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Nathan Schaefer

@nkschaefer.bsky.social
29 followers 38 following 65 posts

UCSF postdoc, human, mammal

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Nathan Schaefer @nkschaefer.bsky.social · 12/03/2026
It was a pleasure working with this team, and I look forward to learning more from P. formosa, which is an excellent model for learning more about both speciation/hybrid incompatibility and the effects of inbreeding/genetic rescue. Hope you enjoy! www.nature.com/articles/s41...
nature.com
Gene conversion empowers natural selection in a clonal fish species - Nature
Analysis of the asexually reproducing Amazon molly Poecilia formosa and its sexually reproducing progenitors Poecilia mexicana and Poecilia latipinna reveals that it maintains a divergent mutational l...
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Nathan Schaefer @nkschaefer.bsky.social · 12/03/2026
It's therefore possible that expression dynamics strongly influence which genes contribute to hybrid incompatibility, and that noncoding regulatory sequences play a much greater role than coding substitutions.
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Nathan Schaefer @nkschaefer.bsky.social · 12/03/2026
RME allows heterozygous alleles directly to influence cellular phenotype without buffering one other; this can speed selection and cause rapid cross-species divergence. RME genes are also overrepresented at loci where humans have purged archaic hominin ancestry: www.science.org/doi/10.1126/...
science.org
An ancestral recombination graph of human, Neanderthal, and Denisovan genomes
A new heuristic ARG inference tool maps archaic hominin admixture and highlights genomic regions unique to modern humans.
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Nathan Schaefer @nkschaefer.bsky.social · 12/03/2026
We see strong enrichment of genes involved in processes like cell adhesion, cell migration, and cell-cell signaling. These appear to be the same types of genes that others found to undergo random cell-by-cell monallelic expression (RME) in humans. www.cell.com/cell-reports...
cell.com
Random allelic expression in the adult human body
Kravitz et al. develop a method to identify genes that randomly differentially express their parental alleles in human tissues and find that random allelic expression disproportionately impacts genes ...
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Nathan Schaefer @nkschaefer.bsky.social · 12/03/2026
Surprisingly, we see little evidence of this when considering coding mutations: GC rarely touches fixed coding differences between the parental species. What about noncoding sequence? We asked what types of genes tend to be near the noncoding sequences that GC changed the most in frequency.
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Nathan Schaefer @nkschaefer.bsky.social · 12/03/2026
When species diverge, gene networks can accumulate sets of new mutations that are compatible and preserve network function within each species, but which can cause dysfunction when brought together in a hybrid. Does GC help alleviate this problem in P. formosa? www.nature.com/scitable/con...
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Nathan Schaefer @nkschaefer.bsky.social · 12/03/2026
We then looked for evidence of positive selection in noncoding sequence and found that gene conversion copies sequences positively selected (low Tajima's D, high Fay & Wu's H, high Zeng et al's E) in their parental species of origin. P. formosa ends up with the handiest sequences from both parents.
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Nathan Schaefer @nkschaefer.bsky.social · 12/03/2026
We asked what types of mutations gene conversion (GC) is likeliest to copy versus overwrite and found evidence that it aids purifying selection. GC likes to eliminate young mutations and those with high variant impact (likely to be deleterious).
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Nathan Schaefer @nkschaefer.bsky.social · 12/03/2026
Gene conversion events occur at nonrandom sites and are associated with polyA/polyT repeats, which have been implicated in double strand break formation during DNA replication. www.cell.com/cell/fulltex...
cell.com
Dual Roles of Poly(dA:dT) Tracts in Replication Initiation and Fork Collapse
Mammalian replication origins are fragile sites defined by poly-dA/dT stretches that are nucleosome free and devoid of the single-strand DNA-protecting protein RPA.
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Nathan Schaefer @nkschaefer.bsky.social · 12/03/2026
Through phased genome assembly, ancestral genome reconstruction, and population resequencing, we find evidence that gene conversion, in which P. formosa "overwrites" sequence from one of its haplotypes with the other, likely facilitates selection. www.nature.com/articles/d41...
nature.com
How an all-female fish species defies evolutionary expectations
Asexual reproduction should be an evolutionary dead end. A study of asexual fish shows that a genetic ‘copy and paste’ helps to overcome the predicted costs of asexuality.
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Nathan Schaefer @nkschaefer.bsky.social · 12/03/2026
Without meiotic recombination, there is no mechanism for decoupling mildly deleterious mutations from beneficial ones. This should weaken purifying selection, and ultimately cause extinction, in a process known as Muller's Ratchet. Prior modeling work suggested this should have already happened.
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Nathan Schaefer @nkschaefer.bsky.social · 12/03/2026
The Amazon molly (Poecilia formosa) is the first clonal vertebrate species known to science. It was formed via hybridization between sister species P. latipinna and P. mexicana around 100kya; hybridization disrupted meiosis and produced an all-female, asexual lineage. www.nature.com/articles/s41...
nature.com
Clonal polymorphism and high heterozygosity in the celibate genome of the Amazon molly - Nature Ecology & Evolution
Asexual vertebrates are extremely rare. Here, the authors sequence the genome of the Amazon molly, an asexual fish, and find few signs of genetic degeneration but clonal polymorphism and high heterozy...
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Nathan Schaefer @nkschaefer.bsky.social · 12/03/2026
Excited to share a new study co-written with Ed Ricemeyer (LMU Munich), supervised by Manfred Schartl (U. of Würzburg) & Wes Warren (U. of Missouri). We investigated how the Amazon molly has survived for 100,000 years (more generations) despite clonal reproduction. www.nature.com/articles/s41...
nature.com
Gene conversion empowers natural selection in a clonal fish species - Nature
Analysis of the asexually reproducing Amazon molly Poecilia formosa and its sexually reproducing progenitors Poecilia mexicana and Poecilia latipinna reveals that it maintains a divergent mutational l...
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
Thanks for reading, and good luck checking IDs and keeping the rifraff out of your single cell data sets. www.biorxiv.org/content/10.1... github.com/nkschaefer/c...
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
In total, our study demonstrates the need for this set of tools, which provide new functionality, speed, and/or accuracy over existing tools. It also demonstrates the power of pooled single cell studies, including those involving composite cell lines, to discover new and interesting biology.
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
Back-mutations to the ancestral state at this type are uncommon, at a frequency typically seen in mitochondrial protein-coding or disease-implicated mutations. This suggests that this mutation may be one of the changes affecting gene regulation at this locus.
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
The affected locus (MT-ND3/MT-ND4L) was found by others (bmcbiol.biomedcentral.com/articles/10....) to be cleaved by an unknown mechanism at a site that we noticed is next to a fixed, derived human-specific mutation that might affect cleavage rates by altering the 3D shape of the RNA.
bmcbiol.biomedcentral.com
Identification of human mitochondrial RNA cleavage sites and candidate RNA processing factors - BMC Biology
Background The human mitochondrial genome is transcribed as long strands of RNA containing multiple genes, which require post-transcriptional cleavage and processing to release functional gene product...
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
Mitochondrial genes are expressed as polycistronic transcripts, then cleaved and selectively degraded. We looked at species differences in this process, from two causes: nuclear and mitochondrial mutations. Interestingly, the biggest differences we found were compensatory, with little net effect.
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
By finding one fusion line that tended to retain both species’ mitochondria, we were able to hone in on the gene network involved in this process: we can see what was turned up in the unhealthy cells, and what was turned down in those that survived.
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
We think this means incompatibility between allospecific mitochondria that causes gene dysregulation, as well as a nuclear self-destruct mechanism. Interestingly, a prior study also found that human cells have “a suicidal preference for self-mtDNA”: www.molbiolcell.org/doi/10.1091/...
molbiolcell.org
Mechanisms of Human Mitochondrial DNA Maintenance: The Determining Role of Primary Sequence and Length over Function | Molecular Biology of the Cell
Although the regulation of mitochondrial DNA (mtDNA) copy number is performed by nuclear-coded factors, very little is known about the mechanisms controlling this process. We attempted to introduce no...
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
Cells with two species’ mitochondria have significantly altered gene expression related to cell cycle arrest and apoptosis relative to other cells, suggesting they’re in trouble. They also express fewer mitochondrial transcripts overall and have abnormal post-expression transcriptional regulation.
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
After demultiplexing with CellBouncer, we found that composite cells mostly inherit only one species’ mitochondria: human, for human/chimpanzee cells, and bonobo, for chimpanzee/bonobo cells. Not always, though: some cells retained both mitochondria, or those from the less common species.
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
We take CellBouncer for a spin on a cool data set: inter-species composite iPSCs we created by cell fusion (www.nature.com/articles/s41...) for studying species differences in gene regulation. Here, we asked if there were biases in which species’ mitochondria were inherited by the composite cells.
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
doublet_dragon takes assignments from the other programs and infers a global doublet rate that encompasses both homotypic doublets (invisible to individual programs) and heterotypic ones. This can help with QC (given expectation based on cell loading density) and serve as a prior for other tools.
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
demux_tags assigns custom labels (e.g. MULTIseq/HTO data), or sgRNAs (CRISPR guide capture data) to cells. Our method considers the distribution of all tag counts together, rather than considering each tag independently, and handles noisy/low-count data better than some alternatives.
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
bulkprops takes genotypes and bulk data (or single cell data, ignoring cell barcodes) and infers the proportion of each individual in the pool. This can cross-check the other programs, and we provide a method to bootstrap proportions and get p-values when comparing two sets of proportions.
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
Additionally, quant_contam models the genotypic origins of ambient RNA, meaning it can highlight when specific donors or cell lines contribute disproportionately to ambient RNA. If expression data are provided, quant_contam can adjust counts to account for contamination.
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
quant_contam quantifies ambient RNA by measuring how often cells mismatch their expected genotypes. This introduces an external ground truth (genotype data), avoids the need to consider empty droplets, and can find ambient RNA in data lacking cell type diversity.
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
After running demux_mt, we suggest a pipeline that can produce a VCF file of nuclear variants and demultiplex more cells using demux_vcf. While not suited to every data set, we demonstrate this method on whole-cell RNA-seq and single nucleus ATAC data, outperforming competing methods.
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
demux_mt answers this problem by simultaneously clustering mitochondrial haplotypes and inferring the number of individuals in the pool. It takes only a BAM file. There is also a way to plot the haplotypes to see how well clustering worked.
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
If you don’t have preexisting genotype data, there are tools to assign cells to individuals of origin by clustering genotypes (Vireo, souporcell, scSplit, freemuxlet), but there’s not a clear way to check results, and they can make mistakes.
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
demux_vcf assigns cells to individuals using genotypes and is fast, accurate, and robust to deep population structure. It groups SNPs by allelic state in each pair of individuals and compares the likelihood of each pair of IDs for each cell, improving speed over methods that filter or refine SNPs.
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
demux_species uses an alignment-free k-mer counting strategy to save time and memory and assigns cells to species using a statistical model instead of a cutoff. Users can plot the clustered k-mer counts to see if it worked. demux_species also separates reads by species for downstream processing.
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
CellBouncer provides fast, compiled, self-contained, interacting programs with methods to validate results where possible (e.g. you can visually compare two sets of IDs for the same cells, and you can visualize inferred mitochondrial haplotypes to determine how well the clustering worked).
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
…and tools that can identify specific types of cell doublets, but cannot calculate a global doublet rate (which includes droplets containing two cells of the same type).
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
…genotype-free demultiplexing tools that lack a validation method, ambient RNA removal tools that require cell type heterogeneity, custom tag (e.g. MULTIseq, HTO) or sgRNA (e.g. CRISPR guide capture) assignment strategies that fail when data are sparse or noisy, …
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
Useful bioinformatic tools for demultiplexing and QCing pooled data exist. We identified several unmet needs, though, including: no dedicated method for species demultiplexing, slow genotype demultiplexing with large SNP panels, sensitivity to deep population structure in SNP reference panels…
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
Pooling cells from multiple donors, cell lines, or species makes it easy to scale up experiments, incorporate genetic variation, and mitigate technical artifacts, while doing cool things like disentangling the effects of cell-extrinsic from cell-intrinsic variation (www.nature.com/articles/s41...).
nature.com
Human neuronal maturation comes of age: cellular mechanisms and species differences - Nature Reviews Neuroscience
Human cortical neurons undergo a protracted period of postmitotic maturation compared with those of other species. Wallace and Pollen review the cell-intrinsic and cell-extrinsic mechanisms that gover...
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
CellBouncer is described in our new manuscript: www.biorxiv.org/content/10.1... and is available on GitHub: github.com/nkschaefer/c...
biorxiv.org
CellBouncer, A Unified Toolkit for Single-Cell Demultiplexing and Ambient RNA Analysis, Reveals Hominid Mitochondrial Incompatibilities
Pooled processing, in which cells from multiple sources are cultured or captured together, is an increasingly popular strategy for droplet-based single cell sequencing studies. This design allows effi...
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
Introducing CellBouncer, a toolkit for pooled single cell data that assigns cells to species or individual of origin, performs genotype-free ids using mitochondrial haplotypes, assigns sgRNAs and custom tags to cells, and models ambient RNA using external genotype data as a ground truth.
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
Picture this: you are running a single cell sequencing experiment and want to incorporate cells from different species, individuals, and maybe even perturbations. You process everything together to save time while reducing batch artifacts. You collect data. What do you do now?
media.tenor.com
a man in a green shirt is carrying a pink bag
ALT: a man in a green shirt is carrying a pink bag
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
In total, our study demonstrates the need for this set of tools, which provide new functionality, speed, and/or accuracy over existing tools. It also demonstrates the power of pooled single cell studies, including those involving composite cell lines, to discover new and interesting biology.
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Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
Back-mutations to the ancestral state at this type are uncommon, at a frequency typically seen in mitochondrial protein-coding or disease-implicated mutations. This supports the idea that this mutation could be one of the changes affecting gene regulation at this locus.
100
Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
The affected locus (MT-ND3/MT-ND4L) was found by others (bmcbiol.biomedcentral.com/articles/10....) to be cleaved by an unknown mechanism at a site that we noticed is next to a fixed, derived human-specific mutation that might affect cleavage rates by altering the 3D shape of the RNA.
bmcbiol.biomedcentral.com
Identification of human mitochondrial RNA cleavage sites and candidate RNA processing factors - BMC Biology
Background The human mitochondrial genome is transcribed as long strands of RNA containing multiple genes, which require post-transcriptional cleavage and processing to release functional gene product...
100
Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
Mitochondrial genes are expressed as polycistronic transcripts, then cleaved and selectively degraded. We looked at species differences in this, from two causes: nuclear and mitochondrial genome mutations. Interestingly, the biggest differences we found were compensatory, with little net effect.
100
Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
By finding one fusion line that tended to retain both species’ mitochondria, we were also able to hone in on some of the genes involved in this process: we can see what was turned up in the unhealthy cells, and what was turned down in those that survived.
100
Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
We think this means incompatibility between allospecific mitochondria that causes dysregulation, plus a nuclear self-destruct mechanism. Interestingly, a prior study also found that human cells have “a suicidal preference for self-mtDNA”: www.molbiolcell.org/doi/10.1091/...
molbiolcell.org
Mechanisms of Human Mitochondrial DNA Maintenance: The Determining Role of Primary Sequence and Length over Function | Molecular Biology of the Cell
Although the regulation of mitochondrial DNA (mtDNA) copy number is performed by nuclear-coded factors, very little is known about the mechanisms controlling this process. We attempted to introduce no...
100
Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
Cells with two species’ mitochondria have significantly altered gene expression related to cell cycle arrest and apoptosis relative to other cells, suggesting they’re in trouble. They also express fewer mitochondrial transcripts overall and have abnormal post-expression transcriptional regulation.
100
Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
After demultiplexing with CellBouncer, we found that composite cells mostly inherit only one species’ mitochondria: human, for human/chimpanzee cells, and bonobo, for chimpanzee/bonobo cells. Not always, though: some cells retained both mitochondria, or those from the less common species.
100
Nathan Schaefer @nkschaefer.bsky.social · 24/03/2025
We take CellBouncer for a spin on a cool data set: inter-species composite iPSCs we created by cell fusion (www.nature.com/articles/s41...) for studying species differences in gene regulation. Here, we asked if there were biases in which species’ mitochondria were inherited by the composite cells.
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