Following completion of mtDNA sequence analysis as described above, results from the edited and reviewed sequences for a question (Q) and a known (K) sample are compared as illustrated in Figure 10.8 for a portion of HV1. All 610 nucleotides (positions 16024-16365 and 73-340) are normally evaluated between samples being compared.
A comparison of the two sequences in question will either result in a perfect match or not. Samples are termed concordant if they match at every evaluated site.
(a) mtDNA Sequences Aligned with rCRS (positions 16071-16140)
1BCH0 101X1 10110 16120 16130 10140
rCRS ACCGCTATGT A^TfTCGTACA TTACTGCCAG CCACCATGAA TATTGTAqGfc TACCATAAAT
Q ACCGCTATGT AljcJrCGTACA TTACTGCCAG CCACCATGAA TATTGTAdijs TACCATAAAT
K ACCGCTATGT AjcjrCGTACA TTACTGCCAG CCACCATGAA TATTGTACJAfe TACCATAAAT
(b) Reporting Format with Differences from rCRS
(a) Comparison of sequence alignments for hypothetical Q and K samples with (b) conversion to the revised Cambridge Reference Sequence (rCRS) differences for reporting purposes.
However, interpretation of results is not always so cut and dry. Laboratories must develop interpretation guidelines as noted below.
Results can generally be grouped into three categories: exclusion, inconclusive, or failure to exclude. The Scientific Working Group on DNA Analysis Methods (SWGDAM) Guidelines for Mitochondrial DNA (mtDNA) Nucleotide Sequence Interpretation lists the following commonly used recommendations (SWGDAM 2003):
■ Exclusion - if there are two or more nucleotide differences between the questioned and known samples, the samples can be excluded as originating from the same person or maternal lineage.
■ Inconclusive - if there is one nucleotide difference between the questioned and known samples, the result will be inconclusive.
■ Cannot Exclude (Failure to Exclude) - if the sequences from questioned and known samples under comparison have a common base at each position or a common length variant in the HV2 C-stretch, the samples cannot be excluded as originating from the same person or maternal lineage.
A common base is defined as a shared base in the case of ambiguity (e.g., hetero-plasmy) in the sequence (Isenberg 2004). For example, if one sequence possesses heteroplasmy at a site and another does not (see Figure 10.9), then they cannot be excluded from one another. A length variant alone especially in the HV2 homopolymeric C-stretch cannot be used to support an interpretation of exclusion (Stewart et al. 2001, SWGDAM 2003). Several examples are provided in Table 10.4 with their respective interpretations based on the SWGDAM guidelines.
The reason that a single base difference is classified in terms of an 'inconclusive result' is that mutations have been observed between mother and children (Parsons et al. 1997). For example, if a maternal relative is used for a reference
Example mtDNA sequences and interpretations for known (K) and question (Q) sample pairs (adapted from Isenberg 2004).
Sequence Results Observations
Q TATTGTACGG K TATTGTACGG
Q TATTGCACAG K TATTGTACGG
Q TATTNTACGG K TATTGTACGG
Q TATTNTACGG K TATTGTACNG
Q TATTGTACA/GG K TATTGTAC G G
Q TATTGTACA/GG K TATTGTACA/GG
Q TATTGCACGG K TATTGTACGG
Sequences are fully concordant with common bases at every position
Sequences differ at two positions
A single unspecified base in one of the sequences; common base at every position
Ambiguous bases in both sequences at different Cannot Exclude positions; common base at every position
Heteroplasmic mixture at a position in one Cannot Exclude sample that is not present in the other; common base at every position (G in both Q and K)
Heteroplasmic mixture at the same site in Cannot Exclude both sequences; common base at every position
Sequences identical at every position except one; no indication of heteroplasmy
Inconclusive sample, the possibility of a single base difference may exist between two samples that are in fact maternally related. Often additional samples, such as more reference samples, are run if an inconclusive result is obtained in an attempt to clarify the interpretation. Hairs from an individual might be pooled in an attempt to detect heteroplasmy (Isenberg 2004).
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