Supplementary Methods
Exome sequencing
Exome libraries were prepared using the Agilent Sure Select All Exon 50Mb kit (Agilent
Technologies, CA, USA) from isolated DNA. Each library was run in a single lane on a
HiSeq2000 (Illumina Inc. CA, USA) using 100bp PE sequencing, at Canada’s Michael
Smith Genome Sciences Centre.
Exomes were aligned to hg19 using BWA-MEM (Li, 2013). Variants were called using
the Genome Analysis Toolkit (1), and filtered for Mapping Quality ≥ 40, Quality by
Depth ≥ 2.0, and Fisher Strand test ≥ 200. Functional annotations for predicted
pathogenicity were run using snpEff (2).
Mitochondrial Sequence Analysis
In this analysis, the primary allele refers to the most common allele in the read counts,
and the secondary allele refers to the second most common allele in the read counts. The
reference allele refers to the allele of the rCRS reference sequence at a given position,
while the alternate allele refers to the most common allele in the read counts that is not
the reference allele. The total allele count refers to the sum of all alleles at a given
position, excluding any ‘n’ calls.
Mitochondrial reads were extracted from the whole exome sequencing data using
MitoSeek (3), which was modified for analysis of both heteroplasmic and homoplasmic
variants. An hg19 to rCRS conversion step was implemented to ensure that variant
analysis would be performed relative to the rCRS reference sequence (NC_012920). Poor
quality reads were filtered out on the criteria of Mapping Quality Score ≥ 20 and Base
Quality Score ≥ 20. Positions with read depth ≤ 5 were considered unsuitable for variant
analysis and excluded. A minimum secondary allele frequency of 10% and alternate
allele count of 5 were set as the frequency cut-off values. When a potential heteroplasmy
was called, the surrounding nucleotides were manually assessed with consideration to
previously reported sequencing error trends (4,5) to determine whether variants were
located in an ‘error hotspot’.
Heteroplasmic variant calls that were outside of an error hotspot and that passed the 10%
secondary allele cutoff as well as the 5-read alternate allele cutoff were taken as true
heteroplasmies. Positions that did not meet the 10% secondary allele frequency cut-off
were then evaluated as possible homoplasmic variants by comparing the primary allele
with the reference allele at that position. Heteroplasmic frequencies of such variants were
reported as (Alternate Allele Count)/(Total Allele Count). Amino acid differences were
also reported for non-synonymous variants, accounting for the differences in genetic code
between nuclear and mitochondrial DNA.
HaploGrep (6,7) was used to predict the subjects’ haplogroup from the list of variants and
to identify variants not accounted for by the assigned haplogroup. Global minor allele
frequencies of reported variants were estimated using MITOMAP (8), which reported the
frequency of each variant using 26850 mitochondrial GenBank sequences.
Supplementary References
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8.
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