Quantitative Assessment of Heteroplasmy of Mitochondrial Genome: Perspectives in Diagnostics and Methodological Pitfalls

Table 1

Comparison of methods for the quantitative analysis of mtDNA heteroplasmy.

Method	Advantages	Disadvantages
Invasive cleavage of oligonucleotide probe (invader assay)	(1) Allows SNP-analysis in a single reaction tube; (2) Allows the use of automatic setup of the reaction	(1) Requires a long incubation time (3-4 h); (2) Big amount of genomic DNA (20–100 ng) in a single reaction; (3) The laborious selection of allele-specific probes
Pyrosequencing	(1) Allows to estimate the exact nucleotide sequence and its changes; (2) The possibility of automation; (3) Quick performance; (4) Can be used for SNP-analysis; (5) The ability to analyze complex secondary structures	(1) Requires special equipment; (2) Limited read length; (3) The quality and effectiveness of four enzymes used in reactions are critical for the accuracy of measurements; (4) Enzymes can lose activity; (5) Sample dilution may reduce the read length
Real-time amplification refractory mutation system for quantitative PCR analysis	(1) Simple; (2) Single-stage; (3) Fast; (4) Nonradioactive; (5) Sensitive; (6) Specific	(1) It is difficult to find the optimal allele-specific primers; (2) It is difficult to find the optimal mode for RT PCR to ensure proper hybridization of allele-specific primers
454 sequencing (Roche)	(1) High read length; (2) Relatively low cost of the instrument and reagents for the single run of GS junior titanium	(1) High cost of the instrument and reagents for the single run of GS FLX titanium; (2) High cost per 1 Mb read; (3) High consumption of fluids passed through the flow cell
Illumina sequencing with Illumina MiSeq	(1) Relatively low cost of the instrument and reagents for the single run; (2) The lowest cost per 1 Mb read among small platforms; (3) The fastest Illumina run time	(1) Relatively few reads; (2) Higher cost per 1 Mb read as compared to other Illumina platforms
Illumina HiScanSQ	(1) Allows versatile genomic research and is scalable in future	(1) Higher cost per 1 Mb read than HiSeq for large amounts of data
Illumina GAIIx	(1) Lower instrument cost than HiSeq; (2) The large number of publications using this instrument	(1) Higher cost per 1 Mb read than HiSeq
Illumina HiSeq 1000 and 2000	(1) The largest number of reads; (2) Maximum sequencing output per 1 day and 1 run; (3) The lowest price for 1 Gb read among all NGS platforms	(1) High instrument cost; (2) High computation needs
Applied Biosystems SOLiD sequencing	(1) Each lane of Flow-Chip can be run independently; (2) The highest sequencing accuracy; (3) The ability to rescue failed sequencing cycles; (4) 96 validated barcodes per lane; (5) Throughput of 10–15 Gb/day for SOLiD-5500 and 20–30 Gb/day for SOLiD 5500XL	(1) High-performance devices have become available only from 2011; (2) Relatively short reads; (3) Increased number of gaps in the assembly of genomes; (4) High instrument cost for SOLiD-5500XL
HRM analysis	(1) Low consumption of reagents with minor losses: it takes 20 𝜇L of PCR mix for the analysis of each sample, eliminating the need for HPLC-solvents or DGGE-gels; (2) The stage of highly sensitive analysis of the melting curves can be added in the end of the PCR for immediate analysis; (3) Unlike DHPLC, it does not require thermal optimization; (4) Low consumption of samples: after HRM-analysis PCR products can be used for Sanger sequencing; (5) High resolution for accurate and reproducible results	(1) Method is not adapted for the quantitative analysis of mitochondrial mutations
TGGE	(1) Does not require denaturing agents; (2) High reproducibility; (3) The ability to analyze fragments with a size of up to 1000 bp	The exact nature of the nucleotide changes is unknown
HPLC	(1) High accuracy; (2) The ability to analyze very small amounts of DNA; (3) High separation efficiency; (4) Flexibility to alter the conditions of separation; (5) Rapid analysis; (6) Comparative simplicity of instrumentation; (7) Automation ability	(1) High cost of columns; (2) The exact nature of changes in nucleotide sequence remains unknown; (3) Sequencing is required to determine the nature of the mutation; (4) Difficult identification of heterozygotes
Endonuclease method using Surveyor nuclease	(1) Simplicity; (2) Special kits for mutations detection	(1) Qualitative estimation
Sanger sequencing	(1) Using dideoxynucleotides with fluorescent labels with different emission wavelengths allows to carry out the reaction in a single tube; (2) The ability to analyze 500–1000 bp sequence in a single run	(1) Complexity of the electrophoretic separation of fragments; (2) Low throughput compared with NGS methods; (3) The loss of accuracy when reading large fragments
Snapshot	(1) A reliable method to identify uncharacterized nucleotide damage; (2) Allows to subsequently determine the untimely stop codons, missense, or silent mutations; (3) Can be used for SNP analysis; (4) Automation ability	(1) Relativeness of the results; (2) Low accuracy of the data; (3) Requires valuation of results