Imputation and Maximum Likelihood Haplotype Refinement of Simulated Ancient Mitochondrial Genomes

Imputation and Maximum Likelihood Haplotype Refinement of Simulated Ancient Mitochondrial Genomes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Imputation and Maximum Likelihood Haplotype Refinement of Simulated Ancient Mitochondrial Genomes Nathaniel Plummer, Arianna Cozzarelli, Suhail Ghafoor, Nicola Jess Murray, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9406784/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background : Mitochondrial DNA (mtDNA) has long served as a foundational target in ancient DNA (aDNA) and palaeogenomic research, owing to its high copy number and well-resolved phylogenetic structure. Yet, external taphonomic and diagenetic factors, including burial environment, hydrolytic and oxidative damage, microbial colonization, and soil chemistry, promote molecular fragmentation. These factors complicate haplotype determination and raise uncertainty about the minimal sequencing coverage needed for reliable haplogroup assignment and phylogenetic inference. Although aDNA studies often apply thresholds between 2x and 10x depth of coverage, a systematic assessment for mtDNA quality has not yet been undertaken. Moreover, while genotype imputation is routinely employed to recover missing data from genomic DNA, its accuracy for ancient mtDNA remains largely untested. Results : Here, we compiled a reference panel of 46,791 complete human mtDNA genomes and simulated aDNA degradation across coverage depths from 0.25x to 15x (n=3500 mtDNA simulations) using gargammel . Simulated paired-end FASTQ files were processed with the EAGER pipeline (v2.5.2), consensus sequences classified in Haplogrep3 , and then imputed using our novel Hidden-Markov Models (HMM)-based pipeline, MAVEN , alongside an existing k -Nearest Neighbor ( k NN)-based imputation tool, MitoImp . Analyses reveal that a mean depth of 10x or breadth of coverage of 88% is necessary for robust haplogroup assignment in whole mtDNA genomes, with minimal gains in correctness of assignment at coverages greater than 10x. In addition, MAVEN consistently performed better than MitoImp performance at ultra-low coverage (<2x), particularly when using more stringent correct assignment criteria, specifically a Haplogrep3 quality score ≥0.90. Nonetheless, absolute probabilities of correct haplotype classification remained modest at the sub-cluster level, highlighting the inherent difficulties of imputing low-coverage haploid genomes. Conclusion : Our findings establish the first comprehensive evaluation of coverage thresholds for mtDNA analysis and underscore the limitations of applying imputation to highly degraded mtDNA. Our results suggest a minimum depth of coverage of 10x, and breadth of coverage of at least 88% (i.e., no more than 12% missing nucleotides) is required for accurate haplogroup assessment, and that HMM-models outperform unsupervised kNN models at mtDNA imputation. ancient DNA mitochondrial DNA imputation Hidden-Markov Models degraded DNA k-Nearest Neighbor depth of coverage coverage threshold mtDNA quality criteria Figures Figure 4 Full Text Additional Declarations The authors declare no competing interests. Supplementary Files PlummerAdditionalFile1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9406784","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":622473314,"identity":"ef65d3e7-f4b5-4690-9927-0b39689a95bf","order_by":0,"name":"Nathaniel 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4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Mitochondrial DNA (mtDNA) has long served as a foundational target in ancient DNA (aDNA) and palaeogenomic research, owing to its high copy number and well-resolved phylogenetic structure. Yet, external taphonomic and diagenetic factors, including burial environment, hydrolytic and oxidative damage, microbial colonization, and soil chemistry, promote molecular fragmentation. These factors complicate haplotype determination and raise uncertainty about the minimal sequencing coverage needed for reliable haplogroup assignment and phylogenetic inference. Although aDNA studies often apply thresholds between 2x and 10x depth of coverage, a systematic assessment for mtDNA quality has not yet been undertaken. Moreover, while genotype imputation is routinely employed to recover missing data from genomic DNA, its accuracy for ancient mtDNA remains largely untested.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Here, we compiled a reference panel of 46,791 complete human mtDNA genomes and simulated aDNA degradation across coverage depths from 0.25x to 15x (n=3500 mtDNA simulations) using \u003cem\u003egargammel\u003c/em\u003e. Simulated paired-end FASTQ files were processed with the \u003cem\u003eEAGER\u003c/em\u003e pipeline (v2.5.2), consensus sequences classified in \u003cem\u003eHaplogrep3\u003c/em\u003e, and then imputed using our novel Hidden-Markov Models (HMM)-based pipeline, \u003cem\u003eMAVEN\u003c/em\u003e, alongside an existing \u003cem\u003ek\u003c/em\u003e-Nearest Neighbor (\u003cem\u003ek\u003c/em\u003eNN)-based imputation tool, \u003cem\u003eMitoImp\u003c/em\u003e. Analyses reveal that a mean depth of 10x or breadth of coverage of 88% is necessary for robust haplogroup assignment in whole mtDNA genomes, with minimal gains in correctness of assignment at coverages greater than 10x. In addition, \u003cem\u003eMAVEN\u003c/em\u003econsistently performed better than \u003cem\u003eMitoImp\u003c/em\u003e performance at ultra-low coverage (\u0026lt;2x), particularly when using more stringent correct assignment criteria, specifically a \u003cem\u003eHaplogrep3\u003c/em\u003e quality score ≥0.90. Nonetheless, absolute probabilities of correct haplotype classification remained modest at the sub-cluster level, highlighting the inherent difficulties of imputing low-coverage haploid genomes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: Our findings establish the first comprehensive evaluation of coverage thresholds for mtDNA analysis and underscore the limitations of applying imputation to highly degraded mtDNA. Our results suggest a minimum depth of coverage of 10x, and breadth of coverage of at least 88% (i.e., no more than 12% missing nucleotides) is required for accurate haplogroup assessment, and that HMM-models outperform unsupervised kNN models at mtDNA imputation.\u003c/p\u003e","manuscriptTitle":"Imputation and Maximum Likelihood Haplotype Refinement of Simulated Ancient Mitochondrial Genomes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-16 07:00:09","doi":"10.21203/rs.3.rs-9406784/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2fee0735-5536-4e2a-92f4-24b0094c65b4","owner":[],"postedDate":"April 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-22T13:16:49+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-16 07:00:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9406784","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9406784","identity":"rs-9406784","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}