Mitochondrial Mitochondrial DNA DNA What is What is mtDNA Typing? mtDNA Typing? Database and Database and statistical statistical issues issues
Mitochondrial DNA What is mtDNA Typing? What is mtDNA Typing? Database and statistical issues Database and statistical issues.
Mar 26, 2015
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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
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