Welsh Black ANALYSIS SHEET WB3

Table A

Breed identified in GeneClass2 (Rank 1) Code -Log(L) Code -Log(L)

Welsh Black WB WB 8.527

Non-Welsh Black Non-WB 7.214 Non-WB

Likelihood ratio (LR) 1.313

Table B

Welsh Black Non-Welsh Black

Welsh Black x 0.992735849

Non-Welsh Black 0.997262985 x

Result P(belonging to non-target breed, given the claimed origin) = 0.993

Cla

ime

d

Bre

ed

Breed identified in Geneclass2 (Rank 1)

Claimed breed of origin

Welsh Black

Non-Welsh Black

Probability table

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Appendix V

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 32639 & SNP 38045. 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.

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Appendix V

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.

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Appendix V

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

S.scrSNP004

S.scrSNP007

S.scrSNP008

S.scrSNP008

S.scrSNP023

S.scrSNP024

S.scrSNP027

S.scrSNP031

S.scrSNP031

S.scrSNP032

S.scrSNP032

Control tube ID sorted by genotype

Hom1

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

PIGW3 PIGW7 PIGW5 PIGO5 PIGO5 PIGH13 PIGW2 PIGW2 PIGW5 PIGW5

Hom2

PIGW1 PIGW1 PIGW4 PIGW4 PIGO8 PIGW2 PIGW3 PIGW4 PIGW4 PIGW8 PIGW8

PIGW6 PIGW6 PIGW4 PIGO9 PIGO8 PIGO8 PIGW5 PIGW5 PIGW9 PIGO8


S.scrSNP036

S.scrSNP036

S.scrSNP043

S.scrSNP043

S.scrSNP047

S.scrSNP047

S.scrSNP048

S.scrSNP057

S.scrSNP057

S.scrSNP060

S.scrSNP060


Hom1

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

PIGW7 PIGW7 PIGW7 PIGO5 PIGH75 PIGW7 PIGW7 PIGW5 PIGW5

Hom2

PIGW4 PIGW4 PIGW2 PIGW2 PIGO4 PIGO4 PIGW4 PIGO5 PIGO5

PIGW9 PIGW9 PIGW3 PIGW3 PIGO9 PIGO9 PIGW5 PIGO8 PIGO8


S.scrSNP073

S.scrSNP073

S.scrSNP080

S.scrSNP080

S.scrSNP093

S.scrSNP093

S.scrSNP096

S.scrSNP096


Hom1

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

PIGW7 PIGW9 PIGW2 PIGW8 PIGW8 PIGW7 PIGW7

Hom2

PIGO8 PIGO8 PIGW5 PIGW5 PIGW5 PIGW5

PIGO9 PIGO9 PIGW7 PIGW7 PIGW9 PIGO5

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Appendix V

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.

B_tau

SNP

00

71

7

B_tau

SNP

02

70

4

B_tau

SNP

02

70

4

B_tau

SNP

03

88

7

B_tau

SNP

03

88

7

B_tau

SNP

06

72

2

B_tau

SNP

07

09

5

B_tau

SNP

12

18

2

B_tau

SNP

12

18

2

B_tau

SNP

24

44

4

Hom1 (FAM)

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

Hets 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

Hom2 (HEX)

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

B_tau

SNP

24

49

5

B_tau

SNP

24

49

5

B_tau

SNP

24

72

5

B_tau

SNP

24

72

5

B_tau

SNP

32

63

9

B_tau

SNP

33

11

2

B_tau

SNP

33

11

2

B_tau

SNP

33

16

8

B_tau

SNP

36

99

9

B_tau

SNP

38

04

5

Hom1 (FAM)

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

Hets 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

Hom2 (HEX)

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

B_tau

SNP

42

26

1

B_tau

SNP

43

30

3

B_tau

SNP

45

37

0

B_tau

SNP

45

37

0

B_tau

SNP

49

51

0

B_tau

SNP

49

51

0

B_tau

SNP

51

42

1

B_tau

SNP

53

24

8

Hom1 (FAM)

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

Hets

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

Hom2 (HEX)

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

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Appendix V

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.

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Appendix V

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Appendix V

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.

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Appendix V

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Appendix V

Appendix V 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.

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Appendix V

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Appendix V

Appendix VI 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.

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Appendix V

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Appendix V

Appendix VII 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

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Appendix V

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Appendix V

Large Black

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Appendix V

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Appendix V

Appendix VIII 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

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Appendix V

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Appendix V

Traditional Hereford

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Appendix V

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Appendix V

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.

SNP Validation - Cotmore · WB2 SNP 49510 20.0, 10.0, 5.0 & 2. 5ng/µL WB2 (20), WB2 (10), WB2 (5),...

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