mitochondrial dna what is mtdna typing? what is mtdna typing? database and statistical issues...

Mitochondrial DNAMitochondrial DNA

What is mtDNA What is mtDNA Typing?Typing?

Database and Database and statistical issuesstatistical issues

Problematic Positive Controls

Problematic Positive Controls

Problem Calls

Exclusion?

Heteroplasmy

Length Heteroplasmy

Length Heteroplasmy?

VALIDATION OF LABORATORY CONTAMINATION  “Because of the sensitivity of this method. The issue of detection and quantitation of external contamination is important. We have observed low levels of amplified product in negative controls and reagent blanks when amplifying human mtDNA.” (Wilson et al., 1995a, p 667). “Previously sequenced amplicons were quantified by CE and two samples with different mtDNA control region sequences were chosen for this study. Amplified DNA was mixed together from the two samples in ratios of 4:1, 8:1, 12:1 and 16:1. Sequencing reactions were performed on the mixtures as previously described. Editing of the analyzed data from the sequence of the mixed samples was conducted by two individuals. Ambiguous bases (bases which could not be designated) and errors (bases which were called incorrectly) were noted. In the 4:1 mixture, both ambiguities and errors were observed. The 8:1 mixture yielded only two ambiguous calls from both editors at positions that differed in sequence between the mixed templates. No errors or ambiguities were noted at the 8:1 mixture. All base calls were completely consistent with the more abundant sequence at and above 8:1.” (Wilson et al., 1995a, p 667).

LABORATORY CONTAMINATION, CONT.  “In order to confirm that correct typing results can be achieved using this ratio, additional tests were conducted. Amplified DNA samples from a variety of donors were mixed in 10:1 ratios with other amplified DNAs. The dominant and minor samples were blindly reversed to the sequence editors. At the 10:1 ratio in a total of five such tests, all of the base calls from both editors were consistent with the known sequence of the more abundant sample with no errors or ambiguous calls.” (Wilson et al., 1995a, p 668).

SEQUENCE FREQUENCYSEQUENCE FREQUENCY

““Unless the discriminatory Unless the discriminatory potential of a test can be potential of a test can be objectively evaluated, an inclusion objectively evaluated, an inclusion could mean anything. It is could mean anything. It is therefore incumbent on the forensic therefore incumbent on the forensic scientist to determine a means to scientist to determine a means to evaluate and communicate the evaluate and communicate the significance of an mtDNA inclusion significance of an mtDNA inclusion or ‘match’.” Holland & Parsons, or ‘match’.” Holland & Parsons, 1999. Forens. Sci. Intl. 1999. Forens. Sci. Intl.

GroupGroup # Profiles# Profiles

African originAfrican origin 13321332

Caucasian originCaucasian origin 16741674

Hispanic originHispanic origin 686686

Asian originAsian origin 821821Native American Native American originorigin 326326

TotalTotal 48394839

DatabaseDatabase # Profiles# Profiles

African-AmericanAfrican-American 11481148

Sierra LeoneSierra Leone 109109

CaucasianCaucasian 16551655

HispanicHispanic 686686

JapanJapan 163163

KoreaKorea 182182

ThailandThailand 5252

NavajoNavajo 146146

ApacheApache 180180

EgyptEgypt 7575

China/TaiwanChina/Taiwan 329329

GuamGuam 8787

0

5

10

15

20

25

30

AfAm SierraL. Cauc. Hisp. Japan Korean Thai Navajo Apache Egypt China Guam

Mean Number of Differences Between Pairs by Ethnic Group

Mean Number of Differences

Standard Deviation

Percent Matches

Pair-wise Matches in Large Mixed Ethnic Groups

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

African(4x) Caucasian(5x) Hispanic(3x) Asian(3x) NatAm(2x)

Increased Likelihood of Match Using FBI Guidelines

Pro

port

ion

of P

aiw

ise

Com

paris

ons

that

Mat

ch

Profiles with Zero DifferencesProfiles with Zero or One Difference

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Number of Differences

Pro

po

rtio

n o

f Pai

r-W

ise

Co

mp

aris

on

s

African

Caucasian

Hispanic

Asian

Native American

Overall Search Results within Forensic Profiles

Number of DifferencesNumber of DifferencesFrom Search Profile From Search Profile Number Number Frequency Frequency

CumulativeCumulativeNumber Number

CumulativeCumulativeFrequency Frequency

0 0 151 151 0.03650.0365 151151 0.0365 0.0365

1 1 332 332 0.08020.0802 483483 0.1166 0.1166

2 2 277 277 0.06690.0669 760760 0.1835 0.1835

3 3 215 215 0.05190.0519 975975 0.2354 0.2354

4 4 266 266 0.06420.0642 12411241 0.2996 0.2996

5 5 436 436 0.10530.1053 16771677 0.4049 0.4049

> 5 > 5 2465 2465 0.5951 0.5951 4142 4142 1.0000 1.0000

Average Number of Differences = 6.681

African Origin Database(s) within Forensic Profiles

Number of Number of DifferencesDifferences

From Search Profile From Search Profile Number Number Frequency Frequency

CumulativeCumulativeNumber Number

CumulativeCumulativeFrequency Frequency

0 0 5 5 0.0054 0.0054 5 5 0.0054 0.0054

1 1 8 8 0.0086 0.0086 13 13 0.0140 0.0140

2 2 8 8 0.0086 0.0086 21 21 0.0226 0.0226

3 3 8 8 0.0086 0.0086 29 29 0.0313 0.0313

4 4 34 34 0.0366 0.0366 63 63 0.0679 0.0679

5 5 69 69 0.0744 0.0744 132 132 0.1422 0.1422

> 5 > 5 796 796 0.8578 0.8578 928 928 1.0000 1.0000

Average Number of Differences = 9.881

Caucasian Origin Database(s) within Forensic Profiles

Number of DifferencesNumber of DifferencesFrom Search Profile From Search Profile Number Number Frequency Frequency

CumulativeCumulativeNumber Number

CumulativeCumulativeFrequency Frequency

0 0 141 141 0.0795 0.0795 141 141 0.0795 0.0795

1 1 296 296 0.1669 0.1669 437 437 0.2465 0.2465

2 2 243 243 0.1371 0.1371 680 680 0.3835 0.3835

3 3 167 167 0.0942 0.0942 847 847 0.4777 0.4777

4 4 158 158 0.0891 0.0891 1005 1005 0.5668 0.5668

5 5 219 219 0.1235 0.1235 1224 1224 0.6904 0.6904

> 5 > 5 549 549 0.3096 0.3096 1773 1773 1.0000 1.0000

Average Number of Differences = 4.111

Overall Search Results within Forensic Profiles

From Search Profile Number Frequency

CumulativeNumber

CumulativeFrequency

0 11 0.0023 11 0.0023

1 163 0.0337 174 0.0360

2 302 0.0624 476 0.0984

3 251 0.0519 727 0.1502

4 301 0.0622 1028 0.2124

> 4 3811 0.7876 4839 1.0000

Average Number of Differences = 7.913

Search Range(s)Differencesfrom CRS

16024-1636573-340

16189 C263 G

309.1 C309.2 C315.1 C

African Origin Database(s) within Forensic Profiles

Number of DifferencesFrom Search Profile Number Frequency

CumulativeNumber

CumulativeFrequency

0 0 0.0000 0 0.0000

1 8 0.0060 8 0.0060

2 13 0.0098 21 0.0158

3 10 0.0075 31 0.0233

4 17 0.0128 48 0.0360

> 4 1284 0.9640 1332 1.0000

Average Number of Differences = 10.986

Caucasian Origin Database(s) within Forensic Profiles

Number of DifferencesFrom Search Profile

Number Frequency

CumulativeNumber

CumulativeFrequency

0 11 0.0066 11 0.0066

1 148 0.0884 159 0.0950

2 271 0.1619 430 0.2569

3 223 0.1332 653 0.3901

4 161 0.0962 814 0.4863

> 4 860 0.5137 1674 1.0000

Average Number of Differences = 4.947

Asian origin Database(s) within Forensic Profiles

Number of Differences

From Search Profile Numbe

r Frequenc

y

Cumulative

Number

Cumulative

Frequency

0 0 0.0000 0 0.0000

1 0 0.0000 0 0.0000

2 0 0.0000 0 0.0000

3 2 0.0024 2 0.0024

4 49 0.0597 51 0.0621

> 4 770 0.9379 821 1.0000

Average Number of Differences = 7.380

Caucasian origin Database(s) within Forensic Profiles

Number of DifferencesFrom Search Profile

Number Frequency

CumulativeNumber

CumulativeFrequency

0 131 0.0783 131 0.0783

1 264 0.1577 395 0.2360

2 225 0.1344 620 0.3704

3 157 0.0938 777 0.4642

4 147 0.0878 924 0.5520

> 4 750 0.4480 1674 1.0000

Average Number of Differences = 4.216

Search Range(s)Differencesfrom CRS

16024-1636573-340

263 G309.1 C309.2 C315.1 C

Number of Base Pair Differences

0

50

100

150

200

250

300

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Number of Differences

Freq

uenc

y African

Caucasian

Hispanic

Asian

Native

A 95% Upper Confidence Limit Using the Normal Approximation of the Binomial

UCL= P + 1.96 [PQ/N]1/2

where P = X/N, Q = 1-P, N = Database Size, and X = number of times a matching sequence is found in the database.

A 99% Upper Confidence Limit Equals

UCL= P + 2.58 [PQ/N]1/2

where P = X/N, Q = 1-P, N = Database Size, and X = number of times a matching sequence is found in the database.

UPPER CONFIDENCE LIMIT FROM ZERO PROPORTION

P limit = 1 – – 1/N, where N = Database Size

N UCL Maximum Frequency

50 0.058 1 in 17

100 0.030 1 in 34

200 0.015 1 in 67

500 0.006 1 in 167

1000 0.003 1 in 334

5000 0.0006 1 in 1670

Not Excl.Not Excl.Not Excl.Not Excl.Not Excl.Not Excl.Not Excl.Not Excl.Observed

16183-C16189-C16189-C16189-C16189-C/TCRS-HV116189-C/T16189-T16189-C16189-C73-G263-G263-G263-G263-G263-G263-G263-G263-G263-G309.1-C309.1-C309.1-C309.1-C309.1-C309.1-C309.1-C309.1-C309.2-C309.2-C309.2-C309.2-C309.2-C309.2-C309.2-C315.1-C315.1-C315.1-C315.1-C315.1-C315.1-C315.1-C315.1-C

1 diff1 diff0 diff1 diff0 diff1 diff`1 diff1 diff0 diff

FBI Protocol: Many Sequences Other than Exact Matches Would Not be Excluded, Including All Which Differ by Zero or One Base Pair. All Individuals in This Group Would Not Be Ruled Out as Potential Sources of the Evidence at Issue and So Must Be Counted to Estimate the Rarity of Potential Contributors.

Statistics Associated with One Case

Match Criteria Frequency 95%UCL Not Excluded

Zero Differences 0.00725 0.01133 1 in 88 98.9% Excluded

Zero+One 0.09662 0.11085 1 in 9 88.9% Excluded