DNA Short Tandem Repeats Organism DNA Short Tandem Repeats Organ - - PowerPoint PPT Presentation

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DNA Short Tandem Repeats Organism DNA Short Tandem Repeats Organ - - PowerPoint PPT Presentation

Apr 11, 2024 348 likes •1.38k views

DNA Short Tandem Repeats Organism DNA Short Tandem Repeats Organ DNA Short Tandem Repeats Cell Weights 1kg a bag of sugar 1g paper clip 1mg (milligram) 0.001g brain of a bee 1g (microgram) 0.000001g weight of a

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SLIDE 1

DNA Short Tandem Repeats

Organism

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SLIDE 2

DNA Short Tandem Repeats

Organ

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SLIDE 3

DNA Short Tandem Repeats

Cell

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SLIDE 4

Weights

  • 1kg – a bag of sugar
  • 1g – paper clip
  • 1mg (milligram) 0.001g – brain of a bee
  • 1µg (microgram) 0.000001g weight of a

bacterium

  • 1ng (nanogram) 0.000000001g a millionth
  • f a grain of salt - recommended input to

profiling

  • 1pg (picogram) 0.000000000001g 6pg of

DNA from each cell

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SLIDE 5

Cells

  • We lose about 30,000-40,000 skin cells an

hour

  • In a year, you lose about 8lbs of cells
  • “Where do they all go? The dust that collects
  • n your tables, TV, windowsills and on those

picture frames that are so hard to get clean is made mostly from dead human skin cells. In

  • ther words, your house is filled with former

bits of yourself.”

  • About 10,000 will fit on the head of a pin
  • Current DNA technology can profile one cell
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SLIDE 6

DNA Short Tandem Repeats

Nucleus

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SLIDE 7

DNA Short Tandem Repeats

Chromosomes

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SLIDE 8

DNA Short Tandem Repeats

DNA

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SLIDE 9

DNA Short Tandem Repeats

Locus

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SLIDE 10

DNA Short Tandem Repeats

STR

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SLIDE 11

DNA Short Tandem Repeats

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SLIDE 12

DNA Short Tandem Repeats

Allele

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SLIDE 13

DNA Short Tandem Repeats

Allele 5 3

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SLIDE 14

DNA Short Tandem Repeats

Locus is important FGA 3 D3 3

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SLIDE 15

DNA Short Tandem Repeats

A D3 vWA D16 D2 D8 D21 D18 D19 THO1

X Y 17 18 18 11 12 18 24 12 14 29 13 17 14 9 9.3

DNA profile Locus Allele Heterozygote Homozygote

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SLIDE 16

The process

  • Extraction
  • Quantitation
  • Amplification
  • Separation
  • Interpretation
  • Evaluation
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SLIDE 17

Amplification = Multiplication

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SLIDE 18

Raw data

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SLIDE 19

Single source profile

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SLIDE 20

One DNA component from mother, another from father Area of DNA tested Names of DNA components

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SLIDE 21

Why statistics?

  • DNA is NOT unique
  • We look at only a few areas
  • Need to know what the probability
  • f finding the profile by chance is

(i.e. to give an idea of how many

  • ther people may have been the

source of the profile)

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SLIDE 22

Statistical estimates

= 0.1

1 in a billion 1 in 10 1 in 111 1 in 20 1 in 22,200

x x

1 in 100 1 in 14 1 in 81 1 in 113,400

x x

1 in 116 1 in 17 1 in 16 1 in 31,552

x x

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SLIDE 23

Probability

  • Black hair
  • Blue eyes
  • Beard
  • Gold tooth

0.6 0.25 0.01 0.001

Probability= 0.6 x 0.25 x 0.01 x 0.001

= 0.0000015 = 1 in 666,666

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SLIDE 24

Random Match Probability

R B f 0.1 0.1

RB = 0.1 x 0.1 = 0.02 = 2 in 100 x 2 = 1 in 50

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SLIDE 25

Mixtures

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SLIDE 26

Mixtures

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SLIDE 27

?

Mixtures

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SLIDE 28

?

Mixtures

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SLIDE 29

?

Mixtures

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SLIDE 30

?

Mixtures

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SLIDE 31

Mixtures

RB RY RG BY BG GY

= 6 ‘suspect’ profiles that ‘cannot be excluded’ as contributors

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SLIDE 32

How many suspects?

  • With 6 possibilities at each of 15 areas
  • There are 6x6x6x6x6x6x6x6x6x6x6x6x6x6x6=
  • More than 60 million suspect profiles
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SLIDE 33

Alleles observed on ‘outside’

D8 D21 D7 CSF D3 THO1 D13 D16 D2 D19 vWA TPOX D18 D5 FGA

13 31.2 8 10 11 10 11 12 16 17 18 6 9 9.3 11 12 11 12 13 14 17 19 25 13 14 14 15 16 8 11 12 14 15 16 12 13 21 22 24 25 13 29 31.2 32.2 8 10 11 12 11 12 16 18 6 7 8 9.3 11 12 13 9 12 13 14 17 25 13 14 14 16 18 8 11 14 16 12 13 20 21 24

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SLIDE 34

D8 D21 D7 CSF D3 THO1 D13 D16 D2 D19 vWA TPOX D18 D5 FGA

13 29 31.2 32.2 8 10 11 12 10 11 12 16 17 18 6 7 8 9 9.3 11 12 13 9 11 12 13 14 17 19 25 13 14 14 15 16 18 8 11

12

14 15 16 12 13 20 21 22 24

25

Alleles observed on ‘outside’

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SLIDE 35
  • No. of alleles at each locus

D8 D21 D7 CSF D3 THO1 D13 D16 D2 D19 vWA TPOX D18 D5 FGA

13 29 31.2 32.2 8 10 11 12 10 11 12 16 17 18 6 7 8 9 9.3 11 12 13 9 11 12 13 14 17 19 25 13 14 14 15 16 18 8 11

12

14 15 16 12 13 20 21 22 24

25

1 3 4 3 3 5 3 5 3 2 4 3 3 2 5

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SLIDE 36

No of ‘suspect’ profiles

D8 D21 D7 CSF D3 THO1 D13 D16 D2 D19 vWA TPOX D18 D5 FGA

13 29 31.2 32.2 8 10 11 12 10 11 12 16 17 18 6 7 8 9 9.3 11 12 13 9 11 12 13 14 17 19 25 13 14 14 15 16 18 8 11

12

14 15 16 12 13 20 21 22 24

25

1 3 4 3 3 5 3 5 3 2 4 3 3 2 5 1 3 6 3 3 10 3 10 3 1 6 3 3 1 10

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SLIDE 37

D8 D21 D7 CSF D3 THO1 D13 D16 D2 D19 vWA TPOX D18 D5 FGA

13 29 31.2 32.2 8 10 11 12 10 11 12 16 17 18 6 7 8 9 9.3 11 12 13 9 11 12 13 14 17 19 25 13 14 14 15 16 18 8 11

12

14 15 16 12 13 20 21 22 24

25

1 3 4 3 3 5 3 5 3 2 4 3 3 2 5 1 x3 x6 x3 x3 x10 x3 x10 x3 x1 x6 x3 x3 x1 x10

No of ‘suspect’ profiles

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SLIDE 38

D8 D21 D7 CSF D3 THO1 D13 D16 D2 D19 vWA TPOX D18 D5 FGA

13 29 31.2 32.2 8 10 11 12 10 11 12 16 17 18 6 7 8 9 9.3 11 12 13 9 11 12 13 14 17 19 25 13 14 14 15 16 18 8 11

12

14 15 16 12 13 20 21 22 24

25

1 3 4 3 3 5 3 5 3 2 4 3 3 2 5 1 x3 x6 x3 x3 x10 x3 x10 x3 x1 x6 x3 x3 x1 x10 = 78,732,000 ‘suspect profiles

No of ‘suspect’ profiles

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SLIDE 39

D8

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SLIDE 40

D8

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SLIDE 41

D8

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SLIDE 42

Adding ‘new’ alleles at D8

D8 D21 D7 CSF D3 THO1 D13 D16 D2 D19 vWA TPOX D18 D5 FGA

9 11 13 14 29 31.2 32.2 8 10 11 12 10 11 12 16 17 18 6 7 8 9 9.3 11 12 13 9 11 12 13 14 17 19 25 13 14 14 15 16 18 8 11

12

14 15 16 12 13 20 21 22 24

25

4 3 4 3 3 5 3 5 3 2 4 3 3 2 5 6 3 6 3 3 10 3 10 3 1 6 3 3 1 10

472,392,000 (470m) ‘suspect’ profiles

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SLIDE 43

D21

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SLIDE 44

D21 ‘zoom’

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SLIDE 45

D21

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SLIDE 46

D8 D21 D7 CSF D3 THO1 D13 D16 D2 D19 vWA TPOX D18 D5 FGA

9 11 13 14 28 29 30 31.2 32.2 8 10 11 12 10 11 12 16 17 18 6 7 8 9 9.3 11 12 13 9 11 12 13 14 17 19 25 13 14 14 15 16 18 8 11

12

14 15 16 12 13 20 21 22 24

25

4 5 4 3 3 5 3 5 3 2 4 3 3 2 5

D8 D21 D7 CSF D3 THO1 D13 D16 D2 D19 vWA TPOX D18 D5 FGA

19 11 13 14 28 29 30 31.2 32.2 8 10 11 12 10 11 12 16 17 18 6 7 8 9 9.3 11 12 13 9 11 12 13 14 17 19 25 13 14 14 15 16 18 8 11

12

14 15 16 12 13 20 21 22 24

25

4 5 4 3 3 5 3 5 3 2 4 3 3 2 5 6 10 6 3 3 10 3 10 3 1 6 3 3 1 10 1,574,640,000 (1.5 billion) ‘suspect profiles

Adding ‘new’ alleles at D21

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SLIDE 47

D8 D21 CSF D3 THO1 D13 D19 TPOX D18 D5

IN 13 14 31.2 10 16 6 12 13 14 11 13 20 OUT 13 29 31.2 32.2 10 11 12 16 17 18 6 7 8 9 9.3 11 12 13 13 14 8 11 12 14 15 16 20 21 22 24 25

Alleles on inside & outside

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SLIDE 48

The Likelihood Ratio = LR

Probability of this evidence if the DNA came from Mr X + unknown Probability of this evidence if it came from 2 unknowns

LR = Probability of E given Hpros Probability of E given Hdef

“… times more likely”

e.g. LR = 1/10 1/100 = 0.1 0.001 = 10

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SLIDE 49

LR = 1 (1/frequency)

For single source profiles

=frequency e.g. 1/(1/10) = 10

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SLIDE 50

Mixtures

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SLIDE 51

R B Y G f 0.25 0.25 0.25 0.25 X p(Hp) p(Hd) LR RB 0.125 0.0469 2.67 RY 0.125 0.0469 2.67 RG 0.125 0.0469 2.67 BY 0.125 0.0469 2.67 BG 0.125 0.0469 2.67 YG 0.125 0.0469 2.67 “Mr X + unknown rather than two unknowns”

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SLIDE 52

R B Y G f 0.1 0.1 0.25 0.25 Mr X p(Hp) p(Hd) LR RB 0.125 0.0075 16.67 RY 0.05 0.0075 6.67 RG 0.05 0.0075 6.67 BY 0.05 0.0075 6.67 BG 0.05 0.0075 6.67 YG 0.02 0.0075 2.67 “Mr X + unknown rather than two unknowns”

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SLIDE 53

R B Y G f 0.1 0.1 0.25 0.25 Mr X p(Hp) p(Hd) LR RB 0.125 0.0075 16.67 RY 0.05 0.0075 6.67 RG 0.05 0.0075 6.67 BY 0.05 0.0075 6.67 BG 0.05 0.0075 6.67 YG 0.02 0.0075 2.67 “Mr X + unknown rather than two unknowns”

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SLIDE 54

RG 33.33

“Mr X + unknown rather than two unknowns”

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SLIDE 55

R B Y G f 0.01 0.1 0.2 0.5 Mr X p(Hp) p(Hd) LR RB 0.2 0.0012 166.67 RY 0.1 0.0012 83.33 RG 0.04 0.0012 33.33 BY 0.01 0.0012 8.33 BG 0.004 0.0012 3.33 YG 0.002 0.0012 1.67

“Mr X + unknown rather than two unknowns”

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SLIDE 56

R B Y G f 0.01 0.1 0.2 0.5 Mr X p(Hp) p(Hd) LR RB 0.2 0.0012 166.67 RY 0.1 0.0012 83.33 RG 0.04 0.0012 33.33 BY 0.01 0.0012 8.33 BG 0.004 0.0012 3.33 YG 0.002 0.0012 1.67

“Mr X + unknown rather than two unknowns”

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SLIDE 57

More complicated mixture

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SLIDE 58

Second area

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SLIDE 59

Second area

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SLIDE 60

A B A C D D B C

Second area (locus)

A B C D

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SLIDE 61

AB AC AD BC BD CD

= 6 ‘suspect’ profiles that ‘cannot be excluded’ as contributors

Second area only

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SLIDE 62

AB AC AD BC BD CD RB RY RG BY BG YG 444

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SLIDE 63

AB AC AD BC BD CD RB RY RG BY BG YG 444 889 444

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SLIDE 64

AB AC AD BC BD CD RB RY RG BY BG YG 1,778 889 1,778 444 889 444

“X + unknown rather than two unknowns”

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SLIDE 65

AB AC AD BC BD CD RB RY RG BY BG 44,444 22,222 44,444 11,111 22,222 11,111 YG 1,778 889 1,778 444 889 444

“X + unknown rather than two unknowns”

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SLIDE 66

AB AC AD BC BD CD RB 88,889 44,444 88,889 22,222 44,444 22,222 RY RG BY BG 44,444 22,222 44,444 11,111 22,222 11,111 YG 1,778 889 1,778 444 889 444

“X + unknown rather than two unknowns”

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SLIDE 67

AB AC AD BC BD CD RB 88,889 44,444 88,889 22,222 44,444 22,222 RY 3,556 1,778 3,556 889 1,778 889 RG 8,889 4,444 8,889 2,222 4,444 2,222 BY 17,778 8,889 17,778 4,444 8,889 4,444 BG 44,444 22,222 44,444 11,111 22,222 11,111 YG 1,778 889 1,778 444 889 444

“X + unknown rather than two unknowns”

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SLIDE 68

Stochastic variation

Examples so far assume allele calls are certain, but low template samples cause new problems because of stochastic variation.

  • Stochastic variation is random

variation

  • Failure to reproduce results
  • Leads to uncertainty
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SLIDE 69
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SLIDE 70
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SLIDE 71
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SLIDE 73
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SLIDE 74

The crimestain

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SLIDE 75

Standard technique

Enough sample so that no dropout is expected and peak height represents amount of DNA present (i.e. not variable)

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SLIDE 76
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SLIDE 77
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SLIDE 78
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SLIDE 79
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SLIDE 80
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SLIDE 81
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SLIDE 82
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SLIDE 83

Low Template Sample

  • Stochastic variation is random

variation

  • Failure to reproduce results
  • Leads to uncertainty
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SLIDE 84
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SLIDE 85
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SLIDE 86
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SLIDE 87
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SLIDE 88
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SLIDE 89
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SLIDE 90
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SLIDE 91

A B C D E F G H I

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SLIDE 92
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SLIDE 93
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SLIDE 94
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SLIDE 95
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SLIDE 96
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SLIDE 97
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SLIDE 98

A B C D E F

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SLIDE 99

Dropout or dropin?

D8 D21 D7 CSF D3 THO1 D13 D16 D2 D19 vWA TPOX D18 D5 FGA

13 31.2 8 10 11 10 11 12 16 17 18 6 9 9.3 11 12 11 12 13 14 17 19 25 13 14 14 15 16 8 11 12 14 15 16 12 13 21 22 24 25 13 29 31.2 32.2 8 10 11 12 11 12 16 18 6 7 8 9.3 11 12 13 9 12 13 14 17 25 13 14 14 16 18 8 11 14 16 12 13 20 21 24

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SLIDE 100

Probability of dropout and dropin

p(D) Is the probability that an allele is really there but you have not detected it. p(C) Is the probability that an allele you have detected is not from the crimestain – it is contamination

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SLIDE 101

FST statistic

  • FST is the programme used to

calculate the LR in this case

  • Statistic depends on

– Probability of dropout which is

  • Dependent usually on the weight of DNA
  • Which is unknown for the minor

contributors

– And the validation data do not support any p(D) for any weight of DNA – The LR being correct

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SLIDE 102

Low Template Sample

  • Identified by variable results, NOT

the amount of DNA

  • Causes problems in;

– Identifying ‘true’ sample alleles – Using peak height information

  • Inclusion/exclusion of people
  • Number of contributors