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. Author manuscript; available in PMC: 2017 Dec 29.
Published in final edited form as: Neurosci Lett. 2016 Feb 10;625:47–55. doi: 10.1016/j.neulet.2016.02.013

Table 1. An overwiew of sequencing technologies that could be utilized to study mtDNA methylation in neurodegenerative diseases.

Generation Method General Overview Advantages Disadvantages
Next MeDIP- & hMeDIP-Seq Methylated DNA Immunoprecipitation sequencing (MeDIP-Seq) is an immunoprecipitation based method which uses monoclonal antibodies against 5-mC. In brief, purified genomic DNA is sheared by sonication to produce random fragments. These fragments are then denatured and immunoprecipitated, followed by PCR amplification. Using high-throughput sequencing at a depth of two Gigabases, around 70-80% of CpGs in the human genome can be identified 1.

  1. Staining with anti-5-hmC antibodies allows for the analysis 5-hmC Stroud et al. 2, which is not only present in the brain and significantly reduced in AD 3, but is also present in brain mitochondria 4

  2. Sequencing produces files typically less than 6GB (fasta format), which make alignments and bioinformatic analysis less computationally exhaustive.

  3. Cost-effective relative to most bisulfite based approaches 5.

  4. Use of antibody means that only regions of interest are investigated.

  1. Investigation of both 5-mC and 5-hmC would be costly and would require several micrograms of mtDNA as typical hMeDIP- Seq experiments require 4 – 5 μg of DNA 2,6.

  2. Requirement of antibody binding reduces resolution to methylated windows. Analysis of single cytosine methylation sites is not possible 7.

  3. Count based data means that reads must be normalised for CpG density and total read counts 8, although pipelines are now more capable of accounting for this 9.
Custom Capture Custom capture kits available allow for a custom design of a library for the enrichment of specific DNA target regions.
In brief, DNA undergoes standard NGS pipelines approaches of sonication, end repair, A-tailing and in this case, methyl-adaptor ligation before being bound by custom baits, bisulfite treated and amplified. Samples can then be run on a sequencer such as the Illumina HiSeq.

  1. Single base resolution for DNA methylation analysis

  2. Customized library to capture 100% of mitochondrial genome leads to further mtDNA enrichment and reducing risk of NUMT amplification.

  3. Can use as little as 1μg mtDNA (Nimblegen).

  4. Small target genome would likely generate small output files from sequencing and could allow for rapid, less computationally exhaustive alignment.

  5. Simultaneous analysis of CpG and non-CpG methylation.

  6. Small size of the genome allows for ample space on a Mi-Seq lane for high (>1000×) coverage of over 100 samples at a time, potentially important given the multi-copy nature of the genome.

  1. Currently the process is not supported by Agilent.

  2. Typically cost exceeds that of non- bisulfite experiments.

  3. Reduced complexity of sequence due to bisulfite conversion 10.
RRBS & ERRBS Reduced Representation BS-Seq (RRBS) makes use of MspI restriction enzymes for selective digestion of genomic DNA. This produces fragments of genomic regions enriched for CpG sites, which can be bisulfite treated and sequenced irrespective of their methylation status.
This way, many samples can be processed efficiently and inexpensively 11.

  1. Requires much less DNA input than WGS-BS, and most other techniques, with RRBS requiring as little as 10ng 12 and Enhanced RRBS requiring around 50ng or less 13

  2. Can provide coverage of CpG promoters and CpG islands at a fraction of the cost of WGS-BS.

  3. Single base resolution

  4. Has been modified to allow for single cell epigenomic analysis 14,15.

  1. Targets CpG promoters and CpG islands, however the mitochondrial genome contains no CpG islands, therefore could give a very poor coverage of the mitochondrial methylome.

  2. Reduced complexity of sequence due to bisulfite conversion 10.
WGS-BS & OxBS-Seq The whole genome shotgun bisulfite sequencing (WGS-BS) technique converts fragmented DNA cytosines to uracil bases by sodium bisulfite treatment, while (hydroxy)methylcytosines remain unmodified. After PCR amplification, methylcytosines will be read as thymine in a sequencer, and can thus be distinguished from unmethylated sites. The reads can then be aligned to recreate the DNA sequence 16.
The recent development of the Oxidative Bisulfite (OxBS) method allows for quantification of hydroxymethylation by converting a 5hmC base to 5fC before bisulfite treatment. This enables a direct measurement of 5mC, and an indirect measurement of 5hmC at base-pair resolution 17.

  1. Single base resolution for DNA methylation analysis

  2. Simultaneous analysis of CpG and non-CpG methylation

  3. The development of OxBS-Seq allows for the identification of both 5-hmC and 5-mC when used in combination with standard BS-Seq 18,19.

  4. OxBS-seq can be further combined with reduced Bisulfite Sequencing to allow for a characterisation of 5-mC, 5-hmC and 5-formylcytosine 20, allowing for an in depth characterization of the mitochondrial methylome.

  1. Sequences whole genome, including many repetitive, non-methylated AT-rich regions.

  2. Vast quantities of data generated leads to massive (100GB+ files) following sequencing, leading to time consuming processing 21.

  3. High cost of sequencing to improve sensitivity makes it unsuitable for large studies 10.

  4. Reduced complexity of sequence due to bisulfite conversion 10
Generation 2.5 PacBio Single-molecule real-time sequencing (SMRT) by Pacific Biosciences is achieved using zero-mode waveguide (ZMW) array technology. This technique uses a single DNA polymerase molecule attached to the bottom of a ZMW hole (tens of nanometers in diameter). By illuminating only the bottom 30nm of the ZMW with a laser, single nucleotide addition to the DNA can be measured. Each nucleotide fluoresces when bound to the DNA polymerase, which is detected by a camera before being cleaved off. Bases can be identified by corresponding fluorescent colours 22.

  1. Single molecule resolution technology avoids potential PCR bias by using pre-amplified DNA as an input for sequencing 23.

  2. Optimised for circular genomes, making the potential use of mtDNA analysis an interesting possibility.

  3. Increased library complexity to bisulfite based methods as DNA modifications are determined based upon changes to polymerase kinetics specific to modification present 24. Potentially, this may be developed to accurately characterise all methylation marks in one simultaneous run.

  4. Longer read length could lead to improved coverage and accuracy for processes such as variant calling 25.

  5. Ideal for de novo sequencing as capable of reads at a length of 8kbp 26

  6. Can detect strand specific patterns of 5hmC, after selective chemical labelling, in a high throughput manner 27

  1. The RSII platform is capable of producing continuous long reads and circular consensus reads, at present, these reads are typically associated with high error rates 28. However, given the random nature of the errors, with sufficient depth SMRT can give an accurate read of the genome if sequenced at high coverage 29.

  2. The cost and size of PacBio RSII, coupled with the need for specialist technical and bioinformatic support may limit much research to outsourcing data to specialist sequencing services.
Third Generation Nanopore The nanopore technique makes use of either biological nanopores or solid state nanopores, which are embedded in a membrane immersed in salt solution. To create a flow of ions through the pore, an electrical current can be applied. A single stranded DNA molecule passing through the pore will create measurable changes in the intensity of the current, and different DNA bases could be distinguished by the degree and duration of modulation of the current 30.

  1. Although still in alpha testing, nanopore technologies such as Oxford's MinION™ allow for sequencing analysis to be done on a portable, benchtop device in real-time 31.

  2. Identification of 5-hmC and 5mC

  3. Systems like the Oxford MinION™ allows for the activation of hundreds of nanopores in parallel to reduce time of sequencing large genomes versus single pore technologies.

  4. Sample preparation steps are reduced as no amplification or cloning is necessary, and use of enzymes is limited. This in turn reduces costs of sequencing 32.

  1. Observed per base error rates for applications such as DNA sequencing have reduced in recent years, but are still very high, recently reported at 30% 31.

  2. Some nanopore technologies utilise the binding of MBD1 to 5mC, altering current flow through the nanopore, to detect the methylation mark 33; MBD1 has not yet been identified in mitochondria.

  3. Although nanopore sequencing could in principle require less than 1 μg of genomic DNA, an estimated 700 μg is needed due to the concentration-limited rate at which DNA molecules can be captured 32

Abbreviations: Methylated DNA Immunoprecipitation sequencing (MeDIP-Seq), Hydroxymethylated DNA Immunoprecipitation sequencing (hMeDIP-Seq), Nuclear mitochondria pseudogenes (NUMTs), Next Generation Sequencing (NGS), Reduced Representation Bisulfite Sequencing (RRBS), Enhanced Reduced Representation Bisulfite Sequencing (ERRBS), Whole Genome Shotgun Bisulfite Sequencing (WGS-BS), 5-hydroxymethylcytosine (5-hmC), -methylcytosine (5-mC), Single-molecule real-time sequencing (SMRT), Zero-mode Waveguide (ZMW), Methyl DNA binding protein 1 (MBD1)

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2

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8

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9

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10

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11

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12

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13

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Guo, H., Zhu, P., Guo, F., Li, X., Wu, X., Fan, X., Wen, L. and Tang, F. (2015) Profiling DNA methylome landscapes of mammalian cells with single-cell reduced-representation bisulfite sequencing. Nature protocols, 10, 645-659.

16

Krueger, F., Kreck, B., Franke, A. and Andrews, S.R. (2012) DNA methylome analysis using short bisulfite sequencing data. Nat Meth, 9, 145-151.

17

Stewart, S.K., Morris, T.J., Guilhamon, P., Bulstrode, H., Bachman, M., Balasubramanian, S. and Beck, S. (2015) oxBS-450K: A method for analysing hydroxymethylation using 450K BeadChips. Methods, 72, 9-15.

18

Booth, M.J., Branco, M.R., Ficz, G., Oxley, D., Krueger, F., Reik, W. and Balasubramanian, S. (2012) Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution. Science, 336, 934-937.

19

Booth, M.J., Ost, T.W., Beraldi, D., Bell, N.M., Branco, M.R., Reik, W. and Balasubramanian, S. (2013) Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine. Nature protocols, 8, 1841-1851.

20

Booth, M.J., Marsico, G., Bachman, M., Beraldi, D. and Balasubramanian, S. (2014) Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution. Nature chemistry, 6, 435-440.

21

Xi, Y. and Li, W. (2009) BSMAP: whole genome bisulfite sequence MAPping program. BMC bioinformatics, 10, 232.

22

Schadt, E.E., Turner, S. and Kasarskis, A. (2010) A window into third-generation sequencing. Human molecular genetics, 19, R227-R240.

23

Davis, B.M., Chao, M.C. and Waldor, M.K. (2013) Entering the era of bacterial epigenomics with single molecule real time DNA sequencing. Current opinion in microbiology, 16, 192-198.

24

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25

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26

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27

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28

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29

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30

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31

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32

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33

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