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developed, including PCR-RFLP analysis , allele-specific oligonucleotide dot-blot analysis [185] , real-time
amplification refractory mutation system quantitative PCR [186] , and pyrosequencing technique [187] . However,
these applications could only detect a single or a few candidate mutations.

The emergence of NGS technologies has rapidly reduced the cost and time spent with a substantial
improvement in the detection ability that allows for a wide-scale detection of genome changes [180,184] . The
introduction of whole-exome sequencing (WES) and whole-genome sequencing (WGS) technologies
increased the mutational detection rate in mitochondrial disease diagnosis. This effect was evidenced by the
percentage of disease-causing mutations identified during the pre-NGS era (10%-20%) compared to after
NGS era (30%-50% in some cohorts) [188-190] . Typically, there are two workflows for the detection of mtDNA
mutations [184,191] : (1) direct analysis (detection of mtDNA sequence from samples that are enriched with
mtDNA apart from the cellular DNA; and (2) indirect analysis (the mtDNA mutations are obtained as by-
products of the high-throughput sequencing reads).

Direct detection of mtDNA is usually done by adding a technique to purify or isolate the mitochondria
before the NGS workflow [184,191] , such as ultracentrifugation (a density gradients isolation) or the biochemical
or mechanical isolation of the organelles. Another approach is to use specific probes or primers to isolate
mtDNA, such as in microarray hybridization and PCR-based enrichment methods [184,192,193] . However, it is
important to note that using a primer-based method often results in large overlapping regions, and these
regions must be removed before the variant calling analysis. The main advantage of this direct method
is the elimination of the DNA regions homologous with mtDNA sequences or those known as nuclear
mitochondrial DNAs (NUMTs), which exist in various sizes as clones of genuine mtDNA and can be specific
to some populations [194] . Therefore, the findings from the direct mtDNA analysis are usually more reliable.

A typical workflow for the indirect mtDNA analysis is through the by-product annotation of the sequencing
reads from the WES and WGS. From these WES or WGS sequencing reads, the annotation process also
includes a step to map the reads to the mitochondrial genome. Since the average coverage of this mtDNA
fraction sequence is higher than the normally targeted gene regions due to the high copy number of
mtDNA per cells, the mapping results in good quality data [195] . Because of this high-quality data and the
cost-effectiveness of the NGS technologies, the indirect mtDNA analysis has become a favorite tool for
mitochondrial disease diagnosis, due to a simple workflow. However, this indirect mtDNA technique has
one problem with false-positive results due to NUMTs [184,191] . The inability to confirm whether the mtDNA
reads from the WES or WGS sequences are from the nuclear or mitochondrial genome can cause ambiguity
of the findings. The simple method to eliminate this issue is to align the raw reads first to the mitochondrial
genome and filter the non-aligned sequences, though some NUMTs do exist in the mtDNA genome
database; thus, false heteroplasmy can be introduced [184,191] . New software such as MitoSeek can help to
address this issue, where this program can extract the mtDNA mutation and heteroplasmy information from
WES data [196] . Furthermore, the existence of the databases such as MSeqDR [197] , MITOMAP [198] , HmtVar [199] ,
HmtDB 2016 [200] , Leigh Map [201] and others provide the comprehensive mutation-phenotype relationships
to allow the interpretation of the WES and WGS analyses and thereby unravel any novel mutations in the
patients. The mitochondrial disease-specific detection kits or panels are already on the market to improve
diagnosis [202-205] . By combining the databases and NGS technologies, there is a continuous discovery of many
mutations responsible for various mitochondrial diseases.

TRANSCRIPTOMICS
Although genomic NGS techniques are powerful enough to diagnose mitochondrial diseases, the rate of
detection for disease-causing mutations are only 25%-50% of cases [180,184,192] . To improve this detection rate, an
approach to employ the whole transcriptome sequencing technologies such as the RNA-seq by prioritizing
the candidate genes (i.e., those genes that are involved in the oxidative phosphorylation pathways, etc.) to