Biology subjects

Gallego Romero, I.

Publications and source records attributed to Gallego Romero, I..

At least 19 recordsLinked to original sources

bioRxiv2026

Massively parallel characterization reveals context-dependent and non-additive regulatory effects of closely spaced variant pairs

Precise control of gene expression relies in part on cis-regulatory elements (CREs), including enhancers. Genetic variation within enhancers can alter their regulatory activity and contribute to variation in gene expression, yet the effects of multiple nearby variants within the same enhancer remain poorly understood. To address this, we designed a massively parallel reporter assay (MPRA) to test the regulatory effect of 7,285 pairs of close-proximity single-nucleotide variants (SNVs) selected from blood-specific and broadly active enhancers. To enrich for functionality, we required at least one variant in each pair to be annotated as an eQTL in eQTLGen dataset. We measured the regulatory activity of all four haplotypes in K562 leukemia cells, where 57% of tested variant pairs had at least one derived haplotype that differed significantly in activity from the ancestral haplotype. The effects of individual variants frequently depended on the allelic background provided by the neighboring variant, with some variants showing opposite effects in different allelic backgrounds. Among a smaller, high-confidence subset selected for analysis of additivity, 59% (105/178) of variant pairs showed non-additive effects, and non-additive pairs were located closer together than additive pairs. Among pairs in which both single-derived haplotypes increased activity, the double-derived haplotype generally showed a smaller effect than expected under additivity. Together, our results demonstrate that nearby variants within the same enhancer can jointly shape regulatory activity and highlight the importance of considering local allelic context when interpreting the functional effects of regulatory variation.

genomics↗

bioRxiv2026

Using sequence-to-function models to interpret archaic hominin introgression

Understanding the functional impact of archaic hominin introgression remains challenging due to the poor representation of global introgression in publicly available genomics resources. Sequence-to-function models can predict the effects of any possible variant in the human genome and may fill this gap. Here, we used AlphaGenome to predict the effects of 144,139 introgressed SNPs segregating in present-day individuals of Papuan genetic ancestry. AlphaGenome's chromatin accessibility predictions recapitulate experimentally observed effects, but gene expression performs no better than chance. Predictions correlate more strongly with an independent reporter assay of single-variant activity than with the same variants' effects in live cells, indicating that AlphaGenome captures the regulatory potential of individual variants more reliably. Predictions carry tissue specificity, allowing us to predict specific tissues potentially impacted by introgressed haplotypes. We identify genes, including JAK1 and TAB2, that are associated with haplotypes that contain an excess of variants predicted by AlphaGenome to have large impacts on chromatin accessibility. Finally, we highlight the challenges and limitations associated with using sequence-to-function models for introgressed variant effect prediction, and show that while AlphaGenome's chromatin accessibility predictions can aid in prioritising candidate functional regions, expression predictions and the assignment of variants to target genes remain as open challenges.

genomics↗

bioRxiv2026

Power is a major confounder in the analysis of cross-ancestry 'portability' in human eQTLs

The phenotypic effects of germline variants are often mediated through gene regulation. Expression quantitative trait loci (eQTLs) are genetic variants associated with changes in gene expression. Understanding how eQTLs vary across populations is essential for characterising the genetic and regulatory drivers of trait diversity. Meta-analysing eQTL studies from multiple populations enables more robust detection of eQTLs and can reveal regulatory mechanisms shaped by population-specific environmental or ancestry-related factors. However, across the multi-ancestry eQTL literature, a wide range of methods have been used to quantify eQTL portability across ancestry groups. Because different studies employ different portability metrics, it is challenging to form a coherent view of the regulatory landscape across populations. In this work, we analyse eQTL summary statistics from ten datasets matched on tissue type and sequencing technology. We compare portability metrics used previously and show that they can yield markedly different patterns of apparent regulatory conservation or divergence. We then examine the statistical determinants of portability across metrics and demonstrate that sample size, minor allele frequency, and linkage disequilibrium are major drivers of the observed differences in eQTL portability across studies. These findings highlight that differences in statistical power stemming from factors such as population size and allele frequency must be accounted for when evaluating eQTL portability. To address this issue, we introduce a new approach designed to correct for these factors when calling eQTL portability. Finally, we show that empirical Bayes multivariate adaptive shrinkage provides a powerful framework for meta-analysing multiple eQTL studies, with the ability to pool signals across populations to produce more robust effect-size estimates within each population, and better powered detection of population specific eQTLs. Author SummaryGenetic variants often influence human traits and diseases by altering gene regulation. Expression quantitative trait loci (eQTLs) are variants associated with changes in gene expression. Many eQTLs are not portable -- they fail to reproduce across cohorts and ancestries -- yet studies use many different metrics to decide when an eQTL has reproduced, making it hard to form a coherent picture of gene regulation across populations. Here we compiled eQTL studies from ten cohorts spanning ancestries, tissues, and sequencing technologies, showing that commonly used portability metrics reveal distinct patterns of sharing. We find the main drivers of non-portability are differences in sample size, minor allele frequency, and linkage disequilibrium, and develop a method that separates these power-related confounders from biological differences. Finally, we show that analysing studies together boosts power, increasing portability. Our results provide a framework to interpret multiple eQTL studies for how genetic variation affects gene regulation across cohorts and ancestries.

genomics↗

bioRxiv2026

Ancestral and environmental diversity shape the immune landscape in Indonesia

Island Southeast Asia (ISEA) remains consistently underrepresented in human genomic resources despite its exceptional ancestral and lifestyle diversity. The interplay between the regions complex population history and its environmental variation provide a window into how ancestry and environment jointly shape the human immune system. Here we report the generation of single-cell PBMC profiles from 199 Indonesians sampled across four communities in the islands of Bali and New Guinea. These groups capture diversity in regional genetic ancestries (East Asian-like and Papuan-like) and lifestyle contrasts (urban versus rural communities in Bali; highland versus lowland communities in New Guinea). We identify over 4,000 expression quantitative trait loci (eQTLs) across nine immune cell types, including eQTLs driven by introgression from both Neanderthals and Denisovans at genes such as IL7R, HLA-E, or STAT2. We also find evidence of local ancestry driving gene-by-environment interactions at pathogen receptors such as MARCO, although the majority of gene-by-environment interactions are not driven by differences in genetic structure between populations. Beyond direct genetic effects, we construct gene co-expression networks that consistently identify environmental signatures, as well as T-cell receptor repertoires that distinguish specific communities, with excess representation of interferon-stimulated genes in rural, but not urban samples. This work establishes a framework for population-aware functional genomics in understudied regions and highlights how ancestral and environmental diversity jointly shape human immunity in this globally important yet underrepresented region.

genomics↗

bioRxiv2025

Lift&Add - rapid and robust addition of new species to alignments of conserved non-coding sequences

Identifying sequence constraint across long evolutionary distances is a powerful method for the discovery of functional genomic sequences, especially putative non-coding elements. Conserved elements have been a mainstay of comparative genomic research, and can be further investigated for specific-specific sequence acceleration to dissect the genetic basis of trait evolution. The conclusions of these comparative genomic studies are however contingent in on the number and range of species included in this phylogenetic analysis. One group of species that has been largely under-represented in genomic comparisons are the marsupials, due to the dearth of marsupial genomes in most publicly available whole-genome alignments. In this study, we firstly showed how biased phylogenetic distributions can profoundly affects estimations of conservation/acceleration with a focus on the marsupials. Then we present a bioinformatic workflow that rapidly enabled us to map 13,287 vertebrate conserved elements--a majority of which were intergenic--identified from the 60-species whole-genome alignment of vertebrates (containing only 4 marsupials) to up to 12 new marsupial genomes ("Lift"). Following this, we combined these new marsupials sequences back to multiple species alignments of these conserved elements ("Add"). Lastly, we demonstrate with our test dataset how expanding phylogenetic breadth can change the conclusions of a comparative genomic analysis.

genomics↗

bioRxiv2025

Dissecting functional regulatory convergence over 160 million years of therian evolution

Understanding the molecular mechanisms underpinning convergent traits can provide insight into the predictability of the evolutionary process. The thylacine, an extinct marsupial carnivore, has been long known to have a very similar craniofacial morphology to that of eutherian canids, despite having diverged{approx} 160 million years ago. Using a massively parallel reporter assay (MPRA), we tested the regulatory potential of a set of previously identified highly conserved craniofacial enhancers that showed convergent sequence acceleration in thylacine and wolf (TWARs). We compared orthologous sequences from six different taxa, including outgroup taxa with non-convergent craniofacial phenotypes (Tasmanian devil and giant panda) and ancestral reconstructions for marsupial and placental carnivores (Dasyuromorphia and Carnivora). Dense 10bp tiling allowed us to thoroughly examine features associated with activity, including percentage GC content and transcription factor binding motifs turnover. We identified marked conservation of levels and patterns of regulatory activity across all six taxa, as well as multiple cases of differentially active TWARs within each clade, with both thylacine and wolf driving functional divergence. However, evidence of convergent divergence was limited to a set of neighbouring TWARs near the neural crest gene Phox2b - one of which exhibited reduced activity in the wolf, and the other in the thylacine. Ultimately our findings suggest that shared gene regulatory potential is not a feature of these convergently accelerated regions in the thylacine and wolf.

genomics↗

bioRxiv2025

A global map for introgressed structural variation and selection in humans

Genetic introgression from Neanderthals and Denisovan has shaped modern human genomes; however, introgressed structural variants (SVs [≥]50 base pairs) remain challenging to discover. We integrated high-quality phased assemblies from four new Papua New Guinea (PNG) genomes with 94 published assemblies of diverse ancestry to infer an archaic introgressed SV map. Introgressed SVs are overall enriched in genes (44%, n=1,592), including critical genomic disorder regions, and most abundant in PNG. We identify 11 centromeres likely derived from archaic hominins, adding unexplored diversity to centromere genomics. Pangenome genotyping across 1,363 samples reveals 16 candidate adaptive SVs, many associated with immune-related genes and their expression, in the PNG. We hypothesize that archaic SV introgression contributed to reproductive success, underscoring introgression as a significant force in human adaptive evolution.

evolutionary biology↗

bioRxiv2025

Mapping the gene regulatory landscape of archaic hominin introgression in modern Papuans

Interbreeding between anatomically modern humans and archaic hominins has contributed to the genomes of present-day human populations. However, our understanding of the specific gene regulatory consequences of Neanderthal, and particularly, Denisovan introgression is limited. Here, we used a massively parallel reporter assay to investigate the regulatory effects of 25,869 high-confidence introgressed SNPs segregating in present-day individuals of Papuan genetic ancestry in immune cell types. Overall, 8.22% of Denisovan and 8.58% of Neanderthal sequences showed active regulatory activity, and 9.22% of these displayed differential activity between alleles. We examined the properties of active and differentially active sequences, and found no impact of introgressed allele frequency on the probability of activity, but do observe an association with distance to the nearest transcription start site. Differential activity was also associated with differential transcription factor binding. Genes predicted to be regulated by differentially active sequences included IFIH1 and TNFAIP3, key immune genes and known examples of archaic introgression. Overall, this work provides insight into the regulatory activity of archaic variants in Papuan populations and, more broadly, the contribution of archaic introgression in shaping modern human genetic diversity.

evolutionary biology↗

bioRxiv2024

Public RNA-seq data are not representative of global human diversity

The field of human genetics has reached a consensus that it is important to work with diverse and globally representative participant groups. This diverse sampling is required to build a robust understanding of the genomic basis of complex traits and diseases as well as human evolution, and to ensure that all people benefit from downstream scientific discoveries. While previous work has characterized compositional biases and disparities for public genome-wide association (GWAS), microbiome, and epigenomic studies, we currently lack a comprehensive understanding of the degree of bias for transcriptomic studies. To address this gap, we analyzed the metadata for RNA-seq studies from two public databases--the Sequence Read Archive (SRA), representing 795,071 samples from 21,209 studies, and the Database of Genotypes and Phenotypes (dbGaP), representing 167,389 samples from 649 studies. We also randomly selected 620 studies from SRA for detailed, manual evaluation. We found that 3% of samples in SRA and 21% of individuals described in the literature had population descriptors (race, ethnicity, or ancestry); 28% of samples in dbGaP had paired genotype data that was used to empirically infer ancestry. In SRA, dbGaP, and the literature, race, ethnicity, and ancestry terms were frequently conflated and difficult to disambiguate. After standardizing population descriptors, we observed many clear biases: for example, among samples in SRA that were coded using US Census terms, 69.0% came from white donors, corresponding to an 1.2x overrepresentation of this group relative to the US population. Among samples in SRA coded using continental ancestry labels, 55.6% came from European ancestry donors--an 4.1x overrepresentation of this group relative to the global population. These biases were generally similar across datasets (SRA, dbGaP, literature review), and were comparable to previous reports for other omics data types. However, we note that, relative to other omics data subsets like GWAS, there is considerably less information, of arguably worse quality, about who is participating in RNA-seq studies. Together, these results demonstrate a critical need to improve our thoughtfulness, consistency, and effort around reporting population descriptors in RNA-seq studies, and to more generally strive for greater diversity in this important data type.

genomics↗

bioRxiv2024

Profiling genetically driven alternative splicing across the Indonesian Archipelago

One of the regulatory mechanisms influencing the functional capacity of genes is alternative splicing (AS). Previous studies exploring the splicing landscape of human tissues have shown that AS has contributed to human biology, especially in disease progression and the immune response. Nonetheless, this phenomenon remains poorly characterised across human populations, and it is unclear how genetic and environmental variation contribute to alternative splicing. Here, we examine a set of 115 Indonesian samples from three traditional island populations spanning the genetic ancestry cline that characterizes Island Southeast Asia. We conduct a global AS analysis between islands to ascertain the degree of functionally significant AS events and their consequences. Using a hierarchical event-based statistical model, we detected over 1,000 significant differential AS events across all comparisons. Additionally, we identify over 6,000 genetic variants associated with changes in splicing (splicing quantitative trait loci; sQTLs), some of which are driven by Papuan-like genetic ancestry, and only show partial overlap with other publicly available sQTL datasets derived from other populations. Computational predictions of RNA binding activity revealed that a fraction of these sQTLs directly modulate the binding propensity of proteins involved in the splicing regulation of immune genes. Overall, these results contribute towards elucidating the role of genetic variation in shaping gene regulation in one of the most diverse regions in the world.

genomics↗

bioRxiv2023

De novo transcriptome assembly and genome annotation of the fat-tailed dunnart (Sminthopsis crassicaudata)

Marsupials exhibit highly specialized patterns of reproduction and development, making them uniquely valuable for comparative genomics studies with their sister lineage, eutherian (also known as placental) mammals. However, marsupial genomic resources still lag far behind those of eutherian mammals, limiting our insight into mammalian diversity. Here, we present a series of novel genomic resources for the fat-tailed dunnart (Sminthopsis crassicaudata), a mouse-like marsupial that, due to its ease of husbandry and ex-utero development, is emerging as a laboratory model. To enable wider use, we have generated a multi-tissue de novo transcriptome assembly of dunnart RNA-seq reads spanning 12 tissues. This highly representative transcriptome is comprised of 2,093,982 assembled transcripts, with a mean transcript length of 830 bp. The transcriptome mammalian BUSCO completeness score of 93% is the highest amongst all other published marsupial transcriptomes. Additionally, we report an improved fat-tailed dunnart genome assembly which is 3.23 Gb long, organized into 1,848 scaffolds, with a scaffold N50 of 72.64 Mb. The genome annotation, supported by assembled transcripts and ab initio predictions, revealed 21,622 protein-coding genes. Altogether, these resources will contribute greatly towards characterizing marsupial biology and mammalian genome evolution.

genomics↗

bioRxiv2023

Cis-regulatory landscapes of the fat-tailed dunnart and mouse provide insights into the drivers of craniofacial heterochrony

Marsupials and placental mammals exhibit significant differences in reproductive and life history strategies. Marsupials are born highly underdeveloped after an extremely short period of gestation, leading to prioritization of the development of structures critical for post-birth survival in the pouch. Critically, they must undergo accelerated development of the oro-facial region compared to placentals. Previously we described the accelerated development of the oro-facial region in the carnivorous Australian marsupial, the fat-tailed dunnart Sminthopsis crassicaudata that has one of the shortest gestations of any mammal. By combining genome comparisons of the mouse and dunnart with functional data for the enhancer-associated chromatin modifications, H3K4me3 and H3K27ac, we investigated divergence of craniofacial regulatory landscapes between these species. This is the first description of genome-wide face regulatory elements in a marsupial, with 60,626 putative enhancers and 12,295 putative promoters described. We also generated craniofacial RNA-seq data for the dunnart to investigate expression dynamics of genes near predicted active regulatory elements. While genes involved in regulating facial development were largely conserved in mouse and dunnart, the regulatory landscape varied significantly. Additionally, a subset of dunnart-specific enhancers were associated with genes highly expressed only in dunnart relating to cranial neural crest proliferation, embryonic myogenesis and epidermis development. Comparative RNA-seq analyses of facial tissue revealed dunnart-specific expression of genes involved in the development of the mechanosensory system. Accelerated development of the dunnart sensory system likely relates to the sensory cues received by the nasal-oral region during the postnatal journey to the pouch. Together these data suggest that accelerated face development in the dunnart may be driven by dunnart-specific enhancer activity. Our study highlights the power of marsupial-placental comparative genomics for understanding the role of enhancers in driving temporal shifts in development.

developmental biology↗

bioRxiv2022

The whole blood microbiome of Indonesians reveals that environmental differences shape immune gene expression signatures

Pathogens found within local environments are a major cause of morbidity and mortality. This is particularly true in Indonesia, where infectious diseases such as malaria or dengue are a significant part of the disease burden. Unequal investment in medical funding throughout Indonesia, particularly in rural areas, has resulted in under-reporting of cases, making surveillance challenging. Here, we use transcriptome data from 117 healthy individuals living on the islands of Mentawai, Sumba, and the Indonesian side of New Guinea Island to explore which pathogens are present within whole blood. We detect a range of taxa within RNA-sequencing data generated from whole blood and find that two pathogens--Flaviviridae and Plasmodium--are the most predominantly abundant, both of which are most pronounced in the easternmost island within our Indonesian dataset. We also compare the Indonesian data to two other cohorts from Mali and UK and find a distinct microbiome profile for each group. This study provides a framework for RNA-seq as a possible retrospective surveillance tool and an insight to what makes up the transient human blood microbiome.

genomics↗

bioRxiv2021

Genetic diversity in chimpanzee transcriptomics does not represent wild populations

Chimpanzees (Pan troglodytes) are a genetically diverse species, consisting of 4 highly distinct subspecies. As humans closest living relative they have been a key model organism in the study of human evolution, and comparisons of human and chimpanzee transcriptomes have been widely used to characterise differences in gene expression levels that could underlie the phenotypic differences between the two species. However, the subspecies from which these transcriptomic datasets have been derived is not recorded in metadata available in the public NCBI Sequence Read Archive (SRA). Furthermore, labelling of RNA-seq samples is for the most part inconsistent across studies, and the true number of individuals from whom transcriptomic data is available is difficult to ascertain. Thus we have evaluated genetic diversity at the subspecies and individual level in 486 public RNA-seq samples available in the SRA, spanning the vast majority of public chimpanzee transcriptomic data. Using multiple population genetics approaches we find that nearly all samples (96.6%) have some degree of Western chimpanzee ancestry. At the individual donor level, we identify multiple samples that have been repeatedly analysed across different studies, and identify a total of 135 genetically distinct individuals within our data, a number that falls to 89 when we exclude likely first and second-degree relatives. Altogether, our results show that current transcriptomic data from chimpanzees is capturing low levels of genetic diversity relative to what exists in wild chimpanzee populations. These findings provide important context to current comparative transcriptomics research involving chimpanzees.

genomics↗

bioRxiv2021

Transcriptomic profiles of Plasmodium falciparum and Plasmodium vivax-infected individuals in Indonesia

Malaria is one of the leading causes of illness and death globally. The vast majority of transcriptomic studies of the impact of malaria on human hosts have been conducted on populations of African ancestry suffering from Plasmodium falciparum infection, making it unclear whether biological responses observed in these studies can be generalised to other populations. Here, we perform differential expression analysis between healthy controls and malaria-infected patients within Indonesia, a country of over 260 million people which has substantial morbidity due to endemic malaria. We find that in samples infected with P. falciparum and P. vivax, there is an upregulation of genes involved in inflammation, the immediate early immune response, translation, and apoptosis. When comparing these findings to transcriptomic studies conducted in Africa (on P. falciparum) and South America (on P. vivax), we find that many pathways are shared. This is particularly apparent for receptor recognition and inflammation-related genes in P. falciparum and innate immune and chemokine-related genes in P. vivax infection. However, we also find that many genes are unique to the Indonesian population, particularly those involved in RNA processing, splicing, and cell surface receptor genes. This study provides a more comprehensive view of malaria infection outside of Africa and contributes to a better characterisation of malaria pathogenesis within humans across a range of genetic architectures.

genomics↗

bioRxiv2020

Genetic architecture of gene regulation in Indonesian populations identifies QTLs associated with local ancestry and archaic introgression

Lack of diversity in human genomics limits our understanding of the genetic underpinnings of complex traits, hinders precision medicine, and contributes to health disparities. To map genetic effects on gene regulation in the underrepresented Indonesian population, we have integrated genotype, gene expression, and CpG methylation data from 115 participants across three island populations that capture the major sources of genomic diversity on the region. In a comparison with a European dataset, we identify 166 uniquely Indonesia-specific eQTLs, highlighting the benefits of performing association studies on non-European populations. By combining local ancestry and archaic introgression inference eQTLs and methylQTLs, we identify regulatory loci driven by modern Papuan ancestry as well as introgressed Denisovan and Neanderthal variation. GWAS colocalization connects QTLs detected here to hematological traits. Our findings illustrate how local ancestry and archaic introgression drive variation in gene regulation across genetically distinct and in admixed populations.

genomics↗

bioRxiv2019

Genome-wide DNA methylation and gene expression patterns reflect genetic ancestry and environmental differences across the Indonesian archipelago

Indonesia is the worlds fourth most populous country, host to striking levels of human diversity, regional patterns of admixture, and varying degrees of introgression from both Neanderthals and Denisovans. However, it has been largely excluded from the human genomics sequencing boom of the last decade. To serve as a benchmark dataset of molecular phenotypes across the region, we generated genome-wide CpG methylation and gene expression measurements in over 100 individuals from three locations that capture the major genomic and geographical axes of diversity across the Indonesian archipelago. Investigating between- and within-island differences, we find up to 10% of tested genes are differentially expressed between the islands of Mentawai (Sumatra) and New Guinea. Variation in gene expression is closely associated with DNA methylation, with expression levels of 9.7% of genes strongly correlating with nearby CpG methylation, and many of these genes being differentially expressed between islands. Genes identified in our differential expression and methylation analyses are enriched in pathways involved in immunity, highlighting Indonesia tropical role as a source of infectious disease diversity and the strong selective pressures these diseases have exerted on humans. Finally, we identify robust within-island variation in DNA methylation and gene expression, likely driven by very local environmental differences across sampling sites. Together, these results strongly suggest complex relationships between DNA methylation, transcription, archaic hominin introgression and immunity, all jointly shaped by the environment. This has implications for the application of genomic medicine, both in critically understudied Indonesia and globally, and will allow a better understanding of the interacting roles of genomic and environmental factors shaping molecular and complex phenotypes.

genomics↗

bioRxiv2018

Widespread conservation of chromatin accessibility patterns and transcription factor binding in human and chimpanzee induced pluripotent stem cells

Changes in gene regulation have been shown to contribute to phenotypic differences between closely related species, most notably in primates. It is likely that a subset of inter-species regulatory differences can be explained by changes in chromatin accessibility and transcription factor binding, yet there is a paucity of comparative data sets with which to investigate this. Using ATAC-seq, we profiled genome-wide chromatin accessibility in a matched set of 6 human and 6 chimpanzee (Pan troglodytes, our closest living relative) induced pluripotent stem cells from which we have previously collected gene expression data. We examined chromatin accessibility patterns near 20,745 orthologous transcriptions start sites and used a footprinting algorithm to predict transcription factor binding activity in each species. We found that the majority of chromatin accessibility patterns and transcription factor activity are conserved between these two closely related species. Interestingly, interspecies divergence in chromatin accessibility and transcription factor binding in pluripotent cells appear to contribute not to differences in the pluripotent state, but to downstream developmental processes. Put together, our findings suggest that the pluripotent state is extremely stable and potentially subject to stronger evolutionary constraint than other somatic tissues.

genomics↗