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relative to nDNA. However, greater depth of coverage is required for reliable detection of low-heteroplasmy
[76]
variants . Dedicated mtDNA enrichment enables simultaneous analysis of mtDNA, with enhanced
[76]
detection of low heteroplasmy variants, down to 8% . Despite vast progress, however, a substantial
proportion (30%-70%) of patients remain undiagnosed following WES [15-21] . Whilst this may reflect
bioinformatic prioritisation or evolving analytic pipelines, there remain a number of insufficiencies in
[103]
WES: coverage may be non-uniform and importantly limited in certain regions (especially G-C rich)
and indels and copy number variations may not be reliably identified [104] . Furthermore, PCR and mtDNA
enrichment also introduce sequencing error and bias, the nature and extent of which depend on the
selected kit and methods [103,105] . By definition, causative variants in non-coding regions are also omitted by
WES. WGS can overcome all of these limitations to offer further utility, with promising early data in rare
diseases [104] that may justify the modest additional cost.

PCR-free whole genome sequencing avoids sequencing error and biases introduced by library amplification,
[24]
offering more consistent breadth and depth of coverage of coding regions as well as covering the
[82]
extensive non-coding regions. WGS can detect small and large chromosomal copy number variants ,
an increased proportion of single nucleotide variants and structural variants [24,25,104] . It also offers superior
mtDNA coverage (1200-4000× with acceptable coverage depths of the nuclear genome, between 14-30×),
allowing reliable detection of low-heteroplasmy variants, down to 2% or less [26,57] . Whilst analysis of
mitochondrial variants presents unique challenges compared to interpretation of nuclear variants [106] which
have more established bioinformatics pipelines, we have developed a novel dedicated tool, mity to offer
TM
[26]
automated, integrated mtDNA variant calling from WGS data . Nuclear and mtDNA bioinformatics
pipelines may be linked, facilitating simultaneous analysis of both nuclear and mitochondrial genomes
[26]
from a single, minimally-invasive sample . WGS therefore offers comprehensive, simultaneous bigenomic
sequencing with superior mtDNA coverage depth and heteroplasmy sensitivity, whilst reducing introduced
sequencing error and bias, and should therefore be the preferred sequencing option. Early WGS results
from mitochondrial disease studies indicate the yield is at least equivalent for known variants, with
potential for improved yield with novel variant identification and as analysis - especially of non-coding
regions - evolves.

CONCLUSION: A MINIMALLY INVASIVE, STREAMLINED APPROACH TO MITOCHONDRIAL
DISEASE GENETIC DIAGNOSIS
Despite significant advances in technology and understanding of mitochondrial biology over recent
decades, the diagnosis of mitochondrial disease continues to present a challenge to the clinician and a large
proportion of cases remain undiagnosed. Whilst the prevailing diagnostic paradigm advocates a “function-
to-gene” approach centred on muscle biopsy, the substantial benefits of a “genetics-first” approach justify
a paradigm shift. Such an approach, as proposed here, incorporating clinical evaluation, serum biomarker
stratification and early bigenomic WGS, offers the potential to streamline a less invasive diagnostic process
for patients, improve diagnostic yield, inform individual prognosis and the collective understanding of
mitochondrial biology and ultimately pave the way for substantial therapeutic advances.

DECLARATIONS
Authors’ contributions
Made substantial contributions to data interpretation, conception and design of the work, revision of the
manuscript: Watson E, Davis R, Sue CM
Drafting: Watson E
Made technical support: Davis R, Sue CM

Availability of data and materials
Not applicable.