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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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Presentation on theme: "Mitochondrial DNA What is mtDNA Typing? What is mtDNA Typing? Database and statistical issues Database and statistical issues."— Presentation transcript:

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

2 Problematic Positive Controls

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4 Problem Calls

5 Exclusion?

6 Heteroplasmy

7 Length Heteroplasmy

8 Length Heteroplasmy?

9 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).

10 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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12 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, Forens. Sci. Intl.

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

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

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18 Overall Search Results within Forensic Profiles Number of Differences From Search Profile NumberFrequency Cumulative Number Cumulative Frequency > Average Number of Differences = 6.681

19 African Origin Database(s) within Forensic Profiles Number of Differences From Search Profile NumberFrequency Cumulative Number Cumulative Frequency > Average Number of Differences = 9.881

20 Caucasian Origin Database(s) within Forensic Profiles Number of Differences From Search Profile NumberFrequency Cumulative Number Cumulative Frequency > Average Number of Differences = 4.111

21 Overall Search Results within Forensic Profiles From Search Profile NumberFrequency Cumulative Number Cumulative Frequency > Average Number of Differences = Search Range(s) Differences from CRS C 263 G C C C

22 African Origin Database(s) within Forensic Profiles Number of Differences From Search Profile NumberFrequency Cumulative Number Cumulative Frequency > Average Number of Differences =

23 Caucasian Origin Database(s) within Forensic Profiles Number of Differences From Search Profile NumberFrequency Cumulative Number Cumulative Frequency > Average Number of Differences = 4.947

24 Asian origin Database(s) within Forensic Profiles Number of Differences From Search Profile Numbe r Frequenc y Cumulativ e Number Cumulativ e Frequenc y > Average Number of Differences = 7.380

25 Caucasian origin Database(s) within Forensic Profiles Number of Differences From Search Profile NumberFrequency Cumulative Number Cumulative Frequency > Average Number of Differences = Search Range(s) Differences from CRS G C C C

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27 A 95% Upper Confidence Limit Using the Normal Approximation of the Binomial UCL= P [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 [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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29 UPPER CONFIDENCE LIMIT FROM ZERO PROPORTION P limit = 1 – – 1/N, where N = Database Size N UCL Maximum Frequency in in in in in in 1670

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31 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.

32 Statistics Associated with One Case Match CriteriaFrequency 95%UCL Not Excluded Zero Differences in % Excluded Zero+One in % Excluded


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