This document summarizes a study that analyzed X-STR linkage groups to establish maternal relatedness in opposite-sex siblings. The study aimed to address problems with current autosomal STR testing which can produce false positives. It hypothesized that siblings would share more X-STR linkage groups than unrelated individuals. The study analyzed 27 pairs of opposite-sex siblings and 110 unrelated male-female pairs using the Investigator Argus X-12 kit. Results found siblings shared significantly more linkage groups than unrelated pairs. Statistical analysis determined a threshold of 2 or more shared linkage groups could determine relatedness with 100% specificity and 85.16% sensitivity. The study concluded linkage group analysis is an effective complementary test to autosomal STR for determining maternal relationships in opposite

1. Importance of X-STR Linkage Groups in the
Establishment of Maternal Relatedness in
Opposite-Sex Siblings
Md Shamir Montazid
Exam Roll: 305
Session: 2014-15
Registration NO: 2011-612-458
Department of Genetic Engineering & Biotechnology
University of Dhaka

2. Background Stories
Current Problems
Objectives of Study
Materials & Methods
Results
Discussion
Conclusion
Outline

3. What is Sibling Analysis?

4. Short Tandem Repeat (STR)
2-7 bp long repeat motifs present in head to tail fashion in DNA.
---[TCAT] [TCAT] [TCAT] [TCAT] [TCAT]---
5 alleles of STR LocusTH01
Sibling Analysis
Determination of
Full-Siblings &
Half Siblings
Determination of
Unrelated
Analysis of
Short Tandem
Repeats of DNA
Involves
Statistical Analysis

5. Current Method
Calculation of
Sibling Index
(SI)
Combined Sibling
Index (CSI)
CSI= SI1×...× SIn
Calculate
Likelihood
Ration (LR)
Determination
of
Genotype

6. Problems of Current Method
Produces
FALSE POSITIVE result.
Nearly 1 in 200 unrelated
pairs are shown as FULL
SIBLINGS.
Major Problem:
Siblings share No
Obligate Allele.
Results are not 100%
conclusive
Low Specificity
of the autosomal
STR test is the
problem

7. Alternatives to Autosomal STR?

8. Females can not be analyzed!
Y-STR analysis is suitable for two Brothers
Application of Y-STR

9. X-STR analysis is suitable for Two Sisters
Maternal X-Chromosome undergoes recombination.
Application of X-STR

10. So, analysis of maternal X-
Chromosome is the
only alternative option!
No Alternative for Opposite-Sex Sibling Analysis!

11. A Linkage Group is a collection of X-STRs that is inherited
together during recombination.
!X-STRs are present as Linkage Groups
STRs in a Linkage Group (LG) are situated in less than 1cM distance.
Reference: Machado et al (2009)
Linkage Group

12. Figure: X-STR Linkage Groups analyzed by Investigator Argus X-12 Kit.
(Distances from the p-telomere has been shown in Mb)
www.ncbi.nlm.nih.gov/genome/guide/human
X-STR Linkage Group

13. So, What is our Problem?

14. ! Alternative Analysis of
Opposite-Sex Siblings
! Increase the Specificity of test to
exclude unrelated individuals
! Addressing the presence of
Linkage Group
To
By
Problems

15. Establishing Linkage Group of X-STRs as an
alternative test to determine maternal
relatedness of Opposite-Sex Siblings
Our Goal

16. ? Do Siblings share more Linkage Groups than
Unrelated individuals?
? What is the “Power of Discrimination”
of Linkage Group?
? What is the Threshold number of shared Linkage
Group for being Siblings?
Research Queries

17. Materials & Methods…

18. 27 Pairs of
Opposite-
Sex Siblings
(Case)
110 Pairs of
Unrelated
Male-Female
(Control)
Case-
Control
Study
Study Design

19. Step-1
• Sample Collection (Buccal Swab)
Step-2
• DNA Extraction (Chelex Method)
Step-3
• DNA Amplification by PCR (Argus X-12 Kit)
Step-4
• Capillary Electrophoresis and Fluorescent
Detection (Genetic Analyzer)
Step-
4a
Peak Identification
Step-
4b
Peak Separation
Step-
4C
Peak Sizing
GeneMapperID-X
Methods

20. Results

21. sadfadsf
[Electropherogram produced by Genetic Analyzer3500 after capillary electrophoresis for 1300s at 15KV]
6-FAM
BTG
BTY
BTR
DyeLabelsusedinInvestigatorArgusX-12Kit
XX (Female)
11,12
16,18 11,11 13,13 35,38
17, 18 28.2, 30 24, 30
27, 28 20, 20
25, 27.1
14, 14
Electropherogram

22. Linkage Group Locus Haplotype of Brother Genotype of Sister
Amelogenin XY XX
Linkage
Group-01
DXS10074 17 15, 16
DXS10079 19 19, 20
DXS7132 12 13, 15
Linkage
Group-02
DXS10101 31 27, 27
DXS10103 18 16, 18
HPRTB 12 12, 13
Linkage
Group-03
DXS10134 36 36, 35
DXS10146 25 25, 29
DXS7423 16 16,16
Linkage
Group-04
DXS10135 23 23,29
DXS10148 26.1 26.1, 24.1
DXS8378 11 11, 11
The Genotype generated by Gene Mapper ID-X software from the Electropherogram
X-STR Genotype/Haplotype

23. Calculation of Linkage Group
Genotypes/
Haplotypes
Genoproof3
Software
Linkage Group
Calculation

24. Linkage
Group
Locus Haplotype of Brother Genotype of Sister
Amelogenin XY XX
Linkage
Group-01
DXS10074 17 15, 16
DXS10079 19 19, 20
DXS7132 12 13, 15
Linkage
Group-02
DXS10101 31 27, 27
DXS10103 18 16, 18
HPRTB 12 12, 13
Linkage
Group-03
DXS10134 36 36, 35
DXS10146 25 25, 29
DXS7423 16 16,16
Linkage
Group-04
DXS10135 23 23,29
DXS10148 26.1 26.1, 24.1
DXS8378 11 11, 11
Number of Shared Linkage Group: 02 (calculated by Genoproof3)
Calculation of Linkage Group

25. Distribution of Linkage Group
0
2
4
6
8
10
12
14
16
0 1 2 3 4
NoofSiblingPairs
No of shared Linkage Group
0
20
40
60
80
100
120
0 1 2 3 4
NoofUnrelatedPairs
No of shared Linkage Group
Sibling Pair (Case) Unrelated Pairs (Control)
Total: 27 Pairs Total: 110 Pairs
XS= No of Share LG between Siblings XU= No of Share LG between Unrelated

26. Statistical Analysis

27. Determination of Threshold
Calculate Rate of True
vs False Positive
ROC Curve
Select Threshold

28. Figure: Receiver-Operating Characteristic Curve
(generated by MS Excel 2016)
ROC Curve
LG=0
LG=1
LG=2
LG=3
LG=4
0
0.2
0.4
0.6
0.8
1
1.2
-0.2 0 0.2 0.4 0.6 0.8 1 1.2
TruePositive
False Positive
1 0.090909091 0.925925926
2 0 0.851851852
Threshold False Positive True Positive
100% True Positive
0% False Positive

29. Area Under the ROC Curve (W)
0
0.2
0.4
0.6
0.8
1
1.2
-0.2 0 0.2 0.4 0.6 0.8 1 1.2
TruePositive
False Positive
Area Under the Curve, θ= Pr (XS > XU)
[Probability of Linkage Group to Discriminate
between a pair of Siblings & Unrelated]
Calculated W*= 0.9562
*Wilcoxon Method was used

30. 𝑺𝑬 𝑾 =
𝛉 𝟏 − 𝜽 + 𝒏 𝑼 − 𝟏 𝑸 𝟏 − 𝜽 𝟐 + 𝒏 𝑺 − 𝟏 𝑸 𝟐 − 𝜽 𝟐
𝒏 𝑺 𝒏 𝑼
Hanley, J. a & McNeil, B. J (1982)
Standard Error of W
SE(W) = 0.0309 = 3.09%
This small Value of SE(W) denotes high Significance!

31. Discussion

32. Hypothesis Testing
Ho: Area Under the Curve = 0.5; XS and XU are indifferent
H1: Area Under the Curve > 0.5; XS is larger than XU
There are No difference between Siblings & Unrelated
Siblings share more LG than Unrelated
LG=0
LG=1
LG=2
LG=3
LG=4
0
0.2
0.4
0.6
0.8
1
1.2
-0.2 0 0.2 0.4 0.6 0.8 1 1.2
TruePositive
False Positive

33. • θ (area under the curve) follows a normal
distribution under null hypothesis
• W is an estimate of θ under null hypothesis
Hypothesis Testing
𝒁 𝑺𝒄𝒐𝒓𝒆 =
𝑾−𝜽
𝑺𝑬(𝑾)
=
𝟎.𝟗𝟓𝟔𝟐𝟑−𝟎.𝟓
𝟎.𝟎𝟑𝟎𝟗
= 𝟏𝟒. 𝟕𝟔𝟏𝟎𝟏*
Ho is rejected with p-value <0.00001*
H1 is accepted: Siblings share more LG
than Unrelated
*Calculated using R-package

34. Answers to Research Queries
1 Siblings share more Linkage Group than
Unrelated Individuals
2 LG has 95.62% probability to discriminate between
siblings and unrelated (std. error=3.09%)
3 If a pair of male-female shares at least 2 linkage
groups, they can not be excluded from test.
(Specificity 100%, Sensitivity 85.16%)

35. Problems
! Alternative Analysis of
Opposite-Sex Siblings
! Increase the Specificity of test to
exclude unrelated individuals
! Addressing the presence of
Linkage Group
To
By
Problems Solved!

36. This approach can only predict Maternal Relatedness.
Two individuals excluded by this test can still be
paternally related half-siblings.
Limitations of X-STR Linkage Group
The sensitivity of the Linkage Group test was 85.16%
So, there is a 14.84% chance of False Negative Result

37. Conclusion

38. Conclusion:
Maternally
Related
LG ≥ 2
How Many
LGs do they
share?
Further
Analysis
LG < 2
What is the
Conclusion
of the Test?
Exclusion from
Test
Unrelated
Linkage Group Analysis is a Complementary test for Autosomal STR
Two-Tier Test Approach
Pair of Male
and Female
Tier-01:
Analysis of
Autosomal
STR
Tier-02:
Calculation
of X-STR
Linkage
Group
Related

39. Novel Research Queries
? Can X-STR Linkage Group approach be applied in the
cases of Same-Sex Siblings?

40. Once you eliminate the impossible,
whatever remains, no matter how
improbable, must be the truth.
(Sherlock Holmes,
Hounds of Baskerville)

41. Thank You

44. Identification of Long Lost
Relatives
Property Inheritance Issue
Disaster Victim Identification
Forensic Cases
Donor Identification for
Organ Transplantation
Application of Sibling Analysis

45. 𝑇𝑟𝑢𝑒 𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒 =
𝑛(𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑏𝑦 𝑡𝑒𝑠𝑡)
𝑛(𝑆𝑖𝑏𝑙𝑖𝑛𝑔 𝑃𝑎𝑖𝑟𝑠)
True Positive vs False Positive
Threshold False Positive True Positive
0
1
1
0.090909091
1
0.925925926
2 0 0.851851852
3
4
0
0
0.333333333
0.148148148
𝐹𝑎𝑙𝑠𝑒 𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒 =
)𝑛(𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑏𝑦 𝑡𝑒𝑠𝑡
)𝑛(𝑈𝑛𝑟𝑒𝑙𝑎𝑡𝑒𝑑 𝑃𝑎𝑖𝑟𝑠
*Calculated in MS Excel 2016

46. t-test is not suitable as:
• Our distribution is not Normal
• Our data is Ordinal and Discontinuous in
nature
Selection of Test
Our Choice: Mann-Whitney-Wilcoxon Test (MWW)
• Equivalent to t-test
• Two Sample Independent Test
• Wilcoxon Statistic follows Normal Distribution

47. Sampling Distribution
Reference: Hettmansperger, T. & McKean, J. Robust
Nonparametric Statistical Methods. 1998. Arnold, London

48. Parameter Setting
Application Type HID
Capillary Length 36 cm
Polymer POP4
Dye Set BT5
Run Module HID36_POP4
Protocol Name Investigator Argus X-12
Oven Temperature Default
Run Voltage Default
Pre-Run Voltage Default
Injection Voltage 3.0 KV
Run Time 1300 sec
Pre-Run Time Default
Injection Time 8 sec
Data Delay Default
Advanced Options Default
CE Parameters

49. Loci Dye Label
Amelogenin-DXS10103-DXS8378-DXS7132-DXS10134 6-FAM (Blue)
DXS10074-DXS10101-DXS10135 BTG (Green)
DXS7423-DXS10146-DXS10079 BTY (Yellow)
HPRTB-DXS10148 BTR (Red)
Size Standard BTO (Orange)
Dye Label

50. Temperature Time Number of
Cycles
94°C 4 min -
96°C
63°C
72°C
30 sec
120 sec
75 sec
5 cycles
94°C
60°C
72°C
30 sec
120 sec
75 sec
25 cycles
68°C 60 min -
10°C ∞ -
PCR Parameters

51. Allele Sharing in
Tested Individuals
Genotypes X
Full Sibling
Y
Half Sibling
Z
UnrelatedIndividual-1 Individual-2
Share two alleles pq pq 1+p+q+2pq p+q+4pq 8pq
Share two allele
doubly
pp pp (1+p)2 2p(1+p) (2p)2
Share one allele
doubly
pp pq (1+p) (1+2p) 2p
Share one allele pq pr (1+2p) (1+4p) 8p
Share no allele pq/pp rs/rr 1 3 4
Sibling Index (SI) Formula

52. Consent Form