Transcriptome innovations in primates revealed by single-molecule long-read sequencing.

Transcriptomic diversity greatly contributes to the fundamentals of disease, lineage-specific biology, and environmental adaptation. However, much of the actual isoform repertoire contributing to shaping primate evolution remains unknown. Here, we combined deep long- and short-read sequencing complemented with mass spectrometry proteomics in a panel of lymphoblastoid cell lines (LCLs) from human, three other great apes, and rhesus macaque, producing the largest full-length isoform catalog in primates to date. Around half of the captured isoforms are not annotated in their reference genomes, significantly expanding the gene models in primates. Furthermore, our comparative analyses unveil hundreds of transcriptomic innovations and isoform usage changes related to immune function and immunological disorders. The confluence of these evolutionary innovations with signals of positive selection and their limited impact in the proteome points to changes in alternative splicing in genes involved in immune response as an important target of recent regulatory divergence in primates.

The genomics of ecological flexibility, large brains, and long lives in capuchin monkeys revealed with fecalFACS.

Ecological flexibility, extended lifespans, and large brains have long intrigued evolutionary biologists, and comparative genomics offers an efficient and effective tool for generating new insights into the evolution of such traits. Studies of capuchin monkeys are particularly well situated to shed light on the selective pressures and genetic underpinnings of local adaptation to diverse habitats, longevity, and brain development. Distributed widely across Central and South America, they are inventive and extractive foragers, known for their sensorimotor intelligence. Capuchins have among the largest relative brain size of any monkey and a lifespan that exceeds 50 y, despite their small (3 to 5 kg) body size. We assemble and annotate a de novo reference genome for Cebus imitator Through high-depth sequencing of DNA derived from blood, various tissues, and feces via fluorescence-activated cell sorting (fecalFACS) to isolate monkey epithelial cells, we compared genomes of capuchin populations from tropical dry forests and lowland rainforests and identified population divergence in genes involved in water balance, kidney function, and metabolism. Through a comparative genomics approach spanning a wide diversity of mammals, we identified genes under positive selection associated with longevity and brain development. Additionally, we provide a technological advancement in the use of noninvasive genomics for studies of free-ranging mammals. Our intra- and interspecific comparative study of capuchin genomics provides insights into processes underlying local adaptation to diverse and physiologically challenging environments, as well as the molecular basis of brain evolution and longevity.

Genetic data from the extinct giant rat from Tenerife (Canary Islands) points to a recent divergence from mainland relatives.

Evolution of vertebrate endemics in oceanic islands follows a predictable pattern, known as the island rule, according to which gigantism arises in originally small-sized species and dwarfism in large ones. Species of extinct insular giant rodents are known from all over the world. In the Canary Islands, two examples of giant rats, †Canariomys bravoi and †Canariomys tamarani, endemic to Tenerife and Gran Canaria, respectively, disappeared soon after human settlement. The highly derived morphological features of these insular endemic rodents hamper the reconstruction of their evolutionary histories. We have retrieved partial nuclear and mitochondrial data from †C. bravoi and used this information to explore its evolutionary affinities. The resulting dated phylogeny confidently places †C. bravoi within the African grass rat clade (Arvicanthis niloticus). The estimated divergence time, 650 000 years ago (95% higher posterior densities: 373 000-944 000), points toward an island colonization during the Günz-Mindel interglacial stage. †Canariomys bravoi ancestors would have reached the island via passive rafting and then underwent a yearly increase of mean body mass calculated between 0.0015 g and 0.0023 g; this corresponds to fast evolutionary rates (in darwins (d), ranging from 7.09 d to 2.78 d) that are well above those observed for non-insular mammals.

Variation in predicted COVID-19 risk among lemurs and lorises.

The novel coronavirus SARS-CoV-2, which in humans leads to the disease COVID-19, has caused global disruption and more than 2 million fatalities since it first emerged in late 2019. As we write, infection rates are at their highest point globally and are rising extremely rapidly in some areas due to more infectious variants. The primary target of SARS-CoV-2 is the cellular receptor angiotensin-converting enzyme-2 (ACE2). Recent sequence analyses of the ACE2 gene predict that many nonhuman primates are also likely to be highly susceptible to infection. However, the anticipated risk is not equal across the Order. Furthermore, some taxonomic groups show high ACE2 amino acid conservation, while others exhibit high variability at this locus. As an example of the latter, analyses of strepsirrhine primate ACE2 sequences to date indicate large variation among lemurs and lorises compared to other primate clades despite low sampling effort. Here, we report ACE2 gene and protein sequences for 71 individual strepsirrhines, spanning 51 species and 19 genera. Our study reinforces previous results while finding additional variability in other strepsirrhine species, and suggests several clades of lemurs have high potential susceptibility to SARS-CoV-2 infection. Troublingly, some species, including the rare and endangered aye-aye (Daubentonia madagascariensis), as well as those in the genera Avahi and Propithecus, may be at high risk. Given that lemurs are endemic to Madagascar and among the primates at highest risk of extinction globally, further understanding of the potential threat of COVID-19 to their health should be a conservation priority. All feasible actions should be taken to limit their exposure to SARS-CoV-2.

Targeted conservation genetics of the endangered chimpanzee.

Populations of the common chimpanzee (Pan troglodytes) are in an impending risk of going extinct in the wild as a consequence of damaging anthropogenic impact on their natural habitat and illegal pet and bushmeat trade. Conservation management programmes for the chimpanzee have been established outside their natural range (ex situ), and chimpanzees from these programmes could potentially be used to supplement future conservation initiatives in the wild (in situ). However, these programmes have often suffered from inadequate information about the geographical origin and subspecies ancestry of the founders. Here, we present a newly designed capture array with ~60,000 ancestry informative markers used to infer ancestry of individual chimpanzees in ex situ populations and determine geographical origin of confiscated sanctuary individuals. From a test panel of 167 chimpanzees with unknown origins or subspecies labels, we identify 90 suitable non-admixed individuals in the European Association of Zoos and Aquaria (EAZA) Ex situ Programme (EEP). Equally important, another 46 individuals have been identified with admixed subspecies ancestries, which therefore over time, should be naturally phased out of the breeding populations. With potential for future re-introduction to the wild, we determine the geographical origin of 31 individuals that were confiscated from the illegal trade and demonstrate the promises of using non-invasive sampling in future conservation action plans. Collectively, our genomic approach provides an exemplar for ex situ management of endangered species and offers an efficient tool in future in situ efforts to combat the illegal wildlife trade.

Transcriptome and genome sequencing uncovers functional variation in humans.

Genome sequencing projects are discovering millions of genetic variants in humans, and interpretation of their functional effects is essential for understanding the genetic basis of variation in human traits. Here we report sequencing and deep analysis of messenger RNA and microRNA from lymphoblastoid cell lines of 462 individuals from the 1000 Genomes Project--the first uniformly processed high-throughput RNA-sequencing data from multiple human populations with high-quality genome sequences. We discover extremely widespread genetic variation affecting the regulation of most genes, with transcript structure and expression level variation being equally common but genetically largely independent. Our characterization of causal regulatory variation sheds light on the cellular mechanisms of regulatory and loss-of-function variation, and allows us to infer putative causal variants for dozens of disease-associated loci. Altogether, this study provides a deep understanding of the cellular mechanisms of transcriptome variation and of the landscape of functional variants in the human genome.

Specific small-RNA signatures in the amygdala at premotor and motor stages of Parkinson's disease revealed by deep sequencing analysis.

MOTIVATION: Most computational tools for small non-coding RNAs (sRNA) sequencing data analysis focus in microRNAs (miRNAs), overlooking other types of sRNAs that show multi-mapping hits. Here, we have developed a pipeline to non-redundantly quantify all types of sRNAs, and extract patterns of expression in biologically defined groups. We have used our tool to characterize and profile sRNAs in post-mortem brain samples of control individuals and Parkinson's disease (PD) cases at early-premotor and late-symptomatic stages. RESULTS: Clusters of co-expressed sRNAs mapping onto tRNAs significantly separated premotor and motor cases from controls. A similar result was obtained using a matrix of miRNAs slightly varying in sequence (isomiRs). The present framework revealed sRNA alterations at premotor stages of PD, which might reflect initial pathogenic perturbations. This tool may be useful to discover sRNA expression patterns linked to different biological conditions. AVAILABILITY AND IMPLEMENTATION: The full code is available at http://github.com/lpantano/seqbuster CONTACT: lpantano@hsph.harvard.edu or eulalia.marti@crg.eu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Y chromosome sequence and epigenomic reconstruction across human populations.

Recent advances in long-read sequencing technologies have allowed the generation and curation of more complete genome assemblies, enabling the analysis of traditionally neglected chromosomes, such as the human Y chromosome (chrY). Native DNA was sequenced on a MinION Oxford Nanopore Technologies sequencing device to generate genome assemblies for seven major chrY human haplogroups. We analyzed and compared the chrY enrichment of sequencing data obtained using two different selective sequencing approaches: adaptive sampling and flow cytometry chromosome sorting. We show that adaptive sampling can produce data to create assemblies comparable to chromosome sorting while being a less expensive and time-consuming technique. We also assessed haplogroup-specific structural variants, which would be otherwise difficult to study using short-read sequencing data only. Finally, we took advantage of this technology to detect and profile epigenetic modifications among the considered haplogroups. Altogether, we provide a framework to study complex genomic regions with a simple, fast, and affordable methodology that could be applied to larger population genomics datasets.

Past Connectivity but Recent Inbreeding in Cross River Gorillas Determined Using Whole Genomes from Single Hairs.

The critically endangered western gorillas (Gorilla gorilla) are divided into two subspecies: the western lowland (G. g. gorilla) and the Cross River (G. g. diehli) gorilla. Given the difficulty in sampling wild great ape populations and the small estimated size of the Cross River gorilla population, only one whole genome of a Cross River gorilla has been sequenced to date, hindering the study of this subspecies at the population level. In this study, we expand the number of whole genomes available for wild western gorillas, generating 41 new genomes (25 belonging to Cross River gorillas) using single shed hairs collected from gorilla nests. By combining these genomes with publicly available wild gorilla genomes, we confirm that Cross River gorillas form three population clusters. We also found little variation in genome-wide heterozygosity among them. Our analyses reveal long runs of homozygosity (>10 Mb), indicating recent inbreeding in Cross River gorillas. This is similar to that seen in mountain gorillas but with a much more recent bottleneck. We also detect past gene flow between two Cross River sites, Afi Mountain Wildlife Sanctuary and the Mbe Mountains. Furthermore, we observe past allele sharing between Cross River gorillas and the northern western lowland gorilla sites, as well as with the eastern gorilla species. This is the first study using single shed hairs from a wild species for whole genome sequencing to date. Taken together, our results highlight the importance of implementing conservation measures to increase connectivity among Cross River gorilla sites.

Annotation of primate miRNAs by high throughput sequencing of small RNA libraries.

BACKGROUND: In addition to genome sequencing, accurate functional annotation of genomes is required in order to carry out comparative and evolutionary analyses between species. Among primates, the human genome is the most extensively annotated. Human miRNA gene annotation is based on multiple lines of evidence including evidence for expression as well as prediction of the characteristic hairpin structure. In contrast, most miRNA genes in non-human primates are annotated based on homology without any expression evidence. We have sequenced small-RNA libraries from chimpanzee, gorilla, orangutan and rhesus macaque from multiple individuals and tissues. Using patterns of miRNA expression in conjunction with a model of miRNA biogenesis we used these high-throughput sequencing data to identify novel miRNAs in non-human primates. RESULTS: We predicted 47 new miRNAs in chimpanzee, 240 in gorilla, 55 in orangutan and 47 in rhesus macaque. The algorithm we used was able to predict 64% of the previously known miRNAs in chimpanzee, 94% in gorilla, 61% in orangutan and 71% in rhesus macaque. We therefore added evidence for expression in between one and five tissues to miRNAs that were previously annotated based only on homology to human miRNAs. We increased from 60 to 175 the number miRNAs that are located in orthologous regions in humans and the four non-human primate species studied here. CONCLUSIONS: In this study we provide expression evidence for homology-based annotated miRNAs and predict de novo miRNAs in four non-human primate species. We increased the number of annotated miRNA genes and provided evidence for their expression in four non-human primates. Similar approaches using different individuals and tissues would improve annotation in non-human primates and allow for further comparative studies in the future.

Recovering the genomes hidden in museum wet collections.

Natural history museums hold vast collections of biomaterials. The collections in museums, often painstakingly sampled, are largely unexplored treasures that may help us better understand biodiversity on the planet. Museum collections can provide a unique window into the past of species long gone or currently declining due to human activity. From a molecular perspective, however, many museum samples are stored under conditions that hasten the damage of DNA, RNA and proteins. For example, samples in wet collections are those stored in liquid preservatives, typically ethanol. These ethanol-preserved tissues are often, although not always, formalin-fixed prior to storage, which may damage DNA. In this and recent issues of Molecular Ecology Resources, Straube et al (2021), O'Connell et al (2021) and Hahn et al (2022) explore different types of specimens from museum wet collections as new sources of DNA for scientific studies. All three articles found that for wet museum collections, overall specimen condition mattered most for recovering high-quality genomic DNA.

Current challenges and best practices for cell-free long RNA biomarker discovery.

The analysis of biomarkers in biological fluids, also known as liquid biopsies, is seen with great potential to diagnose complex diseases such as cancer with a high sensitivity and minimal invasiveness. Although it can target any biomolecule, most liquid biopsy studies have focused on circulating nucleic acids. Historically, studies have aimed at the detection of specific mutations on cell-free DNA (cfDNA), but recently, the study of cell-free RNA (cfRNA) has gained traction. Since 2020, a handful of cfDNA tests have been approved for therapy selection by the FDA, however, no cfRNA tests are approved to date. One of the main drawbacks in the field of RNA-based liquid biopsies is the low reproducibility of the results, often caused by technical and biological variability, a lack of standardized protocols and insufficient cohorts. In this review, we will identify the main challenges and biases introduced during the different stages of biomarker discovery in liquid biopsies with cfRNA and propose solutions to minimize them.

Y-chromosome target enrichment reveals rapid expansion of haplogroup R1b-DF27 in Iberia during the Bronze Age transition.

The Y chromosome can yield a unique perspective into the study of human demographic history. However, due to the repetitive nature of part of its sequence, only a small set of regions are suitable for variant calling and discovery from short-read sequencing data. These regions combined represent 8.9 Mbp or 0.14% of a diploid human genome. Consequently, investing in whole-genome sequencing to resolve Y-chromosome questions is poorly efficient. Here we use, as an alternative, target enrichment technology to greatly increase sequencing effectiveness, validating and applying the technique to 181 males, for 162 of whom we obtained a positive result. Additionally, 75 samples sequenced for the whole genome were also included, for a total sample size of 237. These samples were chosen for their Y chromosome haplogroup: R1b-DF27. In the context of European populations, and particularly in Iberia, this haplogroup stands out for its high frequency and its demographic history. Current evidence indicates that the diffusion of this haplogroup is related to the population movements that mark the cultural Bronze Age transition, making it remarkably interesting for population geneticists. The results of this study show the effects of the rapid radiation of the haplogroup in Spain, as even with the higher discriminating power of whole sequences, most haplotypes still fall within the R1b-DF27* paragroup rather than in the main derived branches. However, we were able to refine the ISOGG 2019-2020 phylogeny, and its two main subbranches, namely L176.2 and Z272, which present geographical differentiation between the Atlantic and Mediterranean coasts of Iberia.

Insights from the rescue and breeding management of Cuvier's gazelle (Gazella cuvieri) through whole-genome sequencing.

Captive breeding programmes represent the most intensive type of ex situ population management for threatened species. One example is the Cuvier's gazelle programme that started in 1975 with only four founding individuals, and after more than four decades of management in captivity, a reintroduction effort was undertaken in Tunisia in 2016, to establish a population in an area historically included within its range. Here, we aim to determine the genetic consequences of this reintroduction event by assessing the genetic diversity of the founder stock as well as of their descendants. We present the first whole-genome sequencing dataset of 30 Cuvier's gazelles including captive-bred animals, animals born in Tunisia after a reintroduction and individuals from a genetically unrelated Moroccan population. Our analyses revealed no difference between the founder and the offspring cohorts in genome-wide heterozygosity and inbreeding levels, and in the amount and length of runs of homozygosity. The captive but unmanaged Moroccan gazelles have the lowest genetic diversity of all genomes analysed. Our findings demonstrate that the Cuvier's gazelle captive breeding programme can serve as source populations for future reintroductions of this species. We believe that this study can serve as a starting point for global applications of genomics to the conservation plan of this species.

Salmonella enterica from a soldier from the 1652 siege of Barcelona (Spain) supports historical transatlantic epidemic contacts.

Ancient pathogen genomics is an emerging field allowing reconstruction of past epidemics. The demise of post-contact American populations may, at least in part, have been caused by paratyphoid fever brought by Europeans. We retrieved genome-wide data from two Spanish soldiers who were besieging the city of Barcelona in 1652, during the Reapers' War. Their ancestry derived from the Basque region and Sardinia, respectively, (at that time, this island belonged to the Spanish kingdom). Despite the proposed plague epidemic, we could not find solid evidence for the presence of the causative plague agent in these individuals. However, we retrieved from one individual a substantial fraction of the Salmonella enterica serovar Paratyphi C lineage linked to paratyphoid fever in colonial period Mexico. Our results support a growing body of evidence that Paratyphi C enteric fever was more prevalent in Europe and the Americas in the past than it is today.

Genomic Analysis of 18th-Century Kazakh Individuals and Their Oral Microbiome.

The Asian Central Steppe, consisting of current-day Kazakhstan and Russia, has acted as a highway for major migrations throughout history. Therefore, describing the genetic composition of past populations in Central Asia holds value to understanding human mobility in this pivotal region. In this study, we analyse paleogenomic data generated from five humans from Kuygenzhar, Kazakhstan. These individuals date to the early to mid-18th century, shortly after the Kazakh Khanate was founded, a union of nomadic tribes of Mongol Golden Horde and Turkic origins. Genomic analysis identifies that these individuals are admixed with varying proportions of East Asian ancestry, indicating a recent admixture event from East Asia. The high amounts of DNA from the anaerobic Gram-negative bacteria Tannerella forsythia, a periodontal pathogen, recovered from their teeth suggest they may have suffered from periodontitis disease. Genomic analysis of this bacterium identified recently evolved virulence and glycosylation genes including the presence of antibiotic resistance genes predating the antibiotic era. This study provides an integrated analysis of individuals with a diet mostly based on meat (mainly horse and lamb), milk, and dairy products and their oral microbiome.

Variation in predicted COVID-19 risk among lemurs and lorises.

The novel coronavirus SARS-CoV-2, which in humans leads to the disease COVID-19, has caused global disruption and more than 1.5 million fatalities since it first emerged in late 2019. As we write, infection rates are currently at their highest point globally and are rising extremely rapidly in some areas due to more infectious variants. The primary viral target is the cellular receptor angiotensin-converting enzyme-2 (ACE2). Recent sequence analyses of the ACE2 gene predicts that many nonhuman primates are also likely to be highly susceptible to infection. However, the anticipated risk is not equal across the Order. Furthermore, some taxonomic groups show high ACE2 amino acid conservation, while others exhibit high variability at this locus. As an example of the latter, analyses of strepsirrhine primate ACE2 sequences to date indicate large variation among lemurs and lorises compared to other primate clades despite low sampling effort. Here, we report ACE2 gene and protein sequences for 71 individual strepsirrhines, spanning 51 species and 19 genera. Our study reinforces previous results and finds additional variability in other strepsirrhine species, and suggests several clades of lemurs have high potential susceptibility to SARS-CoV-2 infection. Troublingly, some species, including the rare and Endangered aye-aye (Daubentonia madagascariensis), as well as those in the genera Avahi and Propithecus, may be at high risk. Given that lemurs are endemic to Madagascar and among the primates at highest risk of extinction globally, further understanding of the potential threat of COVID-19 to their health should be a conservation priority. All feasible actions should be taken to limit their exposure to SARS-CoV-2.

Maximizing the acquisition of unique reads in noninvasive capture sequencing experiments.

Noninvasive samples as a source of DNA are gaining interest in genomic studies of endangered species. However, their complex nature and low endogenous DNA content hamper the recovery of good quality data. Target capture has become a productive method to enrich the endogenous fraction of noninvasive samples, such as faeces, but its sensitivity has not yet been extensively studied. Coping with faecal samples with an endogenous DNA content below 1% is a common problem when prior selection of samples from a large collection is not possible. However, samples classified as unfavourable for target capture sequencing might be the only representatives of unique specific geographical locations, or to answer the question of interest. To explore how library complexity may be increased without repeating DNA extractions and generating new libraries, in this study we captured the exome of 60 chimpanzees (Pan troglodytes) using faecal samples with very low proportions of endogenous content (<1%). Our results indicate that by performing additional hybridizations of the same libraries, the molecular complexity can be maintained to achieve higher coverage. Also, whenever possible, the starting DNA material for capture should be increased. Finally, we specifically calculated the sequencing effort needed to avoid exhausting the library complexity of enriched faecal samples with low endogenous DNA content. This study provides guidelines, schemes and tools for laboratories facing the challenges of working with noninvasive samples containing extremely low amounts of endogenous DNA.

Comprehensive identification of somatic nucleotide variants in human brain tissue.

BACKGROUND: Post-zygotic mutations incurred during DNA replication, DNA repair, and other cellular processes lead to somatic mosaicism. Somatic mosaicism is an established cause of various diseases, including cancers. However, detecting mosaic variants in DNA from non-cancerous somatic tissues poses significant challenges, particularly if the variants only are present in a small fraction of cells. RESULTS: Here, the Brain Somatic Mosaicism Network conducts a coordinated, multi-institutional study to examine the ability of existing methods to detect simulated somatic single-nucleotide variants (SNVs) in DNA mixing experiments, generate multiple replicates of whole-genome sequencing data from the dorsolateral prefrontal cortex, other brain regions, dura mater, and dural fibroblasts of a single neurotypical individual, devise strategies to discover somatic SNVs, and apply various approaches to validate somatic SNVs. These efforts lead to the identification of 43 bona fide somatic SNVs that range in variant allele fractions from ~ 0.005 to ~ 0.28. Guided by these results, we devise best practices for calling mosaic SNVs from 250× whole-genome sequencing data in the accessible portion of the human genome that achieve 90% specificity and sensitivity. Finally, we demonstrate that analysis of multiple bulk DNA samples from a single individual allows the reconstruction of early developmental cell lineage trees. CONCLUSIONS: This study provides a unified set of best practices to detect somatic SNVs in non-cancerous tissues. The data and methods are freely available to the scientific community and should serve as a guide to assess the contributions of somatic SNVs to neuropsychiatric diseases.

Metagenomic analysis of a blood stain from the French revolutionary Jean-Paul Marat (1743-1793).

The French revolutionary Jean-Paul Marat (1743-1793) was assassinated in 1793 in his bathtub, where he was trying to find relief from the debilitating skin disease he was suffering from. At the time of his death, Marat was annotating newspapers, which got stained with his blood and were subsequently preserved by his sister. We extracted and sequenced DNA from the blood stain and also from another section of the newspaper, which we used for comparison. Results from the human DNA sequence analyses were compatible with a heterogeneous ancestry of Marat, with his mother being of French origin and his father born in Sardinia. Metagenomic analyses of the non-human reads uncovered the presence of fungal, bacterial and low levels of viral DNA. Relying on the presence/absence of microbial species in the samples, we could cast doubt on several putative infectious agents that have been previously hypothesised as the cause of his condition but for which we detect not a single sequencing read. Conversely, some of the species we detect are uncommon as environmental contaminants and may represent plausible infective agents. Based on all the available evidence, we hypothesize that Marat may have suffered from a fungal infection (seborrheic dermatitis), possibly superinfected with bacterial opportunistic pathogens.