Authenticating Samples X
Sponsor			:Sponsor Study Number :SponsorSample-Study-001

Sponsor Representative :Telephone		 :Email			:Sample-Sponsor(111) 111-11111
email@email.com

Testing Facility		:

Crown Bioscience Suzhou
Room 303, Building A6, No. 218
Xinghu Street
Industrial Park
Suzhou City
Jiangsu Province

Crown BioScience
Study Number:

12345

Study Director		:Telephone		 :Email			:Director Name
(415) 444-4444
email@email.com

Study Initiation Date
Study Completion Date
Date of Document
Document Author

1 September 2020
15 September 2020
51 September 2020
Author::::STUDY REPORT
V1.0

1. Overview
1.1. Specimen Description
Species: patient-derived xenograft (PDX) tumor sample dissectedfrom an immunodeficient mouse. Number of Specimens: 1
1.2. Services Performed
Service: Cell Line and Model Authentication - Comprehensive
Service items:• Sample identification by matching to standard PDX and cellline databases• Sample contamination check and contaminant identification		(if the contaminant is already in the standard PDX and cell 		line databases)• Mouse syngeneic model identification (if applicable)• Mouse strain (if applicable)• Viral infection detection• Mycoplasma contamination detection• Gender identification for human• Human-mouse interspecies contamination quantification

2. Result Summary• Sample genetic heterogeneity quantification(See Table below)

Table 1. 1. Specimen Description
QC-ID
20200819-02699

Sample IDSpecies

GA9273-R1P2-20191119-R-37790

Human

Table 2. Result Summary
QC ItemResults

Mouse ratio

28.9%

Matched sample

GA9273Heterogeneity ratio*

1.91%

Contaminant detected

None

Mouse syngeneic modelNA

Mouse strain

NAViral infection

EBV

Mycoplasma contamination

Negative

GenderMale

Genetic admixture (CEU: CHB: YRI) **0.002: 0.002: 0.996*: See definition in Reference 1.
**: The three reference populations are: Han Chinese (CHB), Nigeria Yoruba (YRI) and Utah residents with Northern and Western European
ancestry from the CEPH collection (CEU).

2%)

3. Results
3.1. Mouse Ratio

Empirical Density

Density

0.04
0.03
0.02
0.01

4A below, adopted from Reference [1]), and can reliably detectmouse ratio at ~0.1% [1].

R=0.997, p=1.75e-10

75

0.

50

Density

Shapiro-Wilk p-value1.44e-09
Mean (95% CI)=30.62 (27.69-33.7%)
Median=28.9%

Conclusion: The mouse ratio in Sample GA9273-R1P220191119-R-37790 is 28.9%.

Known Mouse Ratio (%)

Comment: Mouse ratio is estimated based on 108 100-300bp
human-mouse homologous segments that share identicalflanking sequences and are amplified by same primers.

25020

40

60

80

100

0.

0.

000.00

0.

25

50

75

0.

Estimated Mouse Ratio (%)

33.2. Matched Sampleof minor contaminant. In contrast, genotype similarities between
unrelated samples are almost always below 50%. If two samples
are related, for example by deriving from same parental sample,
their genotype similarity can range from about 80% to close to100%, depending on their genetic divergence. Below: Figure 1A
from Reference [1].

Conclusion: Sample GA9273-R1P2-20191119-R-37790 is GA9273
based on its high identity to GA9273P00—the standard reference
sample of PDX model GA9273
Technical note: Genotype similarities calculated from >200 SNPs
are always >90% between identical samples even at the presence

IdenticalMismatch

Related50Density4030201000.25

0.500.75Genotype Similarity

1.00

Reference# Matched SNPs

# Nonmatched SNPs

TotalSNPs

Genotype similarity (%)

GA9273P00

222

5227

97.80

CR5076P00

111

98

209

53.11

LU9342P00

115

103

218

52.75

CL01123

111

100

211

52.61

LU11885P00

109

99

208

52.40

4

3.3. Heterogeneity Ratio

3.4. Contaminant Detection

Conclusion: Sample GA9273-R1P2-20191119-R-37790 has
a heterogeneity ratio 1.91% with the above distribution ofheterogeneity ratios for 227 SNPs. This heterogeneity ratio iswithin the standard value of PDX models with ~30% mouse ratios(see Figure 1C of Reference 1).

Conclusion: No contaminant is detected for sample GA9273R1P2-20191119-R-37790.

Technical note: Heterogeneity ratio is a quantitative measurefor genetic heterogeneity of a sample by using the >200 SNPs
and is defined in Table 1 of Reference [1]. Heterogeneity ratiocomes from genetic heterogeneity and contamination. Highheterogeneity ratio, as well as a two/three-modal distribution,
usually indicates possible contamination. See Reference [1]for details.

Technical note: In this quantile–quantile plot, each dot is areference cell line; theoretical and sample quantiles were
calculated from a beta distribution fitted to genotype similarities
between the test sample and all reference samples. The 99%
confidence band is shaded. A reference sample was denoted as
contaminant if (1) it had the highest genotype similarities, (2) its
genotype similarity was above the 99% confidence upper boundin the quantile–quantile graph and (3) its P-value was <1.0E−6 inthe fitted beta distribution. An example for contaminant is shown
below (Figure 3D from Reference [1]).

Figure 3.3
GA9273-R1P2-20191119-R-37790(GA9273P00)Density

60
4020
00.00

0.05

0.10

0.15

Heterogeneity Ratio

0.20

Figure 3.4
G292CloneA14B1

GA9273-R1P2-20191119-R-37790(GA9273P00)

0.8

Sample Quantiles

Sample Quantiles

0.80
0.75
0.70
0.65
0.60
0.60

0.65

0.70

0.75

Theoretical Quantiles

0.80

0.7

0.6

0.5
0.5

0.6

0.7

Theoretical Quantiles

53.5. Viral Infection & Mycoplasma Contamination

3.6. Gender Identification

Comment: If the read depth is > 1000, a base is accounted as ahigh coverage one.

Comments: (1) If the sum of read depths of the 3 Y-chromosome
SNPs >1000, the sample is identified as “Male”; If the sum if <300,the gender is predicted as “Female”; If the estimated mouse ratio
is over 95%, or the sum of depths is less than 1000 and morethan 300, the gender is not assigned. (2) Because Y chromosome
is frequently lost in cancer genomes, it is advised to use
microscope- based observation to confirm the gender prediction.

CMV: Cytomegalovirus
EBV: Epstein-Barr virus
HBV: Hepatitis B virus

Conclusion: Sample GA9273-R1P2-20191119-R-37790 is male.

HIV: Human immunodeficiency virus

3.7. Genetic Admixture Analysis

HPV: Human papillomavirus
Conclusion: Sample GA9273-R1P2-20191119-R-37790 is
EBV positive, and CMV/HBV/HIV/HPV16/HPV18/Mycoplasma
negative.
Technical note: Multiple pairs of primers were used to detecteach virus, and one pair of universal primers were used to detect
all mycoplasma species. See Reference [1] for details.

Based on the genotyping data of 143 SNPs, the genetic admixtureof this sample is 0.2% CHB, 0.2% CEU, and 99.6% YRI, where CHB
stands for Han Chinese, CEU for Utah residents with Northernand Western European ancestry from the CEPH collection, YRI for
Nigeria Yoruba.
Conclusion: Sample GA9273-R1P2-20191119-R-37790 is
predominantly of YRI origin.

Table 3.5.
Virus & Mycoplasma

Number of high coverage bases

CMV0

EBV

428

HBV0HIV0

HPV160

HPV180Mycoplasma0

Table 3.5.
SNP-anchored Y chromosome segment

Read depth

hg19_chrY_14832620

2713

hg19_chrY_15467824

3723

hg19_chrY_15591537

434264. Signatures
4.1. Sponsor Approval		DateSponsor Representative

4.2. Crown Bioscience (Suzhou) Approval		Date

Crown Bioscience Representative

5. References
1Chen,X., Qian,W., Song,Z., Li,Q. and Guo,S. (2020) Authentication, characterization and contamination detection of cell lines, xenografts and organoids
by barcode deep NGS sequencing. NAR Genomics and Bioinformatics, 2, 3. https://academic.oup.com/nargab/article-abstract/2/3/lqaa060/5893932

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