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The escape of DNA from mitochondria into the nuclear genome (nuclear mitochondrial DNA, NUMT) is an ongoing process. Although pervasively observed in eukaryotic genomes, their evolutionary trajectories in a mammal-wide context are poorly understood. The main challenge lies in the orthology assignment of NUMTs across species due to their fast evolution and chromosomal rearrangements over the past ∼200 million years. To address this issue, we systematically investigated the characteristics of NUMT insertions in 45 mammalian genomes, and established a novel, synteny-based method to accurately predict orthologous NUMTs and ascertain their evolution across mammals. With a series of comparative analyses across taxa, we revealed that NUMTs may originate from non-random regions in mtDNA, tend to locate in transposon-rich and intergenic regions, and unlikely code for functional proteins. Using our synteny-based approach, we leveraged 630 pairwise comparisons of genome-wide microsynteny and predicted the NUMT orthology relationships across 36 mammals. With the phylogenetic patterns of NUMT presence-and-absence across taxa, we constructed the ancestral state of NUMTs given the mammal tree using a coalescent method. We found support on the ancestral node of Fereuungulata within Laurasiatheria, whose subordinal relationships are still controversial. This strongly indicates that NUMT gain-and-loss over evolutionary time provides great insights into mammal evolution. However, we also demonstrated that one should be cautious when using ancestral NUMT trees to infer phylogenetic relationships. This study broadens our knowledge on NUMT insertion and evolution in mammalian genomes and highlights the merit of NUMTs as alternative genetic markers in phylogenetic inference.

Since its initial release in 2000, the human reference genome has covered only the euchromatic fraction of the genome, leaving important heterochromatic regions unfinished. Addressing the remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium presents a complete 3.055 billion–base pair sequence of a human genome, T2T-CHM13, that includes gapless assemblies for all chromosomes except Y, corrects errors in the prior references, and introduces nearly 200 million base pairs of sequence containing 1956 gene predictions, 99 of which are predicted to be protein coding. The completed regions include all centromeric satellite arrays, recent segmental duplications, and the short arms of all five acrocentric chromosomes, unlocking these complex regions of the genome to variational and functional studies.

Sea turtles represent an ancient lineage of marine vertebrates that evolved from terrestrial ancestors over 100 Mya. The genomic basis of the unique physiological and ecological traits enabling these species to thrive in diverse marine habitats remains largely unknown. Additionally, many populations have drastically declined due to anthropogenic activities over the past two centuries, and their recovery is a high global conservation priority. We generated and analyzed high-quality reference genomes for the leatherback (Dermochelys coriacea) and green (Chelonia mydas) turtles, representing the two extant sea turtle families. These genomes are highly syntenic and homologous, but localized regions of noncollinearity were associated with higher copy numbers of immune, zinc-finger, and olfactory receptor (OR) genes in green turtles, with ORs related to waterborne odorants greatly expanded in green turtles. Our findings suggest that divergent evolution of these key gene families may underlie immunological and sensory adaptations assisting navigation, occupancy of neritic versus pelagic environments, and diet specialization. Reduced collinearity was especially prevalent in microchromosomes, with greater gene content, heterozygosity, and genetic distances between species, supporting their critical role in vertebrate evolutionary adaptation. Finally, diversity and demographic histories starkly contrasted between species, indicating that leatherback turtles have had a low yet stable effective population size, exhibit extremely low diversity compared with other reptiles, and harbor a higher genetic load compared with green turtles, reinforcing concern over their persistence under future climate scenarios. These genomes provide invaluable resources for advancing our understanding of evolution and conservation best practices in an imperiled vertebrate lineage. This work was completed in part with resources provided by the University of Massachusetts' Green High Performance Computing Cluster (GHPCC). Funding was provided by the University of Massachusetts Amherst, NSF-IOS (grant #1904439 to L.M.K.), NOAA-Fisheries, National Research Council postdoctoral fellowship program to L.M.K., VGP, Rockefeller University, to E.D.J., HHMI to E.D.J., the Sanger Institute, Max-Planck-Gesellschaft, and grant contributions from Tom Gilbert, Paul Flicek, R.W.M., Karen A. Bjorndal, Alan B. Bolten, Ed Braun, N.J.G., T.M.-B., and A.F.S. We acknowledge CONICYT-DAAD for scholarship support to T.C.-V., the São Paulo Research Foundation to E.K.S.R.–FAPESP (grant #2020/10372-6). BeGenDiv is partially funded by the German Federal Ministry of Education and Research (BMbF, Förderkennzeichen 033W034A). The work of F.T.-N. and P.M. was supported by the Intramural Research Program of the National Library of Medicine, NIH. The work of M.P. was partially funded through the Federal Ministry of Education and Research (grant 01IS18026C). H.P. was supported by a Formació de Personal Investigador fellowship from Generalitat de Catalunya (FI_B100131). M.K. was supported by “la Caixa” Foundation (ID 100010434; code LCF/BQ/PR19/11700002), the Vienna Science and Technology Fund (WWTF), and the City of Vienna (VRG20-001). Funding for green turtle resequencing was provided by a Welsh Government Sêr Cymru II and the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 663830-BU115 and the Sea Turtle Conservancy, Florida Sea Turtle Grants Program (17-033R).

High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are available for only a few non-microbial species1,2,3,4. To address this issue, the international Genome 10K (G10K) consortium5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling highly accurate and nearly complete reference genomes. Here we present lessons learned from generating assemblies for 16 species that represent six major vertebrate lineages. We confirm that long-read sequencing technologies are essential for maximizing genome quality, and that unresolved complex repeats and haplotype heterozygosity are major sources of assembly error when not handled correctly. Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries. These include the identification of many false gene duplications, increases in gene sizes, chromosome rearrangements that are specific to lineages, a repeated independent chromosome breakpoint in bat genomes, and a canonical GC-rich pattern in protein-coding genes and their regulatory regions. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an international effort to generate high-quality, complete reference genomes for all of the roughly 70,000 extant vertebrate species and to help to enable a new era of discovery across the life sciences., We thank them for their permission to publish. A.R., S.K., B.P.W. and A.M.P. were supported by the Intramural Research Program of the NHGRI, NIH (1ZIAHG200398). A.R. was also supported by the Korea Health Technology R&D Project through KHIDI, funded by the Ministry of Health & Welfare, Republic of Korea (HI17C2098). S.A.M., I.B. and R.D. were supported by Wellcome Trust grant WT207492; W.C., M. Smith, Z.N., Y.S., J.C., S. Pelan, J.T., A.T., J.W. and Kerstin Howe by WT206194; L.H., F.M., Kevin Howe and P. Flicek by WT108749/Z/15/Z, WT218328/B/19/Z and the European Molecular Biology Laboratory. O.F. and E.D.J. were supported by Howard Hughes Medical Institute and Rockefeller University start-up funds for this project. J.D. and H.A.L. were supported by the Robert and Rosabel Osborne Endowment. M.U.-S. received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement (750747). F.T.-N., J. Hoffman, P. Masterson and K.C. were supported by the Intramural Research Program of the NLM, NIH. C.L., B.J.K., J. Kim and H.K. were supported by the Marine Biotechnology Program of KIMST, funded by the Ministry of Ocean and Fisheries, Republic of Korea (20180430). M.C. was supported by Sloan Research Fellowship (FG-2020-12932). S.C.V. was funded by a Max Planck Research Group award from the Max Planck Society, and a Human Frontiers Science Program (HFSP) Research grant (RGP0058/2016). T.M.L., W.E.J. and the Canada lynx genome were funded by the Maine Department of Inland Fisheries & Wildlife (F11AF01099), including when W.E.J. held a National Research Council Research Associateship Award at the Walter Reed Army Institute of Research (WRAIR). C.B. was supported by the NSF (1457541 and 1456612). D.B. was funded by The University of Queensland (HFSP - RGP0030/2015). D.I. was supported by Science Exchange Inc. (Palo Alto, CA). H.W.D. was supported by NSF grants (OPP-0132032 ICEFISH 2004 Cruise, PLR-1444167 and OPP-1955368) and the Marine Science Center at Northeastern University (416). G.J.P.N. and the thorny skate genome were funded by Lenfest Ocean Program (30884). M.P. was funded by the German Federal Ministry of Education and Research (01IS18026C). M. Malinsky was supported by an EMBO fellowship (ALTF 456-2016). The following authors’ contributions were supported by the NIH: S. Selvaraj (R44HG008118); C.V.M., S.R.F., P.V.L. (R21 DC014432/DC/NIDCD); K.D.M. (R01GM130691); H.C. (5U41HG002371-19); M.D. (U41HG007234); and B.P. (R01HG010485). D.G. was supported by the National Key Research and Development Program of China (2017YFC1201201, 2018YFC0910504 and 2017YFC0907503). F.O.A. was supported by Al-Gannas Qatari Society and The Cultural Village Foundation-Katara, Doha, State of Qatar and Monash University Malaysia. C.T. was supported by The Rockefeller University. M. Hiller was supported by the LOEWE-Centre for Translational Biodiversity Genomics (TBG) funded by the Hessen State Ministry of Higher Education, Research and the Arts (HMWK). H.C. was supported by the NHGRI (5U41HG002371-19). R.H.S.K. was funded by the Max Planck Society with computational resources at the bwUniCluster and BinAC funded by the Ministry of Science, Research and the Arts Baden-Württemberg and the Universities of the State of Baden-Württemberg, Germany (bwHPC-C5). B.V. was supported by the Biomedical Research Council of A*STAR, Singapore. T.M.-B. was funded by the European Research Council under the European Union’s Horizon 2020 research and innovation programme (864203), MINECO/FEDER, UE (BFU2017-86471-P), Unidad de Excelencia María de Maeztu, AEI (CEX2018-000792-M), a Howard Hughes International Early Career award, Obra Social “La Caixa” and Secretaria d’Universitats i Recerca and CERCA Programme del Departament d’Economia i Coneixement de la Generalitat de Catalunya (GRC 2017 SGR 880). E.C.T. was supported by the European Research Council (ERC-2012-StG311000) and an Irish Research Council Laureate Award. M.T.P.G. was supported by an ERC Consolidator Award 681396-Extinction Genomics, and a Danish National Research Foundation Center Grant (DNRF143). T.W. was supported by the NSF (1458652). J. M. Graves was supported by the Australian Research Council (CEO561477). E.W.M. was partially supported by the German Federal Ministry of Education and Research (01IS18026C). Complementary sequencing support for the Anna’s hummingbird and several genomes was provided by Pacific Biosciences, Bionano Genomics, Dovetail Genomics, Arima Genomics, Phase Genomics, 10X Genomics, NRGene, Oxford Nanopore Technologies, Illumina, and DNAnexus. All other sequencing and assembly were conducted at the Rockefeller University, Sanger Institute, and Max Planck Institute Dresden genome labs. Part of this work used the computational resources of the NIH HPC Biowulf cluster (https://hpc.nih.gov). We acknowledge funding from the Wellcome Trust (108749/Z/15/Z) and the European Molecular Biology Laboratory., With funding from the Spanish government through the "Severo Ochoa Centre of Excellence" accreditation (CEX2018-000792-M).

Homologous chromosomes in the diploid genome are thought to contain equivalent genetic information, but this common concept has not been fully verified in animal genomes with high heterozygosity. Here we report a near-complete, haplotype-phased, genome assembly of the pearl oyster, Pinctada fucata, using hi-fidelity (HiFi) long reads and chromosome conformation capture data. This assembly includes 14 pairs of long scaffolds (>38 Mb) corresponding to chromosomes (2n = 28). The accuracy of the assembly, as measured by an analysis of k-mers, is estimated to be 99.99997%. Moreover, the haplotypes contain 95.2% and 95.9%, respectively, complete and single-copy BUSCO genes, demonstrating the high quality of the assembly. Transposons comprise 53.3% of the assembly and are a major contributor to structural variations. Despite overall collinearity between haplotypes, one of the chromosomal scaffolds contains megabase-scale non-syntenic regions, which necessarily have never been detected and resolved in conventional haplotype-merged assemblies. These regions encode expanded gene families of NACHT, DZIP3/hRUL138-like HEPN, and immunoglobulin domains, multiplying the immunity gene repertoire, which we hypothesize is important for the innate immune capability of pearl oysters. The pearl oyster genome provides insight into remarkable haplotype diversity in animals.

Motivation Long tandem repeat expansions of more than 1000 nt have been suggested to be associated with diseases, but remain largely unexplored in individual human genomes because read lengths have been too short. However, new long-read sequencing technologies can produce single reads of 10 000 nt or more that can span such repeat expansions, although these long reads have high error rates, of 10–20%, which complicates the detection of repetitive elements. Moreover, most traditional algorithms for finding tandem repeats are designed to find short tandem repeats ( Results Here, we report an efficient algorithm for solving this problem that takes advantage of the length of the repeat. Namely, a long tandem repeat has hundreds or thousands of approximate copies of the repeated unit, so despite the error rate, many short k-mers will be error-free in many copies of the unit. We exploited this characteristic to develop a method for first estimating regions that could contain a tandem repeat, by analyzing the k-mer frequency distributions of fixed-size windows across the target read, followed by an algorithm that assembles the k-mers of a putative region into the consensus repeat unit by greedily traversing a de Bruijn graph. Experimental results indicated that the proposed algorithm largely outperformed Tandem Repeats Finder, a widely used program for finding tandem repeats, in terms of sensitivity. Availability and implementation https://github.com/morisUtokyo/mTR.

Estimation of optical aberrations from volumetric intensity images is a key step in sensorless adaptive optics for 3D microscopy. Recent approaches based on deep learning promise accurate results at fast processing speeds. However, collecting ground truth microscopy data for training the network is typically very difficult or even impossible thereby limiting this approach in practice. Here, we demonstrate that neural networks trained only on simulated data yield accurate predictions for real experimental images. We validate our approach on simulated and experimental datasets acquired with two different microscopy modalities and also compare the results to non-learned methods. Additionally, we study the predictability of individual aberrations with respect to their data requirements and find that the symmetry of the wavefront plays a crucial role. Finally, we make our implementation freely available as open source software in Python.

Variant calling has been widely used for genotyping and for improving the consensus accuracy of long-read assemblies. Variant calls are commonly hard-filtered with user-defined cutoffs. However, it is impossible to define a single set of optimal cutoffs, as the calls heavily depend on the quality of the reads, the variant caller of choice and the quality of the unpolished assembly. Here, we introduce Merfin, a k-mer based variant-filtering algorithm for improved accuracy in genotyping and genome assembly polishing. Merfin evaluates each variant based on the expected k-mer multiplicity in the reads, independently of the quality of the read alignment and variant caller's internal score. Merfin increased the precision of genotyped calls in several benchmarks, improved consensus accuracy and reduced frameshift errors when applied to human and nonhuman assemblies built from Pacific Biosciences HiFi and continuous long reads or Oxford Nanopore reads, including the first complete human genome. Moreover, we introduce assembly quality and completeness metrics that account for the expected genomic copy numbers.

Funder: Howard Hughes Medical Institute, Funder: National Science Foundation; Grant(s): DBI:IIBR:CAREER #1943371, A global international initiative such as the Earth BioGenome Project (EBP) requires both agreement and coordination on standards to ensure that the collective effort generates rapid progress towards its goals. To this end, the EBP initiated five technical standards committees comprising volunteer members from the global genomics scientific community: Sample Collection and Processing, Sequencing and Assembly, Annotation, Analysis, and, IT and Informatics. The current versions of the resulting standards documents are available on the EBP website, with the recognition that opportunities, technologies and challenges may improve or change in the future requiring flexibility for the EBP to meet its goals. Here, we describe some highlights from the proposed standards, and areas where additional challenges will need to be met.

Some organisms in nature have developed the ability to enter a state of suspended metabolism called cryptobiosis1when environmental conditions are unfavorable. This state-transition requires execution of a combination of genetic and biochemical pathways1,2,3that enable the organism to survive for prolonged periods. Recently, nematode individuals have been reanimated from Siberian permafrost after remaining in cryptobiosis. Preliminary analysis indicates that these nematodes belong to the generaPanagrolaimusandPlectus4. Here, we present precise radiocarbon dating indicating that thePanagrolaimusindividuals have remained in cryptobiosis since the late Pleistocene (∼46,000 years). Phylogenetic inference based on our genome assembly and a detailed morphological analysis demonstrate that they belong to an undescribed species, which we namedPanagrolaimus n. sp. Comparative genome analysis revealed that the molecular toolkit for cryptobiosis inPanagrolaimus n. sp. and inC. elegansis partly orthologous. We show that biochemical mechanisms employed by these two species to survive desiccation and freezing under laboratory conditions are similar. Our experimental evidence also reveals thatC. elegansdauer larvae can remain viable for longer periods in suspended animation than previously reported. Altogether, our findings demonstrate that nematodes evolved mechanisms potentially allowing them to suspend life over geological time scales.

G.M.H. was funded by a UCD Ad Astra Fellowship. C.L. was funded by a UCD Ad Astra studentship. L.R. was funded by an SFI Centre for Research Training in Genomics Data Science grant (18/CRT/6214). L.M.D. was supported in part by NSF awards 1838273 and 2032063. E.C.T. and T.L. were funded by an SFI Frontiers for the Future Programme grant (19/FFP/6790). Relationships among laurasiatherian clades represent one of the most highly disputed topics in mammalian phylogeny. In this study, we attempt to disentangle laurasiatherian interordinal relationships using two independent genome-level approaches: (1) quantifying retrotransposon presence/absence patterns, and (2) comparisons of exon datasets at the levels of nucleotides and amino acids. The two approaches revealed contradictory phylogenetic signals, possibly due to a high level of ancestral incomplete lineage sorting. The positions of Eulipotyphla and Chiroptera as the first and second earliest divergences were consistent across the approaches. However, the phylogenetic relationships of Perissodactyla, Cetartiodactyla, and Ferae, were contradictory. While retrotransposon insertion analyses suggest a clade with Cetartiodactyla and Ferae, the exon dataset favoured Cetartiodactyla and Perissodactyla. Future analyses of hitherto unsampled laurasiatherian lineages and synergistic analyses of retrotransposon insertions, exon and conserved intron/intergenic sequences might unravel the conflicting patterns of relationships in this major mammalian clade. Publisher PDF