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Long read mitochondrial genome sequencing using Cas9-guided adaptor ligation

Amy R. Vandiver, Brittany Pielstick, Timothy Gilpatrick, Austin N. Hoang, Hillary J. Vernon, Jonathan Wanagat, Winston Timp

Preprint posted on 25 February 2022 https://www.biorxiv.org/content/10.1101/2022.02.23.480720v2

Article now published in Mitochondrion at http://dx.doi.org/10.1016/j.mito.2022.06.003

How many different genomes can one person have? Long-read sequencing clarifies the diversity of mitochondrial genomes found within individual tissues.

Selected by Nate Mullin

Background:

Mitochondria possess a small (1-100s kbp depending on species), semi-autonomous genome that encodes proteins vital to oxidative phosphorylation. Each organelle contains several copies of this genome, and each human cell contains hundreds to thousands of mitochondrial genomes (1). Profiling variants on the mitochondrial chromosome (mtDNA) has several useful applications. First, mtDNA variants are known to cause human disease affecting a variety of organ systems (2). Variants on the mtDNA have also been proposed as a means for retrospective lineage tracing in humans, due to the propensity for accumulation of mutations (3-5) and known lineage-specific segregation of mtDNA variants during early development (6). Specific combinations of these mutations could be used to identify clonally related cells. While determining the percentage of mitochondrial genomes which harbor a mutation has been possible through PCR and short-read sequencing methods, identifying linkage between mtDNA variants is more difficult with these methods (7). Detection of different combinations of mtDNA variants being present on the same mtDNA molecules would enable a better understanding of mtDNA disease and increase the utility of mtDNA variants for lineage tracing.

In this preprint, Vandiver et al introduce a method for sequencing full length mtDNAs. They demonstrate the utility of this technique in understanding the diversity of mtDNA species in human samples.

Figure 1. nCATS experimental schematic and validation by nanopore sequencing. Adapted from Vandiver et al., bioRxiv 2022.02.23.480720; doi: https://doi.org/10.1101/2022.02.23.480720

Main Findings:

1. Nanopore Cas9-targeted sequencing (nCATS) can be applied to mtDNA

Nanopore sequencing allows for analysis of very long stretches of DNA in continuous reads. DNA is passed through a nanopore and the order of bases is detected via changes in current across the pore. As such, DNA fragment length is the only limiting factor in seuqnece read length. In order to be sequenced using nanopore technology, adapters are ligated to the target DNA. Here, the authors use nCATS to enrich for mtDNA by first using dephosphorylation to inhibit nuclear DNA from being ligated to such adapters (8). Then, Cas9 is used to target the mtDNA at a conserved site to open the circular chromosome and allow for adapter ligation (Figure 1A). This process greatly enriches for full length mtDNA molecules in the sequencing library (Figure 1B) while removing contaminating nuclear DNA with homology to mtDNA.

2. Individual patients harbor a diverse collection of mtDNA deletions which grow with age

Vandiver et al. used their nCATS method to sequence mtDNA molecules in samples from 6 patients – three over the age of sixty and three in the third and fourth decade of life. They found that there was a large diversity of mtDNA species (i.e., molecules with unique deletions or mutations) found within individual patients. Further, they show that the older patients had more unique deletions than younger patients, implying a random accumulation of deletion-harboring mtDNA species during human aging.

3. Analysis of multiple variants in long mtDNA reads implies mtDNA recombination or specific sites predisposed to somatic

In addition to identifying the increase in unique mtDNAs harboring deletions with age, the authors also used nCATS to provide potential evidence of recombination between mtDNA. The authors used nCATS to generate long reads spanning the position of two mtDNA variants known to be present in a patient sample. These variants of interest are approximately 5kbp apart. They found that more reads contained only one of the variants, with a minority of total reads containing both variants. These data indicate the presence of at least four unique mtDNA species and imply that either the variants of interest commonly arise from somatic mtDNA mutation, or that these species are the result of mtDNA recombination.

 

What I like about this preprint

This work contributes a novel method for targeted mtDNA sequencing that enables analysis of a new dimension of mitochondrial genetics; linkage between mtDNA variants. Linkage of mtDNA analysis by traditional approaches is problematic due to uniparental inheritance and the intrinsic high copy number of mtDNA. The approach and findings of Vandiver and colleagues allow us to conceive of mtDNA genetics not only in terms of proportion of individual variants, but also in terms of unique mtDNA species co-existing within a cell. This study introduces an elegant new method that is relatively straightforward and provides two new insights into human mtDNA using their method. Primarily, they demonstrate that unique mtDNA deletions accumulate with age and that recombination between mtDNA molecules may be more frequent than previously appreciated. This preprint demonstrates that individuals may harbor more unique genomes than we previously were aware of, and I am excited to see how this more diverse genetic landscape may influence the understanding of human mitochondrial disease.

 

Open Questions

  1. Are these biases for/against specific mtDNA species using this enrichment method? For example, are deletion-containing mtDNAs more likely to be sequenced?
  2. Are there any specific challenges in using long-read sequencing for single nucleotide variants in this context? How is the quality of variant calls assessed?
  3. What further insight does knowledge of phased mtDNA variants give for human mitochondrial disease pathogenesis?

 

References

  1. Satoh M, and Kuroiwa T. Organization of multiple nucleoids and DNA molecules in mitochondria of a human cell. Exp Cell Res. 1991;196(1):137-40.
  2. Stewart JB, and Chinnery PF. The dynamics of mitochondrial DNA heteroplasmy: implications for human health and disease. Nat Rev Genet. 2015;16(9):530-42.
  3. Ludwig LS, Lareau CA, Ulirsch JC, Christian E, Muus C, Li LH, et al. Lineage Tracing in Humans Enabled by Mitochondrial Mutations and Single-Cell Genomics. 2019;176(6):1325-39 e22.
  4. Miller TE, Lareau CA, Verga JA, DePasquale EAK, Liu V, Ssozi D, et al. Mitochondrial variant enrichment from high-throughput single-cell RNA sequencing resolves clonal populations. Nat Biotechnol.
  5. Lareau CA, Ludwig LS, Muus C, Gohil SH, Zhao T, Chiang Z, et al. Massively parallel single-cell mitochondrial DNA genotyping and chromatin profiling. Nat Biotechnol. 2021;39(4):451-61.
  6. Mertens J, Regin M, De Munck N, Deckersberg EC, Belva F, Sermon K, et al. Mitochondrial DNA variants segregate during human preimplantation development into genetically different cell lineages that are maintained postnatally. Hum Mol Genet.
  7. Alkanaq AN, Hamanaka K, Sekiguchi F, Taguri M, Takata A, Miyake N, et al. Comparison of mitochondrial DNA variants detection using short- and long-read sequencing. J Hum Genet. 2019;64(11):1107-16.
  8. Vandiver AR, Pielstick B, Gilpatrick T, Hoang AN, Vernon HJ, Wanagat J, et al. Long read mitochondrial genome sequencing using Cas9-guided adaptor ligation. 2022.

 

Posted on: 29 April 2022

doi: https://doi.org/10.1242/prelights.31896

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