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Mitochondrial
DNA Sequence Analysis
Mitochondrial DNA sequencing has
become a useful tool in biological
and medical disciplines, including
the study of diabetes, cancer, and
forensic identification. These studies
often require sequencing projects
of the mitochondrial genome that
cover both coding regions and non-coding
regions, such as the hypervariable
regions located in the D-loop. However,
the circular nature of the genome
and differences in the mitochondrial
amino acid codons present a challenge
for precise variant calling in mtDNA
sequences. Mutation Surveyor software
offers solutions to these issues
while providing high accuracy and
sensitivity in the detection of
single nucleotide polymorphisms
(SNPs), homozygous and heterozygous
insertions and deletions (indels),
and low-frequency variants such
as heteroplasmy. Along with accuracy
greater than 99% in the bi-directional
analysis mode and sensitivity to
5% of the primary peak, Mutation
Surveyor software automatically
aligns sequences to the revised
Cambridge Reference Sequence (rCRS),
correctly spans the origin (D-loop)
of the mitochondrial genome, and
accounts for the amino acid differences
relative to the vertebrate mitochondrial
genetic code.
Sequencing
Across the Region
Figure
1: The purple bar in the
nucleotide sequence frame represents
the origin of the mtDNA genome.
The hypervariable regions of the
mitochondrial genome are separated
by an area within the control region,
which marks the ending and beginning
of the genome annotation. Mutation
Surveyor software automatically
recognizes a tag in the mtDNA GenBank
file, enabling the program to identify
the origin of the D-loop separating
the HV1 and HV2 regions. When sequences
spanning the origin are loaded into
Mutation Surveyor, nucleotides on
both sides of the origin are aligned,
and the software places a purple
bar in the nucleotide sequence frame
of the Graphical Analysis Display
(GAD) to display the origin between
position 16569 and position 1.
Annotation
of Mitochondrial Genetic Code
Mutation
Surveyor software Recognizes Amino
Acid Translation from Different
Genetic Codes
Figure
2: The ATA codon translated
to Isoleucine in the standard genetic
code is read as Methionine using
the mtDNA GenBank file. A SNP of
C>T is detected at position 6546
and results in an amino acid change
from Leucine (L) to Phenylalanine
(F).
Mutation Surveyor software annotates
the sample sequences according to
the mitochondrial genetic code without
any user intervention by reading
the translation table within the
GenBank file. In this example, the
"ATA" codon that codes
for Isoleucine (I) in the standard
genetic code is translated as Methionine
(M) using the vertebrate mitochondrial
GenBank file. Amino acid changes
caused by detected variants are
also annotated relative to the mtDNA
code, and variants are annotated
by their position in the rCRS.
Robust Sequence
Alignment and Accurate Variant Identification
Figure
3: Variations are detected
on both sides of the origin in a
single sequence. Mutation Surveyor
software accurately aligns highly
variable sequences and detects variations
with greater than 99% accuracy,
and the Custom Report displays mutation
call nomenclature following SWGDAM
guidelines.
Mutation Surveyor software is able
to quickly and accurately align
mtDNA sequence traces to the revised
Cambridge Reference Sequence (rCRS),
including hypervariable sequences
spanning the HV1 and HV2 regions
for identification of variants associated
with human mitochondrial DNA halplogroups.
Variants are detected using the
same "anti-correlation"
technology used with genomic DNA
sequences, providing an increased
level of accuracy and sensitivity
not found in other software. The
Custom Report can display calls
following the SWGDAM guidelines,
such as 152G, 309.1C, and 522d for
substitution, insertion, and deletion,
respectively.
Download application note
Mitochondrial
DNA Sequence Analysis using Mutation
Surveyor® Software
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