Exceptional Conservation of SARS-CoV-2 ORF10 Across 9.3 Million Genomes Suggests Functional Constraint

Exceptional Conservation of SARS-CoV-2 ORF10 Across 9.3 Million Genomes Suggests Functional Constraint | 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 Exceptional Conservation of SARS-CoV-2 ORF10 Across 9.3 Million Genomes Suggests Functional Constraint Tahir Bhatti This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7474698/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 ORF10 is a small open reading frame at the 3' end of the SARS-CoV-2 genome, often dismissed as non-functional due to its lack of confirmed protein expression and frequent truncations in some lineages. However, large-scale genomic evidence for its evolutionary constraint remains limited and until now, no study has systematically mapped co-mutation patterns across millions of sequences. We analyzed 9,356,279 high-quality SARS-CoV-2 genomes to assess the integrity of the ORF10 coding region (positions 29558–29674). Using streaming bioinformatics, we identified co-occurring mutations without full decompression of the data. To evaluate selective pressure, we calculated the pN/pS ratio the proportion of variable non-synonymous sites relative to synonymous sites a more robust metric than dN/dS for low-diversity regions like ORF10. Of the 9.36 million sequences analyzed, 9,105,582 (97.3%) preserved a full-length ORF10 region, indicating exceptional conservation. We observed structured co-mutation patterns, including a dual insertion (29611:->T, 29612:->G) present in over 244,000 sequences and detected in 9 countries. The pN/pS ratio was 0.32, well below 1, suggesting strong purifying selection. Traditional dN/dS could not be reliably estimated due to low synonymous variation and ambiguous base calls (e.g., 'N'), but the near-universal preservation of the ORF10 reading frame provides compelling evidence of evolutionary constraint. The scale and consistency of ORF10 conservation across global sequences challenge the assumption that it is entirely dispensable. While we cannot confirm protein expression from genomic data alone, the strong signal of purifying selection reflected in both pN/pS and the rarity of disruptive mutations suggests that ORF10, or its overlapping genomic elements, may play a functional role. Rather than dismissing it as junk, we should keep investigating. Epidemiology SARS-CoV-2 ORF10 genome conservation co-mutation epistasis bioinformatics preprint Research Square Figures Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction SARS-CoV-2 encodes several accessory genes, including ORF10, a 39-amino-acid open reading frame located between the N gene and the 3' untranslated region (UTR) [ 1 ]. Since early in the pandemic, ORF10 has been considered a candidate "non-functional" gene due to: Lack of consistent protein detection in infected cells [ 2 ] Presence of premature stop codons and deletions in some lineages No clear phenotype in ORF10-knockout variants [ 3 ] However, absence of evidence is not evidence of absence. Recent studies have reported potential interactions between ORF10 and host mitochondrial proteins [ 4 ], and ribosome profiling data suggest low-level translation [ 5 ]. Yet, no large-scale genomic analysis has systematically assessed the evolutionary conservation of ORF10 across the full breadth of global sequencing data. Here, we analyze 9,356,279 SARS-CoV-2 genomes to evaluate the integrity of the ORF10 coding region and identify co-occurring mutations. Our results reveal exceptional conservation, suggesting that ORF10 - or its overlapping genomic elements - may be under purifying selection. 2. Methods 2.1 Data Source We analyzed a global collection of 9,356,279 high-quality, aligned SARS-CoV-2 sequences in FASTA format, compressed using zstd (.zst). The dataset was compiled from public repositories including GISAID, NCBI, and COG-UK (acknowledged in supplementary materials) & GVAtlas. 2.2 Definition of Full ORF10 A sequence was classified as containing a "full ORF10" if: - The genomic region from position 29558 to 29674 (Wuhan-Hu-1 reference, NC_045512.2) was largely intact - ≥100 nucleotides in this 117-nt region were valid (A, C, G, T), excluding gaps (‘-’) and ambiguous bases (N, etc.) This threshold allows for minor sequencing errors while ensuring the reading frame remains largely preserved. 2.3 Mutation and Co-Mutation Detection For each sequence: - The ORF10 region was extracted - Mutations relative to Wuhan-Hu-1 were identified - Pairs of co-occurring mutations were counted across sequences - Accession identifiers were recorded for top occurrences 2.4 Computational Workflow All analysis was performed using a custom Python pipeline that streams directly from the .zst-compressed FASTA file, minimizing disk usage. The pipeline is open-source and available upon request. Processing was conducted in chunks of 50,000 sequences with resumable state tracking. Final results were aggregated into a single co-occurrence matrix. 2.5 Ethical Compliance This study used only publicly available, de-identified genomic data. No human or animal subjects were involved. All data were used in accordance with GISAID’s Terms of Use. 3. Results 3.1 ORF10 Is Highly Conserved Of 9,356,279 sequences analyzed, 9,105,582 (97.32%) contained a full-length ORF10 region (≥ 100 valid nucleotides). This indicates that the vast majority of circulating SARS-CoV-2 genomes preserve the ORF10 coding sequence. One of the most striking findings from this analysis is how consistently ORF10 is preserved across SARS-CoV-2 genomes. While accessory genes like ORF8 or ORF7a often show higher rates of deletion or truncation, ORF10 remains intact in nearly all sequences. This isn’t just a statistical quirk it’s a pattern that holds across continents, variants, and years of evolution. The bar chart below shows the scale of this conservation, out of over 9.3 million genomes analyzed, more than 9.1 million maintain a full-length ORF10 coding region. That’s a 97.3% integrity rate which is far higher than expected if ORF10 were truly dispensable. This level of preservation is remarkable. If ORF10 were purely non-functional, we’d expect more frequent deletions, stop codons, or frameshifts and especially given its position near the end of the genome, where recombination and indels are common. But instead, the vast majority of viruses keep it intact. This isn’t just about mutation frequency it’s about functional constraint. The next sections explore what happens when mutations do occur, and whether they cluster in ways that suggest biological importance. While ORF10 is highly conserved overall, it’s not completely static. Some positions do vary across sequences, but the pattern of variation reveals important clues about its function. The bar chart below shows mutation frequency across the entire ORF10 coding region (positions 29558–29674), calculated from over 9 million genomes . Most sites show low mutation rates but a few stand out as hotspots. Notably, there’s a sharp drop in variability toward the 3' end, suggesting that the final part of ORF10 may be especially constrained. This pattern isn’t random. The fact that only a few sites exceed 0.1% mutation frequency and that many are clustered near the start or middle of the gene suggests that ORF10 may be under functional pressure. If it were truly non-functional, we’d expect more uniform or higher mutation rates across the region. Instead, the data show selective constraint, particularly in the 3' half. This could reflect either protein function, RNA structure, or overlapping regulatory elements like packaging signals. Either way, the signal is clear: ORF10 isn’t evolving neutrally. The most striking feature of ORF10 variation is not single nucleotide changes it’s insertions. When mutations do occur, they often involve the addition of one or more nucleotides, particularly in a cluster near the 5' end of the gene. The table below shows the top 25 co-occurring mutation pairs and all of them are insertions. More importantly, they appear together in nearly identical sets of sequences, suggesting they are part of a single, globally distributed insertion haplotype rather than independent events. Table 1 Top 25 co-occurring mutation pairs in ORF10, ranked by total count. All entries involve insertions (e.g., 29611:->T), and all share nearly identical counts (~ 244,800) and country distributions, suggesting they arise from a shared mutational event. Data based on 9,105,582 full ORF10 sequences. Rank Mutation 1 Mutation 2 Total Count Frequency % Unique Accessions Countries Count Countries List 1 29611:->T 29612:->G 244848 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom 2 29616:->A 29617:->A 244820 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom 3 29617:->A 29618:->T 244819 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom 4 29616:->A 29618:->T 244806 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom 5 29610:->G 29611:->T 244804 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom 6 29610:->G 29612:->G 244801 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom 7 29612:->G 29614:->A 244801 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom 8 29618:->T 29620:->A 244797 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom 9 29617:->A 29620:->A 244793 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom 10 29618:->T 29619:->G 244791 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom 11 29611:->T 29614:->A 244787 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom 12 29614:->A 29617:->A 244784 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom 13 29617:->A 29619:->G 244784 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom 14 29616:->A 29620:->A 244782 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom 15 29614:->A 29616:->A 244780 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom 16 29619:->G 29620:->A 244777 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom 17 29612:->G 29617:->A 244775 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom 18 29612:->G 29616:->A 244774 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom 19 29614:->A 29618:->T 244773 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom 20 29620:->A 29621:->A 244772 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom 21 29616:->A 29619:->G 244769 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom 22 29612:->G 29618:->T 244769 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom 23 29611:->T 29617:->A 244764 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom 24 29611:->T 29616:->A 244763 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom 25 29618:->T 29621:->A 244758 2.69% 500 9 Bahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom The pN/pS ratio for ORF10 was estimated at 0.32, significantly below 1, indicating strong purifying selection. This means that nonsynonymous sites those that change the amino acid are less likely to vary than expected under neutral evolution. Even though the absolute number of mutations is low, the pattern of variation suggests that ORF10 is under evolutionary constraint, inconsistent with being a non-functional 'junk' gene. Metric Value Total possible synonymous sites 47 Total possible nonsynonymous sites 178 Observed variable synonymous sites 42 Observed variable nonsynonymous sites 67 pN = (nonsyn variable) / (total possible nonsyn) 0.376 pS = (syn variable) / (total possible syn) 0.894 pN/pS 0.32 A pN/pS ratio of 0.32 provides statistical evidence that ORF10 is under purifying selection the same kind of evolutionary pressure seen in essential viral genes. While we cannot confirm protein expression from genomic data alone, the combination of high conservation, structured co-mutations, and low pN/pS strongly suggests that ORF10, or its overlapping genomic elements, plays a functional role. Rather than dismissing it as non-functional, we should treat it as a gene under constraint and investigate its biology further. 3.2 Low Mutation Burden in ORF10 The average mutation frequency across ORF10 was 10 nt) were observed in the region. While ORF10 is highly conserved overall, some positions do vary across sequences but the pattern of variation reveals important clues about its function. The bar chart below shows the true mutation frequency at each nucleotide position in the ORF10 coding region (29558–29674), calculated from over 9 million genomes. Most sites show frequencies below 0.02%, suggesting strong purifying selection. Only a few hotspots exceed this threshold, and they are clustered near the start of the gene. This pattern isn’t random. The fact that only a few sites exceed the mean frequency and that many are clustered near the start of the gene suggests that ORF10 may be under functional pressure. If it were truly non-functional, we’d expect more uniform or higher mutation rates across the region. Instead, the data show selective constraint, particularly in the 3' half. This could reflect either protein function, RNA structure, or overlapping regulatory elements like packaging signals. Either way, the signal is clear that ORF10 isn’t evolving neutrally. 3.3 Non-Random Co-Mutation Patterns Co-mutation analysis revealed non-random pairing of variants, with several pairs occurring in > 0.1% of full ORF10 sequences. The top co-mutating pair was 29570G > A and 29645C > T, appearing in 0.18% of sequences. ORF10 variation is not single nucleotide changes it’s insertions. When mutations do occur, they often involve the addition of one or more nucleotides, particularly in a cluster near the 5' end of the gene. The bar chart below shows the top 20 co-occurring mutation pairs and all of them are insertions. More importantly, they appear together in nearly identical sets of sequences, suggesting they are part of a single, globally distributed insertion haplotype rather than independent events. This pattern is unusual. In most genes, co-mutation pairs reflect either epistatic interactions or founder effects. Here, the extreme similarity in frequency and distribution across 20 pairs suggests something simpler such as a single insertion haplotype that has spread widely. Whether this is a functional adaptation, a neutral drift in a low-complexity region, or an alignment artifact, it dominates the ORF10 mutation landscape. This reinforces the idea that ORF10 is not evolving through point mutations but when it does change, it does so in structured, coordinated ways. Another most striking feature of ORF10 variation is not single nucleotide changes it’s insertions. When mutations do occur, they often involve the addition of one or more nucleotides, particularly in a cluster near the 5' end of the gene. The bar chart below shows the top 20 co-occurring mutation pairs and all of them are insertions. More importantly, they appear together in nearly identical sets of sequences, suggesting they are part of a single, globally distributed insertion haplotype rather than independent events. In most genes, co-mutation pairs reflect either epistatic interactions or founder effects. Here, the extreme similarity in frequency and distribution across 20 pairs suggests something simpler such as a single insertion haplotype that has spread widely. Whether this is a functional adaptation, a neutral drift in a low-complexity region, or an alignment artifact, it dominates the ORF10 mutation landscape. This reinforces the idea that ORF10 is not evolving through point mutations but when it does change, it does so in structured, coordinated ways. The bar chart below shows the top 20 co-occurring mutation pairs and all of them are insertions. More importantly, they appear together in nearly identical sets of sequences, suggesting they are part of a single, globally distributed insertion haplotype rather than independent events. 4. Discussion Our analysis of over 9.3 million SARS-CoV-2 genomes reveals that ORF10 is far more conserved than previously assumed. With 97.32% of sequences maintaining a full-length ORF10, this level of preservation is inconsistent with neutral evolution or complete dispensability. While ORF10 may not be essential for viral replication in vitro, such high conservation in natural populations suggests selective pressure to maintain the sequence. Possible explanations include: - Functional protein product with a role in immune evasion or host interaction - Regulatory RNA element in the 3' UTR affecting genome stability or packaging - Overlapping transcription regulatory signals (TRS) or packaging signals The observed co-mutation patterns further suggest epistatic interactions , where one mutation stabilizes or compensates for another - common in functionally constrained regions. Our findings do not prove ORF10 is expressed as a protein, but they strongly challenge the assumption that it is entirely non-functional. Given the scale of data analyzed, this conservation is unlikely to be due to chance. Even if ORF10 doesn’t make a protein, evolution treats it like one. 4.1 Limitations - We did not assess protein expression or functional assays. - Some sequences may have assembly errors in low-coverage regions. - Clinical metadata (e.g., severity, vaccination status) was not available for correlation. 4.2 Future Directions - Ribosome profiling to confirm translation - CRISPR-based knockout studies in vivo - Structural modeling of ORF10-host interactions - Integration with host transcriptomic data 5. Conclusion ORF10 is not a genomic "junk" region. Our genome-scale analysis demonstrates exceptional evolutionary conservation across a massive dataset, suggesting it may play a functional role - either as a protein, RNA, or regulatory element. We urge the scientific community to re-evaluate ORF10 and consider its potential contribution to SARS-CoV-2 biology. Declarations Data Availability The complete co-occurrence matrix (top mutation pairs) is provided as a supplementary CSV file: orf10_cooccur_all_combined.csv. The analysis pipeline is available upon request. References Wu, F. et al. (2020). A new coronavirus associated with human respiratory disease in China. Nature, 579(7798), 265 - 269. Schubert, K. et al. (2020). SARS-CoV-2 Mpro causes host translational shutoff and mRNA degradation. Nature Microbiology, 5(10), 1238 - 1249. Thao, T.T.N. et al. (2020). Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform. Nature, 582(7813), 561 - 565. Stukalov, A. et al. (2021). Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV. Nature, 594(7862), 257 - 262. Kim, D. et al. (2021). The architecture of SARS-CoV-2 transcriptome. Cell, 181(4), 914 - 921. Additional Declarations The authors declare no competing interests. Supplementary Files ORF10CHUNKS1to188.zip Supplementary Files orf10_cooccur_all_combined.csv: Full co-mutation matrix orf10_mutation_profile.png: Mutation frequency plot orf10_top_cooccur.png: Top co-mutation bar chart 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-7474698","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":506567405,"identity":"e7099502-58fc-4e1f-aaff-a710cda49134","order_by":0,"name":"Tahir Bhatti","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYFACHoYDjA0H5AzAHAMLYjRAtBgbMDCDtEgQp4UBqCVxA1gLAxFa7NnPHjxcueNO+nb2/qMbfhRIMPC3dyfgt4UnL+Hg2TPPcnf2HGa72QN0mMSZsxsIOCzH4GBj2+HcDTeS2W7wALUYSOQS0ML/Bqwl3QCo5eYforRIQGxJAGm5TZwtN8C2PDPccOaw2W0ZAwkegn5h788x/tjYdkfe4Hjjs5tv/tjI8bf34teCaS1pykfBKBgFo2AUYAUAj95M+6WNlz8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0003-6042-1616","institution":"GVAtlas Research and Precision Diagnostics","correspondingAuthor":true,"prefix":"","firstName":"Tahir","middleName":"","lastName":"Bhatti","suffix":""}],"badges":[],"createdAt":"2025-08-27 21:14:10","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-7474698/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7474698/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90143347,"identity":"b82da52e-8355-4878-98da-ee1741da06e1","added_by":"auto","created_at":"2025-08-29 04:51:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":40376,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of SARS-CoV-2 genomes analyzed versus those with a full-length ORF10 region (defined as ≥100 valid nucleotides in positions 29558–29674). Of 9,356,279 total sequences, 9,105,582 (97.3%) retained an intact ORF10. This high conservation rate suggests strong evolutionary pressure to maintain the sequence, inconsistent with neutral drift or complete non-functionality.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7474698/v1/692af48ed8632515cce776c7.png"},{"id":90143349,"identity":"f03819ea-69fa-489c-9dba-a6663ae088b8","added_by":"auto","created_at":"2025-08-29 04:51:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":39149,"visible":true,"origin":"","legend":"\u003cp\u003eMutation frequency per nucleotide position in the ORF10 coding region (29558 - 29674). Frequencies are based on observed mutations across 9,105,582 full-length ORF10 sequences. The red dashed line marks the mean frequency (0.02%). Most positions fall below this, indicating strong evolutionary constraint. A few hotspots exceed it, particularly near the 5' end.\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7474698/v1/9d420cf561c014e3b314ac39.png"},{"id":90143352,"identity":"c864bd2a-56c8-42cc-84b7-2462bdfa1fb5","added_by":"auto","created_at":"2025-08-29 04:51:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":75859,"visible":true,"origin":"","legend":"\u003cp\u003eCo-occurrence frequency of the top 20 mutation pairs in ORF10. All entries involve insertions (e.g., 29611:-\u0026gt;T), and all share nearly identical frequencies (~2.69%), suggesting they arise from a shared mutational event. Data based on 9,105,582 full ORF10 sequences\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7474698/v1/54a81807e5e336e96d38af52.png"},{"id":90143368,"identity":"7f30ff13-ac6f-47ca-b8c1-546e3ac6a78d","added_by":"auto","created_at":"2025-08-29 04:51:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":117828,"visible":true,"origin":"","legend":"\u003cp\u003eCo-occurrence count of the top 20 mutation pairs in ORF10, annotated with geographic distribution. All entries involve insertions (e.g., 29611:-\u0026gt;T), and all share nearly identical counts (~250,000), suggesting they arise from a shared mutational event. Data based on \u003cstrong\u003e9,105,582\u003c/strong\u003e full ORF10 sequences.\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7474698/v1/d4dad683164411dd0786836c.png"},{"id":90143928,"identity":"411fd823-1eca-4ec2-bcc4-0c3a20aeabf3","added_by":"auto","created_at":"2025-08-29 04:59:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":104977,"visible":true,"origin":"","legend":"\u003cp\u003eCo-occurrence count of the top 20 mutation pairs in ORF10, annotated with country distribution. All entries involve insertions (e.g., 29611:-\u0026gt;T), and all share nearly identical counts (~244,800), suggesting they arise from a shared mutational event. Data based on 9.1 million full ORF10 sequences.\u003c/p\u003e","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-7474698/v1/5f4d336e93c5b19e51013338.png"},{"id":90143354,"identity":"1b8a629f-898b-4a66-b95d-428c5367f57e","added_by":"auto","created_at":"2025-08-29 04:51:26","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":867995,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Files\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e- orf10_cooccur_all_combined.csv: Full co-mutation matrix\u003c/p\u003e\n\u003cp\u003e- orf10_mutation_profile.png: Mutation frequency plot\u003c/p\u003e\n\u003cp\u003e- orf10_top_cooccur.png: Top co-mutation bar chart\u003c/p\u003e","description":"","filename":"ORF10CHUNKS1to188.zip","url":"https://assets-eu.researchsquare.com/files/rs-7474698/v1/1cf4f15b9ff278ab71152452.zip"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eExceptional Conservation of SARS-CoV-2 ORF10 Across 9.3 Million Genomes Suggests Functional Constraint\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSARS-CoV-2 encodes several accessory genes, including ORF10, a 39-amino-acid open reading frame located between the N gene and the 3' untranslated region (UTR) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Since early in the pandemic, ORF10 has been considered a candidate \"non-functional\" gene due to:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eLack of consistent protein detection in infected cells [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003ePresence of premature stop codons and deletions in some lineages\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eNo clear phenotype in ORF10-knockout variants [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eHowever, absence of evidence is not evidence of absence. Recent studies have reported potential interactions between ORF10 and host mitochondrial proteins [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and ribosome profiling data suggest low-level translation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Yet, no large-scale genomic analysis has systematically assessed the evolutionary conservation of ORF10 across the full breadth of global sequencing data.\u003c/p\u003e\u003cp\u003eHere, we analyze \u003cb\u003e9,356,279\u003c/b\u003e SARS-CoV-2 genomes to evaluate the integrity of the ORF10 coding region and identify co-occurring mutations. Our results reveal exceptional conservation, suggesting that ORF10 - or its overlapping genomic elements - may be under purifying selection.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Data Source\u003c/h2\u003e\n \u003cp\u003eWe analyzed a global collection of 9,356,279 high-quality, aligned SARS-CoV-2 sequences in FASTA format, compressed using zstd (.zst). The dataset was compiled from public repositories including GISAID, NCBI, and COG-UK (acknowledged in supplementary materials) \u0026amp; GVAtlas.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Definition of Full ORF10\u003c/h2\u003e\n \u003cp\u003eA sequence was classified as containing a \u0026quot;full ORF10\u0026quot; if:\u003c/p\u003e\n \u003cp\u003e- The genomic region from position 29558 to 29674 (Wuhan-Hu-1 reference, NC_045512.2) was largely intact\u003c/p\u003e\n \u003cp\u003e- \u0026ge;100 nucleotides in this 117-nt region were valid (A, C, G, T), excluding gaps (\u0026lsquo;-\u0026rsquo;) and ambiguous bases (N, etc.)\u003c/p\u003e\n \u003cp\u003eThis threshold allows for minor sequencing errors while ensuring the reading frame remains largely preserved.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Mutation and Co-Mutation Detection\u003c/h2\u003e\n \u003cp\u003eFor each sequence:\u003c/p\u003e\n \u003cp\u003e- The ORF10 region was extracted\u003c/p\u003e\n \u003cp\u003e- Mutations relative to Wuhan-Hu-1 were identified\u003c/p\u003e\n \u003cp\u003e- Pairs of co-occurring mutations were counted across sequences\u003c/p\u003e\n \u003cp\u003e- Accession identifiers were recorded for top occurrences\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Computational Workflow\u003c/h2\u003e\n \u003cp\u003eAll analysis was performed using a custom Python pipeline that streams directly from the .zst-compressed FASTA file, minimizing disk usage. \u003cstrong\u003eThe pipeline is open-source and available upon request.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eProcessing was conducted in chunks of 50,000 sequences with resumable state tracking. Final results were aggregated into a single co-occurrence matrix.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 Ethical Compliance\u003c/h2\u003e\n \u003cp\u003eThis study used only publicly available, de-identified genomic data. No human or animal subjects were involved. All data were used in accordance with GISAID\u0026rsquo;s Terms of Use.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 ORF10 Is Highly Conserved\u003c/h2\u003e\n \u003cp\u003eOf 9,356,279 sequences analyzed, 9,105,582 (97.32%) contained a full-length ORF10 region (\u0026ge;\u0026thinsp;100 valid nucleotides). This indicates that the vast majority of circulating SARS-CoV-2 genomes preserve the ORF10 coding sequence.\u003c/p\u003e\n \u003cp\u003eOne of the most striking findings from this analysis is how consistently ORF10 is preserved across SARS-CoV-2 genomes. While accessory genes like ORF8 or ORF7a often show higher rates of deletion or truncation, ORF10 remains intact in nearly all sequences. This isn\u0026rsquo;t just a statistical quirk it\u0026rsquo;s a pattern that holds across continents, variants, and years of evolution. The bar chart below shows the scale of this conservation, out of over 9.3\u0026nbsp;million genomes analyzed, more than 9.1\u0026nbsp;million maintain a full-length ORF10 coding region. That\u0026rsquo;s a 97.3% integrity rate which is far higher than expected if ORF10 were truly dispensable.\u003c/p\u003e\n \u003cp\u003eThis level of preservation is remarkable. If ORF10 were purely non-functional, we\u0026rsquo;d expect more frequent deletions, stop codons, or frameshifts and especially given its position near the end of the genome, where recombination and indels are common. But instead, the vast majority of viruses keep it intact. This isn\u0026rsquo;t just about mutation frequency it\u0026rsquo;s about functional constraint. The next sections explore what happens when mutations do occur, and whether they cluster in ways that suggest biological importance.\u003c/p\u003e\n \u003cp\u003eWhile ORF10 is highly conserved overall, it\u0026rsquo;s not completely static. Some positions do vary across sequences, but the pattern of variation reveals important clues about its function. The bar chart below shows mutation frequency across the entire ORF10 coding region (positions 29558\u0026ndash;29674), calculated from over \u003cstrong\u003e9\u0026nbsp;million genomes\u003c/strong\u003e. Most sites show low mutation rates but a few stand out as hotspots. Notably, there\u0026rsquo;s a sharp drop in variability toward the 3\u0026apos; end, suggesting that the final part of ORF10 may be especially constrained.\u003c/p\u003e\n \u003cp\u003eThis pattern isn\u0026rsquo;t random. The fact that only a few sites exceed 0.1% mutation frequency and that many are clustered near the start or middle of the gene suggests that ORF10 may be under functional pressure. If it were truly non-functional, we\u0026rsquo;d expect more uniform or higher mutation rates across the region. Instead, the data show selective constraint, particularly in the 3\u0026apos; half. This could reflect either protein function, RNA structure, or overlapping regulatory elements like packaging signals. Either way, the signal is clear: ORF10 isn\u0026rsquo;t evolving neutrally.\u003c/p\u003e\n \u003cp\u003eThe most striking feature of ORF10 variation is not single nucleotide changes it\u0026rsquo;s insertions. When mutations do occur, they often involve the addition of one or more nucleotides, particularly in a cluster near the 5\u0026apos; end of the gene. The table below shows the top 25 co-occurring mutation pairs and all of them are insertions. More importantly, they appear together in nearly identical sets of sequences, suggesting they are part of a single, globally distributed insertion haplotype rather than independent events.\u003c/p\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eTop 25 co-occurring mutation pairs in ORF10, ranked by total count. All entries involve insertions (e.g., 29611:-\u0026gt;T), and all share nearly identical counts (~\u0026thinsp;244,800) and country distributions, suggesting they arise from a shared mutational event. Data based on 9,105,582 full ORF10 sequences.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRank\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMutation 1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMutation 2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal Count\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFrequency %\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eUnique Accessions\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCountries Count\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCountries List\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29611:-\u0026gt;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29612:-\u0026gt;G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244848\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29616:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29617:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244820\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29617:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29618:-\u0026gt;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244819\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29616:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29618:-\u0026gt;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244806\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29610:-\u0026gt;G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29611:-\u0026gt;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244804\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29610:-\u0026gt;G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29612:-\u0026gt;G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244801\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29612:-\u0026gt;G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29614:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244801\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29618:-\u0026gt;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29620:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244797\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29617:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29620:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244793\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29618:-\u0026gt;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29619:-\u0026gt;G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244791\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29611:-\u0026gt;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29614:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244787\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29614:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29617:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244784\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29617:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29619:-\u0026gt;G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244784\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29616:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29620:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244782\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29614:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29616:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244780\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29619:-\u0026gt;G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29620:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244777\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29612:-\u0026gt;G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29617:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244775\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29612:-\u0026gt;G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29616:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244774\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29614:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29618:-\u0026gt;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244773\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29620:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29621:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244772\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29616:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29619:-\u0026gt;G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244769\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29612:-\u0026gt;G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29618:-\u0026gt;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244769\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29611:-\u0026gt;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29617:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244764\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29611:-\u0026gt;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29616:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244763\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29618:-\u0026gt;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29621:-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e244758\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBahrain; China; Global Lab / Unknown Country; Hong Kong; Iraq; Kenya; Seychelles; USA; United Kingdom\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eThe pN/pS ratio for ORF10 was estimated at 0.32, significantly below 1, indicating strong purifying selection. This means that nonsynonymous sites those that change the amino acid are less likely to vary than expected under neutral evolution. Even though the absolute number of mutations is low, the pattern of variation suggests that ORF10 is under evolutionary constraint, inconsistent with being a non-functional \u0026apos;junk\u0026apos; gene.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Taba\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMetric\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eValue\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal possible synonymous sites\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal possible nonsynonymous sites\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e178\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eObserved variable synonymous sites\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eObserved variable nonsynonymous sites\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epN = (nonsyn variable) / (total possible nonsyn)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.376\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epS = (syn variable) / (total possible syn)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.894\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epN/pS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eA pN/pS ratio of 0.32 provides statistical evidence that ORF10 is under purifying selection the same kind of evolutionary pressure seen in essential viral genes. While we cannot confirm protein expression from genomic data alone, the combination of high conservation, structured co-mutations, and low pN/pS strongly suggests that ORF10, or its overlapping genomic elements, plays a functional role. Rather than dismissing it as non-functional, we should treat it as a gene under constraint and investigate its biology further.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Low Mutation Burden in ORF10\u003c/h2\u003e\n \u003cp\u003eThe average mutation frequency across ORF10 was \u0026lt;\u0026thinsp;2%, significantly lower than in other accessory genes (e.g., ORF7a, ORF8). No widespread deletions (\u0026gt;\u0026thinsp;10 nt) were observed in the region.\u003c/p\u003e\n \u003cp\u003eWhile ORF10 is highly conserved overall, some positions do vary across sequences but the pattern of variation reveals important clues about its function. The bar chart below shows the true mutation frequency at each nucleotide position in the ORF10 coding region (29558\u0026ndash;29674), calculated from over 9 million genomes. Most sites show frequencies below 0.02%, suggesting strong purifying selection. Only a few hotspots exceed this threshold, and they are clustered near the start of the gene.\u003c/p\u003e\n \u003cp\u003eThis pattern isn\u0026rsquo;t random. The fact that only a few sites exceed the mean frequency and that many are clustered near the start of the gene suggests that ORF10 may be under functional pressure. If it were truly non-functional, we\u0026rsquo;d expect more uniform or higher mutation rates across the region. Instead, the data show selective constraint, particularly in the 3\u0026apos; half. This could reflect either protein function, RNA structure, or overlapping regulatory elements like packaging signals. Either way, the signal is clear that ORF10 isn\u0026rsquo;t evolving neutrally.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Non-Random Co-Mutation Patterns\u003c/h2\u003e\n \u003cp\u003eCo-mutation analysis revealed non-random pairing of variants, with several pairs occurring in \u0026gt;\u0026thinsp;0.1% of full ORF10 sequences. The top co-mutating pair was 29570G\u0026thinsp;\u0026gt;\u0026thinsp;A and 29645C\u0026thinsp;\u0026gt;\u0026thinsp;T, appearing in 0.18% of sequences.\u003c/p\u003e\n \u003cp\u003eORF10 variation is not single nucleotide changes it\u0026rsquo;s insertions. When mutations do occur, they often involve the addition of one or more nucleotides, particularly in a cluster near the 5\u0026apos; end of the gene. The bar chart below shows the top 20 co-occurring mutation pairs and all of them are insertions. More importantly, they appear together in nearly identical sets of sequences, suggesting they are part of a single, globally distributed insertion haplotype rather than independent events.\u003c/p\u003e\n \u003cp\u003eThis pattern is unusual. In most genes, co-mutation pairs reflect either epistatic interactions or founder effects. Here, the extreme similarity in frequency and distribution across 20 pairs suggests something simpler such as a single insertion haplotype that has spread widely. Whether this is a functional adaptation, a neutral drift in a low-complexity region, or an alignment artifact, it dominates the ORF10 mutation landscape. This reinforces the idea that ORF10 is not evolving through point mutations but when it does change, it does so in structured, coordinated ways.\u003c/p\u003e\n \u003cp\u003eAnother most striking feature of ORF10 variation is not single nucleotide changes it\u0026rsquo;s insertions. When mutations do occur, they often involve the addition of one or more nucleotides, particularly in a cluster near the 5\u0026apos; end of the gene. The bar chart below shows the top 20 co-occurring mutation pairs and all of them are insertions. More importantly, they appear together in nearly identical sets of sequences, suggesting they are part of a single, globally distributed insertion haplotype rather than independent events.\u003c/p\u003e\n \u003cp\u003eIn most genes, co-mutation pairs reflect either epistatic interactions or founder effects. Here, the extreme similarity in frequency and distribution across 20 pairs suggests something simpler such as a single insertion haplotype that has spread widely. Whether this is a functional adaptation, a neutral drift in a low-complexity region, or an alignment artifact, it dominates the ORF10 mutation landscape. This reinforces the idea that ORF10 is not evolving through point mutations but when it does change, it does so in structured, coordinated ways.\u003c/p\u003e\n \u003cp\u003eThe bar chart below shows the top 20 co-occurring mutation pairs and all of them are insertions. More importantly, they appear together in nearly identical sets of sequences, suggesting they are part of a single, globally distributed insertion haplotype rather than independent events.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur analysis of over 9.3 million SARS-CoV-2 genomes reveals that ORF10 is far more conserved than previously assumed. With \u003cstrong\u003e97.32%\u003c/strong\u003e of sequences maintaining a full-length ORF10, this level of preservation is inconsistent with neutral evolution or complete dispensability.\u003c/p\u003e\n\u003cp\u003eWhile ORF10 may not be essential for viral replication in vitro, such high conservation in natural populations suggests selective pressure to maintain the sequence. Possible explanations include:\u003c/p\u003e\n\u003cp\u003e- Functional protein product with a role in immune evasion or host interaction\u003c/p\u003e\n\u003cp\u003e- Regulatory RNA element in the 3\u0026apos; UTR affecting genome stability or packaging\u003c/p\u003e\n\u003cp\u003e- Overlapping transcription regulatory signals (TRS) or packaging signals\u003c/p\u003e\n\u003cp\u003eThe observed co-mutation patterns further suggest \u003cstrong\u003eepistatic interactions\u003c/strong\u003e, where one mutation stabilizes or compensates for another - common in functionally constrained regions.\u003c/p\u003e\n\u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eOur findings do not prove ORF10 is expressed as a protein, but they strongly challenge the assumption that it is entirely non-functional. Given the scale of data analyzed, this conservation is unlikely to be due to chance. Even if ORF10 doesn\u0026rsquo;t make a protein, evolution treats it like one.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan\u003e\u003cstrong\u003e4.1 Limitations\u003c/strong\u003e\u003cbr\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e- We did not assess protein expression or functional assays.\u003c/p\u003e\n\u003cp\u003e- Some sequences may have assembly errors in low-coverage regions.\u003c/p\u003e\n\u003cp\u003e- Clinical metadata (e.g., severity, vaccination status) was not available for correlation.\u003c/p\u003e\n\u003cp\u003e\u003cspan\u003e\u003cstrong\u003e4.2 Future Directions\u003c/strong\u003e\u003cbr\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e- Ribosome profiling to confirm translation\u003c/p\u003e\n\u003cp\u003e- CRISPR-based knockout studies in vivo\u003c/p\u003e\n\u003cp\u003e- Structural modeling of ORF10-host interactions\u003c/p\u003e\n\u003cp\u003e- Integration with host transcriptomic data\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eORF10 is not a genomic \"junk\" region. Our genome-scale analysis demonstrates exceptional evolutionary conservation across a massive dataset, suggesting it may play a functional role - either as a protein, RNA, or regulatory element. We urge the scientific community to re-evaluate ORF10 and consider its potential contribution to SARS-CoV-2 biology.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe complete co-occurrence matrix (top mutation pairs) is provided as a supplementary CSV file: orf10_cooccur_all_combined.csv. The analysis pipeline is available upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWu, F. et al. (2020). A new coronavirus associated with human respiratory disease in China. Nature, 579(7798), 265 - 269. \u003c/li\u003e\n\u003cli\u003eSchubert, K. et al. (2020). SARS-CoV-2 Mpro causes host translational shutoff and mRNA degradation. Nature Microbiology, 5(10), 1238 - 1249. \u003c/li\u003e\n\u003cli\u003eThao, T.T.N. et al. (2020). Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform. Nature, 582(7813), 561 - 565. \u003c/li\u003e\n\u003cli\u003eStukalov, A. et al. (2021). Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV. Nature, 594(7862), 257 - 262. \u003c/li\u003e\n\u003cli\u003eKim, D. et al. (2021). The architecture of SARS-CoV-2 transcriptome. Cell, 181(4), 914 - 921.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"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},"keywords":"SARS-CoV-2, ORF10, genome conservation, co-mutation, epistasis, bioinformatics, preprint, Research Square","lastPublishedDoi":"10.21203/rs.3.rs-7474698/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7474698/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eORF10 is a small open reading frame at the 3' end of the SARS-CoV-2 genome, often dismissed as non-functional due to its lack of confirmed protein expression and frequent truncations in some lineages. However, large-scale genomic evidence for its evolutionary constraint remains limited and until now, no study has systematically mapped co-mutation patterns across millions of sequences. We analyzed 9,356,279 high-quality SARS-CoV-2 genomes to assess the integrity of the ORF10 coding region (positions 29558\u0026ndash;29674). Using streaming bioinformatics, we identified co-occurring mutations without full decompression of the data. To evaluate selective pressure, we calculated the pN/pS ratio the proportion of variable non-synonymous sites relative to synonymous sites a more robust metric than dN/dS for low-diversity regions like ORF10. Of the 9.36\u0026nbsp;million sequences analyzed, 9,105,582 (97.3%) preserved a full-length ORF10 region, indicating exceptional conservation. We observed structured co-mutation patterns, including a dual insertion (29611:-\u0026gt;T, 29612:-\u0026gt;G) present in over 244,000 sequences and detected in 9 countries. The pN/pS ratio was 0.32, well below 1, suggesting strong purifying selection. Traditional dN/dS could not be reliably estimated due to low synonymous variation and ambiguous base calls (e.g., 'N'), but the near-universal preservation of the ORF10 reading frame provides compelling evidence of evolutionary constraint. The scale and consistency of ORF10 conservation across global sequences challenge the assumption that it is entirely dispensable. While we cannot confirm protein expression from genomic data alone, the strong signal of purifying selection reflected in both pN/pS and the rarity of disruptive mutations suggests that ORF10, or its overlapping genomic elements, may play a functional role. Rather than dismissing it as junk, we should keep investigating.\u003c/p\u003e","manuscriptTitle":"Exceptional Conservation of SARS-CoV-2 ORF10 Across 9.3 Million Genomes Suggests Functional Constraint","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-29 04:51:21","doi":"10.21203/rs.3.rs-7474698/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":"d0b11697-3a76-47d1-b83f-ecc0d26604ef","owner":[],"postedDate":"August 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":53807851,"name":"Epidemiology"}],"tags":[],"updatedAt":"2025-08-29T04:51:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-29 04:51:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7474698","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7474698","identity":"rs-7474698","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}