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Mitochondrial DNA What is mtDNA Typing? What is mtDNA Typing? Database and statistical issues Database and statistical issues

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Problematic Positive Controls

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Problem Calls

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Exclusion?

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Heteroplasmy

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Length Heteroplasmy

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Length Heteroplasmy?

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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).

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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).

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

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Group # Profiles African origin 1332 Caucasian origin 1674 Hispanic origin 686 Asian origin 821 Native American origin 326 Total4839

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Database # Profiles African-American1148 Sierra Leone 109 Caucasian1655 Hispanic686 Japan163 Korea182 Thailand52 Navajo146 Apache180 Egypt75 China/Taiwan329 Guam87

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Overall Search Results within Forensic Profiles Number of Differences From Search Profile NumberFrequency Cumulative Number Cumulative Frequency 01510.03651510.0365 13320.08024830.1166 22770.06697600.1835 32150.05199750.2354 42660.064212410.2996 54360.105316770.4049 > 5 24650.595141421.0000 Average Number of Differences = 6.681

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African Origin Database(s) within Forensic Profiles Number of Differences From Search Profile NumberFrequency Cumulative Number Cumulative Frequency 050.005450.0054 180.0086130.0140 280.0086210.0226 380.0086290.0313 4340.0366630.0679 5690.07441320.1422 > 5 7960.85789281.0000 Average Number of Differences = 9.881

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Caucasian Origin Database(s) within Forensic Profiles Number of Differences From Search Profile NumberFrequency Cumulative Number Cumulative Frequency 01410.07951410.0795 12960.16694370.2465 22430.13716800.3835 31670.09428470.4777 41580.089110050.5668 52190.123512240.6904 > 5 5490.309617731.0000 Average Number of Differences = 4.111

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Overall Search Results within Forensic Profiles From Search Profile NumberFrequency Cumulative Number Cumulative Frequency 0110.0023110.0023 11630.03371740.0360 23020.06244760.0984 32510.05197270.1502 43010.062210280.2124 > 438110.787648391.0000 Average Number of Differences = 7.913 Search Range(s) Differences from CRS 16024-16365 73-340 16189 C 263 G 309.1 C 309.2 C 315.1 C

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African Origin Database(s) within Forensic Profiles Number of Differences From Search Profile NumberFrequency Cumulative Number Cumulative Frequency 000.00000 180.00608 2130.0098210.0158 3100.0075310.0233 4170.0128480.0360 > 412840.964013321.0000 Average Number of Differences = 10.986

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Caucasian Origin Database(s) within Forensic Profiles Number of Differences From Search Profile NumberFrequency Cumulative Number Cumulative Frequency 0110.0066110.0066 11480.08841590.0950 22710.16194300.2569 32230.13326530.3901 41610.09628140.4863 > 48600.513716741.0000 Average Number of Differences = 4.947

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Asian origin Database(s) within Forensic Profiles Number of Differences From Search Profile Numbe r Frequenc y Cumulativ e Number Cumulativ e Frequenc y 000.00000 10 0 20 0 320.00242 4490.0597510.0621 > 47700.93798211.0000 Average Number of Differences = 7.380

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Caucasian origin Database(s) within Forensic Profiles Number of Differences From Search Profile NumberFrequency Cumulative Number Cumulative Frequency 01310.07831310.0783 12640.15773950.2360 22250.13446200.3704 31570.09387770.4642 41470.08789240.5520 > 47500.448016741.0000 Average Number of Differences = 4.216 Search Range(s) Differences from CRS 16024-16365 73-340 263 G 309.1 C 309.2 C 315.1 C

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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.

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UPPER CONFIDENCE LIMIT FROM ZERO PROPORTION P limit = 1 – – 1/N, where N = Database Size N UCL Maximum Frequency 500.058 1 in 17 1000.030 1 in 34 2000.015 1 in 67 5000.006 1 in 167 10000.003 1 in 334 50000.0006 1 in 1670

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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.

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Statistics Associated with One Case Match CriteriaFrequency 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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