for Mitochondrial DNA (mtDNA) Nucleotide Sequence Interpretation
Scientific Working Group on DNA Analysis Methods (SWGDAM)
interpretation of mitochondrial DNA (mtDNA) nucleotide sequencing
results in casework is a matter of professional judgement and expertise.
Although there are general guidelines for mtDNA analysis (Budowle
et al. 1999; Carracedo et al. 2000; Holland and Parsons 1999; Tully
et al. 2001), not every situation can or should be covered by a
preset rule. It is important that each laboratory develop and implement
written guidelines for the interpretation of analytical results.
This document provides a framework for the laboratory to develop
mitochondrial DNA (mtDNA) interpretation guidelines. The laboratory's
interpretation guidelines must be based on validation studies conducted
according to the Quality Assurance Standards for Forensic DNA
Testing and Convicted Offender Databasing Laboratories. The
SWGDAM Validation Guidelines and data from the scientific literature
(such as Wilson et al. 1995a) should also be consulted.
Assurance / Quality Control
essential practice for mtDNA typing is to minimize and monitor contamination
within the laboratory. Because of the sensitivity of detection with
mtDNA analysis, low levels of exogenous DNA contamination and/or
background is sometimes observed. However, reliable results can
be obtained using appropriate quality control measures.
must be monitored, and each laboratory must have a method to define
and quantify contamination. Laboratories must determine the maximum
allowable threshold for contamination through internal validation
studies. The laboratory must have standard operating procedures
in place to address contamination (Wilson et al. 1995b). Each laboratory
must establish evaluation criteria for controls, including but not
limited to a positive control, a negative control, and a reagent
blank control. Each of these controls must be processed through
sequencing along with the sample. In addition, typing of laboratory
personnel is highly recommended for elimination purposes in order
to trace potential sources of contamination.
positive control is a sample of known mtDNA sequence used to monitor
the success of the analysis. The positive control must be processed
starting at amplification (e.g., DNA purified from the HL60 cell
line is required as a positive control for inclusion of mtDNA forensic
data into the Combined DNA Index System [CODIS]).
blanks and negative controls are used to monitor levels of contamination.
Reagent blanks monitor contamination from extraction to final sequence
analysis. Negative controls monitor contamination from amplification
to final sequence analysis. If the reagent blank and/or the negative
control of a particular amplification results in a sequence that
is the same as that of the sample, all data for the sample must
be rejected. The analysis must be repeated, starting with the extraction
of the sample. If contamination in the reagent blank and/or negative
control is present above the threshold set by the laboratory, then
the data cannot be used for interpretative purposes. The samples
must be re-amplified or re-extracted.
sequence data obtained from population database samples must include
a minimum of HV1 (positions 16024-16365) and HV2 (positions 73-340).
Nuceleotide sequence from known (K) samples should include HV1 (positions
16024-16365) and HV2 (positions 73-340). There are no minimum length
requirements for nucleotide sequence data obtained from questioned
(Q) samples. Both strands of the amplified product must be sequenced
to reduce ambiguities in sequence determination.
laboratory must establish criteria to assign nucleotide base calls
to appropriate peaks or bands and to determine whether the results
are of sufficient quality for interpretation purposes. The overall
quality of the electropherogram data must be assessed. The results
must be examined to determine if they meet the laboratory's analytical
and interpretation threshold(s) established through internal validation
studies. If the overall quality of the electropherogram is not suitable
for analysis, the data should be rejected and the sample should
be re-extracted, re-amplified, and/or re-sequenced.
consensus sequence obtained from the sample will be compared to
the Revised Cambridge Reference Sequence (rCRS) described by Andrews
and co-workers ([Andrews et al. 1999; rCRS replaced the Cambridge
Reference Sequence (CRS) described by Anderson and co-workers (Anderson
et al. 1981]). Differences between the reference sequence and the
sample sequence will be noted as polymorphisms. The nucleotide position
and the DNA base difference from the reference will be noted (e.g.,
16089 C). (N.B. listing of polymorphisms relative to rCRS is required
for inclusion of mtDNA data into CODIS).
are described by noting the site immediately 3' to the insertion
with respect to the light strand of the rCRS followed by a point
and a '1' for the first inserted base, with sequential numbering
for each inserted base thereafter. With homopolymeric regions, the
insertion is placed at the highest-numbered end of the homopolymeric
region with respect to rCRS. Insertions should not alter subsequent
numbering of the sequence. Variants from rCRS should be coded in
accordance with the guidelines proposed by Wilson et al. 2002a and
Wilson et al. 2002b.
base call designation should be based on the nomenclature system
set forth by the International Union of Pure and Applied Chemistry
At confirmed positions of ambiguity, the following IUPAC codes
should be used:
conservative approach to listing ambiguities is to call them as
'N'. Deletions should be marked as ' - '.
relevant sequence traces must be imported into a software program
for analysis and alignment. The heavy strand sequences should be
reverse-complemented so that the bases are aligned in the light
strand orientation. Strands are compared and bases designated. Heteroplasmy
is defined as more than one mtDNA type present in an individual
that can be detected at an operational level. Heteroplasmy can be
observed as point heteroplasmy where two DNA bases are observed
at the same nucleotide position. Heteroplasmy can also be seen as
length heteroplasmy, which typically is observed as a variation
in the number of bases in a homopolymeric stretch of bases (i.e.,
C-stretch). Each laboratory should define heteroplasmy within the
operational limits of the system used for sequencing. When the specimens
under consideration differ by a single nucleotide, additional samples
should be run in order to attempt to resolve the interpretation.
stretches of the same nucleotide are referred to as homopolymeric
tracts. In HV1, the homopolymeric C-stretch region typically starts
at nucleotide position 16184, in HV2 the homopolymeric tract is
found between nucleotide positions 303 to 315. Homopolymeric tracts
can differ in length within the same individual. In most cases,
no attempt will be made to determine the exact number of bases in
an HV1 C-stretch; however, laboratories must develop their own interpretation
guidelines for HV2 length variants. A common length variant usually
can be determined in the HV2 homopolymeric tract. A length variant
alone cannot be used to support an interpretation of exclusion (Stewart
et al. 2001).
laboratory must define conditions under which the data would lead
to the conclusion that an individual can or cannot be eliminated
as a possible source of the mtDNA. In addition, laboratories should
develop guidelines for evaluation of cases where heteroplasmy may
have occurred. This may be accomplished by an examination of the
number, position, and nucleotide composition of polymorphic sites.
following guidelines may be used in most cases:
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.
there is one nucleotide difference between the questioned and
known samples, the result will be inconclusive.
ExcludeIf 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.
mtDNA profile of a reference sample and an evidence sample that
cannot be excluded as potentially originating from the same source
can be searched in a population database. The population database(s)
used to assess the weight of forensic evidence such as the mtDNA
population database or CODIS (Miller and Budowle 2001; Monson et
al. 2002) must be documented.
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