The Ultimate Guessing Game: Intra-Tissue Genetic Diversity

Our bodies contain trillions of unique cellular genomes. We’re just starting to appreciate the significance of this diversity in development and disease.

59 min watch

The Ultimate Guessing Game: Intra-Tissue Genetic Diversity

Key Takeaways

  • As a consequence of imperfect cell division and DNA repair, each one of the cells in our bodies has a unique genome – and some cell types, like neurons, immune cells, and cancer cells, can have even greater genetic diversity.
  • To fully understand the nature and consequences of this cellular diversity in both development and disease, researchers need better technologies for single-cell genomic sequencing.
  • A new approach to single-cell whole-genome amplification, Primary Template-directed Amplification (PTA), solves multiple technical challenges that have limited the use of single-cell genomic sequencing.
  • With PTA, researchers and clinicians can thoroughly characterize genetically heterogeneous cell populations for applications such as pre-implantation genetic testing, cancer research, immunology research, and neuroscience.

 

Primary Template-directed Amplification: An Elegant Solution to a Classic Puzzle

You may have encountered this guessing game in doctors’ waiting rooms, pubs, or back-to-school events. A large jar is filled with candies and you’re challenged to guess the number of candies just by looking. How do you approach the problem? What if you furthermore needed to guess the number of blue candies in the assortment?

As it turns out, the candy jar makes a great metaphor for intra-tissue genetic diversity, or the surprising degree of genetic variation that exists between individual cells within the same organism and even within the same organ. If the jar represents a sample of cells – say, a biopsy specimen – and each candy represents a single cell, how many unique genomes are present? 

We Contain Multitudes

The answer is not in fact “one,” as your high school biology class may have taught. As Charles Gawad, MD, PhD, explains in this recent webinar , both healthy tissues and cancer tumors possess more genetic diversity than previously appreciated. This diversity arises from mutations acquired during cell division and error-prone DNA damage repair mechanisms.

Recent studies have shown that human cells acquire approximately one mutation per round of division. Some cell types, such as neurons and B-lymphocytes, exhibit especially high rates of mutation in the course of normal development (Werner et al, 2020). Considering that the adult human body contains about thirty-seven trillion cells, each one the result of a sequence of cell divisions, the likely number of mutations present in a healthy adult is staggering (Bianconi et al., 2013).

In a cancerous tumor, where cell division is unchecked, and DNA repair mechanisms are often compromised, the mutational load can be much higher. The genetic diversity within a typical tumor creates the opportunity for the tumor to evolve rapidly in response to treatment. A few hardy surviving cells bearing an advantageous mutation are all it takes for the cancer to recur at a later date, this time resistant to the treatment that worked previously.

These facts have led an increasing number of researchers and clinicians to appreciate the importance of studying intra-tissue genetic diversity in both development and disease. While it’s true that your cells are more genetically similar to one another than different, small genetic differences may have a broad range of effects on a cell’s phenotype. These effects can be advantageous, as when naïve B-cells edit their genomes in response to pathogen exposure, or deleterious, as when cancer cells gain the ability to invade surrounding tissues. It all depends on the cell type, the nature of the mutation, and the physiological context.

The Problem with the Candy Jar Challenge

The historical approach to understanding how genetics contribute to development and disease has been bulk whole-genome sequencing, which pools genetic material from all the cells in the sample. The resulting data yield a clear, detailed picture of the average cell in the population. However, just as very few if any of the sweets in the jar are the size, shape, and color of an average candy, most of the cells don’t actually match this average genetic profile. And if you’re looking for blue candies, or cells bearing a rare but physiologically significant mutation, they must represent a fairly large fraction of the sample in order to be detectable.

This means that if you want to know the number of unique candies in the jar – or, even trickier, the number of blue candies – with absolute confidence, you need to examine each piece individually. Researchers and clinicians interested in intra-tissue genetic diversity are therefore turning to single-cell whole-genome sequencing (scWGS), which combines physical isolation of individual cells with next-generation sequencing and heavy-duty bioinformatics to analyze the genomes of many cells in parallel.

However, there are technical challenges that have so far limited the use of scWGS. Sequencing requires much more genomic material than can be isolated from a single cell, so it’s necessary to amplify the genome before library prep. Previous PCR-based amplification technologies suffer from biased amplification and a high rate of errors that make it difficult to interpret the sequencing data. If some parts of the genome are disproportionately represented by sequencing reads, how can you detect a copy number variant? If early errors were propagated, how can you distinguish between a real single nucleotide variant and an artifact?

A Novel Approach to Whole-Genome Amplification

Dr. Gawad – a pediatric hematologist-oncologist at Stanford University and a Chan-Zuckerberg Biohub Investigator – and colleagues have developed a technology that overcomes these limitations. Primary Template-directed Amplification (PTA), invented at St. Jude’s Children’s Research Hospital and licensed to BioSkryb Genomics, uses pseudo-linear reaction kinetics, rather than exponential, to suppress errors and ensure uniform amplification across the genome.

The results are remarkable: a recent comparison study sponsored by BioSkryb showed that in terms of coverage uniformity, sensitivity, and precision, PTA outperformed all other single-cell whole-genome amplification technologies tested (Gonzalez-Pena et al., 2021). The quality of sequencing data generated with PTA made it possible to call both copy number variants and single nucleotide variants with high confidence and reproducibility.

These capabilities enable researchers and clinicians to gather more comprehensive, reliable information about how intra-tissue diversity arises, changes over time, and correlates with phenotypic outcomes. One high-impact application for PTA-powered scWGS is monitoring the progression and treatment response of blood cancers. The emerging consensus in this field is that single-cell genomic analysis, combined with transcriptomics, proteomics, and epigenomics, is essential for understanding the rapid evolution of blood cancers and selecting optimal treatments (Nam et al. 2020).

The Ultimate Guessing Game: Intra-Tissue Genetic DiversityDr. Gawad’s own research in pediatric leukemia illustrates the promise of PTA. Following a standard course of treatment for a patient whose cancer exhibited heterozygous mutations in the kinase JAK2, bulk sequencing of the bone marrow did not detect either JAK2 mutation. However, PTA-powered single-cell sequencing found that most of the cells had one or the other – and furthermore, that many of the cells carried secondary mutations in other genes that could contribute to the disease phenotype. This kind of information can be extremely beneficial in treatment planning.

No More Guessing Games

As Dr. Gawad has discovered through collaborations with colleagues at Stanford, the potential for other applications of PTA is enormous. Already, in-vitro fertilization researcher-clinicians have used PTA to test pre-implantation embryos for genetic abnormalities incompatible with life; neurologists have begun to unravel the pathophysiology of intractable seizure disorders; and cardiologists have found that certain cardiac arrythmias can be caused by mutated ion channels in just a small fraction of pacemaker cells. Dr. Gawad presents highlights from these recent studies in the webinar, which can be watched on-demand below.

Whereas bulk whole-genome sequencing yields a very clear picture of the average cell in a sample, and previous whole-genome analysis technologies yield an incomplete or distorted view of each cell, PTA offers a complete, unbiased analysis of every single cell. For researchers and clinicians in any discipline impacted by intra-tissue genetic diversity, the unprecedented quality and quantity of information opens new avenues of inquiry. To find out more about how PTA works and how it can be applied to your research, please visit Bioskryb or email.

Watch the webinar here.

References

Bianconi E, Piovesan A, Facchin F, Beraudi A, Casadei R, Frabetti F, Vitale L, Pelleri MC, Tassani S, Piva F, Perez-Amodio S, Strippoli P, Canaider S. An estimation of the number of cells in the human body. Ann Hum Biol. 2013 Nov-Dec;40(6):463-71. doi: 10.3109/03014460.2013.807878. Epub 2013 Jul 5. Erratum in: Ann Hum Biol. 2013 Nov-Dec; 40(6):471. PMID: 23829164.
Gonzalez-Pena V, Natarajan S, Xia Y, Klein D, Carter R, Pang Y, Shaner B, Annu K, Putnam D, Chen W, Connelly J, Pruett-Miller S, Chen X, Easton J, Gawad C. Accurate genomic variant detection in single cells with primary template-directed amplification. Proc Natl Acad Sci USA. 2021 Jun 15; 118(24):e2024176118. doi: 10.1073/pnas.2024176118. PMID: 34099548; PMCID: PMC8214697.
Nam AS, Chaligne R, Landau DA. Integrating genetic and non-genetic determinants of cancer evolution by single-cell multi-omics. Nat Rev Genet. 2021 Jan;22(1):3-18. doi: 10.1038/s41576-020-0265-5. Epub 2020 Aug 17. PMID: 32807900; PMCID: PMC8450921.
Werner B, Case J, Williams MJ, Chkhaidze K, Temko D, Fernández-Mateos J, Cresswell GD, Nichol D, Cross W, Spiteri I, Huang W, Tomlinson IPM, Barnes CP, Graham TA, Sottoriva A. Measuring single cell divisions in human tissues from multi-region sequencing data. Nat Commun. 2020 Feb 25; 11(1):1035. doi: 10.1038/s41467-020-14844-6. PMID: 32098957; PMCID: PMC7042311.

Speaker: Dr. Charles Gawad, MD, PhD

Dr. Charles Gawad is a co-Founder of BioSkryb Genomics, Chan Zuckerberg Biohub Investigator, as well as an Associate Professor of Pediatrics at Stanford University where he has an active research lab and is a practicing pediatric oncologist. He was previously at St. Jude Children’s Research Hospital where they invented primary template-directed amplification (PTA) technology that was licensed to BioSkryb. Dr. Gawad received his medical degree from the University of Arizona and a PhD in cancer biology from Stanford. Among a number of honors, he is a recipient of a Career Award for Medical Scientists from the Burroughs Wellcome Fund, as well as an NIH Director’s New Innovator Award.

Dr. Charles Gawad

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