Efficient variant phasing utilizing a replication cycle reaction system

Efficient variant phasing utilizing a replication cycle reaction system | 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 Article Efficient variant phasing utilizing a replication cycle reaction system Tatsushi Toda, Akihiko Mitsutake, Hiroyuki Ishiura, Takashi Matsukawa, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4025036/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 When two heterozygous variants are detected in genes for diseases with autosomal recessive inheritance, determining whether the two variants are located in cis or in trans is crucial. Subcloning long-range PCR products or cDNA is limited by factors such as the distance between variants (up to 10 kb) and cDNA availability. Droplet digital PCR, effective up to distances of 100 kb, faces challenges in specific probe design. We utilized replication cycle reaction (RCR) for amplifying large genomic DNA segments with multiple heterozygous variants. RCR is an in vitro replication cycle based on chromosome replication in Escherichia coli . Circular DNA molecules were generated by combining CRISPR/Cas9-cleaved genomic DNA fragments with an oriC – AmpR cassette, followed by amplification through RCR. Various molar ratios of gDNA to the oriC – AmpR cassette were evaluated to optimize the ligation step. We analyzed gDNAs from seven patients carrying two heterozygous pathogenic variants with distances ranging from 4.3 to 152 kb. A genomic region up to 104 kb could be amplified by RCR. A higher input of the oriC – AmpR cassette resulted in a higher rate of successful RCR amplification and a lower rate of successful monoallelic amplification. Monoallelic clonal amplification occurred in six patients, facilitating a rapid determination of variant phases. A haplotype was successfully reconstructed using an SNP located 78 kb away from the variant in one patient with two variants separated by a 152 kb distance. Our method proves particularly valuable for phasing multiple heterozygous variants separated over long genomic distances. Biological sciences/Genetics/Sequencing/DNA sequencing Health sciences/Diseases/Neurological disorders/Spinocerebellar ataxia Figures Figure 1 Figure 2 Introduction When two heterozygous variants are detected in genes for diseases with autosomal recessive inheritance, it is necessary to determine whether the two variants are located in cis or in trans . Although the genomic deoxyribonucleic acids (gDNAs) of the parents are usually analyzed, we face difficulties when parental DNAs are unavailable. Subcloning of long-range polymerase chain reaction (PCR) products obtained from gDNAs is useful, but long-range PCR 1 fails to amplify segments usually exceeding 10 kb. Subcloning of complementary DNA (cDNA) is another option, but its success largely depends on the availability of cells expressing the relevant messenger ribonucleic acid (mRNA). In addition, PCR amplification-based subcloning can occasionally yield inconsistent phase results potentially attributed to incomplete elongation and mispriming on the heterologous allele producing PCR chimera. 2 Droplet digital PCR is useful when these methods cannot be applied. 3 This method isolates gDNA into 30,000–50,000 droplets in such a way that a single DNA molecule in each droplet is amplified with allele-specific fluorescence-labeled probes. In principle, an appropriate combination of allele-specific fluorescent probes will produce double-positive droplets when DNA fragments containing physically linked variants are present in the same droplets. 3 This method can be applied when the distance between variants is up to 100 kb, 3 although designing and optimizing allele-specific probes sometimes remain challenging. Recently, Su’etsugu et al . have successfully reconstituted the entire replication cycle of E. coli in vitro . 4 This replication cycle reaction (RCR) propagates amplified circular DNA in an isothermal reaction. 4 This method can amplify a very large circular DNA carrying the oriC sequence up to 0.2 Mb. Furthermore, it is characterized by a high accuracy of replication, approximately ∼1.2 × 10 − 8 per base per replication cycle. By combining the oriC sequence and the excised gDNA fragment, we considered that we could amplify a genomic region too large to be amplified by PCR (Fig. 1 a). To accomplish the amplification of a large genomic DNA fragment carrying two heterozygous variants, we employed RCR and tried to determine whether the heterozygous variants are located in cis or in trans . First, we determined the optimal conditions for the RCR amplification of the excised gDNA fragment. We then investigated haplotyping through RCR amplification using DNA samples already analyzed by long-read sequencing, demonstrating successful haplotyping. Finally, we examined clinical samples in actual clinical practice. Materials and Methods Samples In this study, we utilized eight gDNA samples that had already been confirmed to carry two heterozygous variants on the basis of whole-exome or whole-genome sequence analyses. This study was approved by the Institutional Review Board of the University of Tokyo Hospital (G1396). Written informed consent was obtained from all the patients. Digestion of gDNA gDNAs were extracted from peripheral blood leukocytes following a standard procedure. Extracted gDNAs were digested using a CRISPR/Cas9 system. CHOPCHOP ( https://chopchop.cbu.uib.no/ ) was used to design the CRISPR RNA (crRNA) sequence, a 20-nucleotide sequence upstream of the PAM sequence (5’-NGG-3’). Then, we synthesized a single guide RNA (sgRNA) from target-specific oligonucleotides with an EnGen sgRNA Synthesis Kit (New England Biolabs, Ipswich, MA. #E2233S) following the manufacturer’s instructions. After the purification of sgRNA using an RNeasy Plus Mini Kit (QIAGEN Venlo, The Netherlands), 10 µg of gDNA was cleaved with Cas9 Nuclease, S. Pyogenes (New England Biolabs. #M0386S). 5 µL of NEBuffer r3.1, 5 µL of sgRNA (5 µM), 1 µL of Cas9 nuclease (20 µM), and nuclease-free water were mixed in a total volume of 50 µL. The mixture was incubated for 1 h at 37°C and then for 20 min at 65°C. The cleaved gDNA was purified with AmPure XP (Agencourt Bioscience Corp., Beverly, MA). The quality of gDNA was evaluated using a 4150 TapeStation (Agilent Technologies, Santa Clara, CA). RCR amplification of gDNA fragment An oriC cassette contained in the OriCiro Cell-Free Cloning System (OriCiro Genomics, Tokyo, Japan) was subcloned into pBR322 downstream of the ampicillin resistance gene using an In-Fusion® HD Cloning Kit (Takara Bio, Otsu, Japan). The nucleotide sequence of the plasmid is shown in Supplementary Methods 1. With the vector as a template, the region containing the cassette with rop , ori , AmpR , and oriC cassettes ( oriC – AmpR cassette) was amplified by PCR using primer pairs containing 60 nt overlapping sequences with the target sequence located on the 5’-side, followed by PCR product purification. This procedure was conducted for the subsequent transformation of RCR products. The detailed PCR procedure and the primer sequences are described in Supplementary Methods 2. Then, we ligated the cleaved gDNA fragment and PCR-amplified oriC – AmpR cassette using the 2xRA mix contained in the OriCiro Cell-Free Cloning System to form a circular DNA molecule. The reaction conditions are as follows. The cleaved gDNA fragment and oriC-AmpR cassette were mixed in a total of 2.4 µL at various molar ratios of gDNA to the oriC-AmpR cassette (1:10, 1:100, 1:1000, and 1:10000). The amount of gDNA input was fixed at 100 ng (1.4 µL), and various amounts of oriC – AmpR cassette (1.6 pg, 16 pg, 160 pg, and 1.6 ng) were added to a total volume of 1.1 µL. After adding 2.5 µL of 2x RA Mix, the mixture was incubated for 1 hour at 42°C to form circular DNA molecules by an enzyme-based annealing mechanism using 2xRA Mix. 4 Afterward, we mixed 0.5 µL of the product with 1 µL of 5x Buffer I, 1 µL of 5x Buffer II, 0.5 µL of 10x RE mix, and 2 µL of nuclease-free water. The resulting mixture was then incubated for 16 hours at 33°C for the amplification of circular DNA molecules by RCR. The successful amplification by RCR was confirmed by agarose gel electrophoresis (0.6%) of RCR products (100 V, 1 h) after digestion with specific restriction enzymes. Determination of the optimal molar ratio of gDNA to oriC cassette Various molar ratios of gDNA to the oriC-AmpR cassette were evaluated to determine the optimal conditions for the ligation step. In this experiment, we used a gDNA sample (#9565) already analyzed by long-read sequencing. Whole-genome sequence analysis was conducted using a long-read sequencer, PacBio RSII (Pacific Biosciences), utilizing gDNAs extracted from lymphoblastoid cells. 5 Circular consensus sequence (CCS) reads were generated from the subreads using the ccs application SMRT Link version 6.0.0.47841 provided by PacBio ( https://ccs.how/ ). The reads were aligned to GRCh38/hg38 using Minimap2. 6 The result was visualized using Integrated Genome Viewer (IGV) (version 2.8.6). 7 Three regions (chr5:149,503,216–149,513,393; chr10:79,309,011–79,336,568; chr14:88,450,095–88,455,957) containing heterozygous single nucleotide variants (SNVs), which were phased by the long-read sequencing, were selected, digested with sgRNA, and then amplified by RCR. Heterozygous SNVs located in the regions are listed in Table 1 . Each sample was amplified by RCR in 8 independent tubes for each molar ratio. The successful amplification by RCR was confirmed by electrophoresis after digestion with specific restriction enzymes. After confirming successful amplification, the RCR products were then subjected to direct nucleotide sequence analysis to examine whether one or two alleles were amplified in each tube after RCR amplification. The phase of the variants was determined using RCR products in which one allele was amplified. Table 1 Amplified genomic region and heterozygous SNPs in each region No. region size (bp) SNP1 SNP2 1 chr5:149,503,216–149,513,393 10,178 chr5:149,511,792 chr5:149,512,042 A/G A/G 2 chr10:79,309,011–79,336,568 27,558 chr10:79,316,010 C/G 3 chr14:88,450,095–88,455,957 5,863 chr14:88,453,186 chr14:88,453,278 A/G G/T Direct nucleotide sequence analysis of RCR products The obtained RCR products were purified employing ExoSAP-IT for PCR Product Clean-Up (Affymetrix, Santa Clara, CA), with one cycle of 30 min at 37°C followed by another cycle of 15 min at 80°C. The purified RCR products were subjected to nucleotide sequence analysis employing the ABI PRISM BigDye 3.1 terminator method (Applied Biosystems, Foster City, CA) and the ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems). The sequences of the primers are shown in Supplementary Methods 3. Analysis of gDNAs from patients carrying two heterozygous variants in a clinical setting In this experiment, we analyzed seven gDNA samples already analyzed by whole-exome sequence analysis. gDNA samples were from seven patients carrying two heterozygous variants in SYNE1 , CYP27A1 , ATP7B , COQ4 , or CLCN2 . The variants are shown in Table 2 . All the variants were classified as pathogenic or likely pathogenic according to the American College of Medical Genetics and Genomics (ACMG) guidelines. 8 The distances between the two variants ranged from 4.3 to 152 kb (Table 2 ). Table 2 Summary of identified heterozygous variants, distance between variants in each patient, and results of RCR amplification 1 (#10614) CYP27A1 c.410G > A c.1421G > A 5 kb chr2: 219,674,341–219,680,561 4/2 (NM_000784.4) (6.2 kb) 2 (#3946) SYNE1 c.21250C > T c.22622_22623delAA 18 kb chr6: 152,527,031–152,546,879 5/0 (NM_033071.4) (20 kb) 3 (JCAT0069) SYNE1 c.19943_19944delAG c.21100C > T 11 kb chr6: 152,545,700–152,558,998 8/0 (NM_033071.4) (13 kb) 4 (#11528) ATP7B c.2810delT c.2975C > T 3.3 kb chr13: 52,506,042–52,586,194 6/0 (NM_000053.4) (80 kb) 5 (C0438) SYNE1 c.4640_4643dup c.11576A > C 73 kb chr6: 152,599,149–152,671,977 6/0 (73 kb) (NM_033071.4) c.11576A > C c.18325C > T 79 kb chr6: 152,670,346–152,754,123 5/0 (84 kb) 6 (#12162) COQ4 c.238C > T c.718C > T 8.4 kb chr9:131,084,924 − 131,096,154 0/8 (NM_016035.5) (11 kb) 7 (#12302) CLCN2 c.61dupC c.983 + 2T > A 4.3 kb chr3:184,074,010–184,081,131 5/0 (NM_004366.6) (7.1 kb) For Patient 1, RCR products in which two alleles were amplified in a single tube were transformed in HST08 Premium Competent Cells (Takara Bio). The resultant colonies were picked, and plasmid DNA was extracted using a QIAprep Spin Miniprep Kit (QIAGEN). Direct nucleotide sequence analysis of plasmid DNA was performed. Results Optimization of molar ratio of gDNA to oriC-AmpR cassette and RCR success rate We used a gDNA sample (#9565) already analyzed by a long-read sequencer. First, we tried to amplify the genomic region of approximately 100 kb. We fixed the molar ratio of gDNA to the oriC – AmpR cassette at 1:100. We successfully amplified the region chr15:44852987–44956565 (104 kb) (Fig. 1 b). We assumed that two alleles were amplified in each tube (biallelic amplification). However, we observed that the amplification of only one allele (monoallelic amplification) sometimes occurred (Fig. 1 c). We examined whether the molar ratios of gDNA to the oriC – AmpR cassette at the ligation step affected the rate of successful RCR amplification and biallelic/monoallelic amplification. Thus, three regions on chromosomes 5, 10, and 14 (Table 1 ) were amplified at various molar ratios from 1:10 to 1:10000. As a result, we found that the molar ratio of gDNA to the oriC – AmpR cassette considerably affected the rate of successful RCR amplification (Table 3 ). When the input oriC-AmpR cassette amount was low (molar ratio of 1:10, 1.6 pg), the rate of successful RCR amplification was low (4/24), and only one allele was amplified (4/4). In contrast, when the input oriC – AmpR cassette amount was high (molar ratio of 1:1000, 1.6 ng), the rate of successful RCR amplification was high (20/24), and two alleles were amplified (18/20). Using the RCR product with monoallelic amplification, we were able to determine the phases of the variants in each region, which were consistent with those obtained by long-read sequencing. Table 3 Molar ratio of gDNA to OriC-AmpR cassette and its effect on success rate of RCR amplification and amplified alleles chr5:149,503,216–149,513,393 chr10:79,309,011–79,336,568 chr14:88,450,095–88,455,957 gDNA: oriC success rate of RCR amplification amplified alleles 1 allele/2 alleles success rate of RCR amplification amplified alleles 1 allele/2 alleles success rate of RCR amplification amplified alleles 1 allele/2 alleles 1:10 1/8 1/0 3/8 3/0 0/8 0/0 1:100 2/8 2/0 7/8 3/4 1/8 1/0 1:1000 8/8 0/8 7/8 0/7 3/8 2/1 1:10000 8/8 0/8 8/8 0/8 4/8 2/2 Determination of the phase of pathogenic variants by RCR amplification in a clinical setting We used gDNA samples from patients already analyzed by whole-exome sequence analysis. In these patients, multiple heterozygous variants were detected in genes for diseases with autosomal recessive inheritance. The variants are listed in Table 2 . All the variants were classified as pathogenic or likely pathogenic. Nucleotide sequence analysis of the RCR products was conducted to determine the phase in seven cases that require the confirmation of compound heterozygosity. We observed the monoallelic amplification of one of two variants in six of the seven cases (Table 2 ), for which the phasing of the two variants was immediately accomplished except for Patient 6 (Fig. 2 a, Supplementary Fig. 1). For Patient 1, four RCR products showed monoallelic amplification (only one allele was amplified in a tube), and in the other two PCR products (products 5 and 6), DNA molecules originating from both alleles were amplified (Fig. 2 a). The phase was determined using four RCR products with monoallelic amplification, but we further examined whether alleles could be separated by transforming the RCR products with biallelic amplification. Both alleles were almost equally amplified in product 6, and this product was transformed. Direct nucleotide sequence analysis of the plasmid DNA confirmed the phasing (Fig. 2 a). Thus, the transformation of the RCR products enabled us to separate the alleles and determine the phase. For Patient 5, the distance between two variants was 152 kb (Fig. 2 b). Although we first tried to amplify the region containing both variants (152 kb), it was impossible to amplify the entire region. WES showed that there was a heterozygous SNV located 78 kb away from the variant (c.11576A > C). We were also able to reconstruct the haplotype by utilizing this SNV (Fig. 2 b). We were able to amplify the region spanning variant 1 and the SNP, and the region spanning the SNP and variant 2, separately, which eventually enabled us to determine the phase. For Patient 6, two alleles were simultaneously amplified in each tube by RCR. The molar ratio of the gDNA: oriC – AmpR cassette was changed to 1:10 to facilitate monoallelic amplification. We achieved monoallelic amplification at this molar ratio, resulting in the confirmation of the compound heterozygosity (Supplementary Fig. 1e). Discussion In this study, we successfully amplified excised gDNA fragments up to 104 kb using RCR. The analysis of RCR products proved to be valuable for phasing multiple heterozygous variants. In fact, we successfully determined the phase of the variants in seven patients with various diseases with autosomal recessive inheritance. Increasing the amount of the oriC - AmpR cassette increases the rate of successful RCR amplification; however, this also increases the possibility of two alleles being amplified in a single tube. Conversely, reducing the amount of the oriC – AmpR cassette decreases the rate of successful RCR amplification but lowers the possibility of both alleles being amplified in a single tube, which means there is an increased possibility of monoallelic amplification. Thus, even when amplifying the same region, the number of amplified alleles varied depending on the conditions of the ligation step. Of note, achieving a monoallelic or skewed amplification was useful for the prompt phasing of the two variants. Maintaining a low concentration of the oriC – AmpR cassette is preferable for monoallelic amplification. However, if this proves challenging, increasing its concentration may improve the rate of successful RCR amplification. Furthermore, our method enables the transformation of RCR products in E. coli , which is also useful for phasing (Fig. 2 a). In Patient 1, a previous study of PCR amplification-based subcloning yielded inconsistent phasing results, presumably owing to incomplete elongation and mispriming on the heterologous allele. On the other hand, RCR products with monoallelic amplification showed consistent phase results in 16 individual RCR amplification processes. The design of sgRNA necessary for cleaving gDNA varies depending on the region of interest. However, when the entire gene can be amplified by RCR, the same sgRNA can be used for that gene, regardless of the variants of interest in the gene. In this study, we were able to design the sgRNA for the RCR amplification of the entire ATP7B as done for Patient 4. Thus, the phasing of any variants in this gene can be performed using the same sgRNA and oriC – AmpR cassette for patients harboring multiple heterozygous variants in ATP7B . As compared with other methods for phasing, the study reveals that the RCR method is simple and effective. First, no chimeric sequences were observed as in PCR-based methods. Second, this method has an advantage over ddPCR in that it does not require specialized devices. Third, designing sgRNA suitable for RCR is far more flexible than designing allele-specific probes and optimizing conditions in ddPCR. Fourth, although long-read sequencing presents another option for phasing, it is still costly. Lastly, the RCR method provides actual DNA fragments, which can be used for downstream functional analyses such as splicing assays. In summary, we established an RCR method using OriCiro technology, enabling long-range amplification. Depending on the molar ratio of the oriC-AmpR cassette to gDNA, we found that the success rate varied, and monoallelic or biallelic amplification occurred. Determining the phase is one of the burdens in the diagnosis of autosomal recessively inherited diseases, particularly when DNA samples of parents are unavailable. Thus, we propose a new method for phase determination in this study. Declarations Data Availability Data are available on request from the authors. Acknowledgments We thank OriCiro Genomics for helpful comments. Funding The work was supported by the Japan Agency for Medical Research and Development (grant numbers JP22ek0109491 and JP23ek0109673). Competing Interests None. Ethics Approval This study was approved by the Institutional Review Board of the University of Tokyo Hospital (G1396). Written information about the study was obtained from all the patients. References Jia H, Guo Y, Zhao W, Wang K. Long-range PCR in next-generation sequencing: comparison of six enzymes and evaluation on the MiSeq sequencer. Sci Rep. 2014;4:5737. Jansen R, Ledley FD. Disruption of phase during PCR amplification and cloning of heterozygous target sequences. Nucleic Acids Res. 1990;18:5153–5156. Regan JF, Kamitaki N, Legler T, Cooper S, Klitgord N, Karlin-Neumann G,et al. A rapid molecular approach for chromosomal phasing. PLoS One. 2015;10:e0118270. Su’etsugu M, Takada H, Katayama T, Tsujimoto H. Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle. Nucleic Acids Res. 2017;45:11525–11534. Kubota A, Ishiura H, Porto KJL, Tanaka M, Mitsui J, Unuma A, et al. DMD exon 2 duplication due to a complex genomic rearrangement is associated with a somatic mosaicism. Neuromuscul Disord. 2022;32:263–269. Li H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018; 34:3094–3100. Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, et al. Integrative genomics viewer. Nat Biotechnol. 2011;29:24–26. Richards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17:405–424. Additional Declarations There is no duality of interest Supplementary Files oriCsupple.docx oriCSupplementaryFigure.pptx Supplementary Fig. 1. RCR amplification and determination of the phase in Patients 2 –7 b. Patient 2. In these five RCR products, only one allele is amplified. c. Patient 3. In these eight RCR products, only one allele is amplified. d. Patient 4. In these six RCR products, only one allele is amplified. e. Patient 5. The region containing variant 1 (c.18325C>T, p.Q6109*) and heterozygous SNP (c.11576A>C) is amplified. In these six RCR products, only one allele is amplified. Similarly, the region containing heterozygous SNP (c.11576A>C) and variant 2 (c.4640_4643dup, p.Q1548Hfs*19) is amplified. In these five RCR products, only one allele is amplified. f. Patient 6. In these eight RCR products, two alleles are amplified. Then, the molar ratio of gDNA to oriC – AmpR cassette is changed from 1:100 to 1:10. In these six RCR products, only one allele is amplified in five products. g. Patient 7. In these five RCR products, only one allele is amplified. 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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-4025036","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":280048747,"identity":"c1beed35-1207-41bb-9e33-09824d92afe7","order_by":0,"name":"Tatsushi Toda","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYHACA4YPEAYzTCQBr3oeoBbGGSAWGylamHnQtOAH9uzNGz/b5jAkNsg3HzbmqWCQ529gePYAry08x4qlc7cBtbCxJSfznGEwnHGAId0ArxaJHAOwlv3HeIwP87YxMG5gYEiTIKDF+Lcl2BaIFntitJhJM0K1JAO1JBLWcuZYmWXvNgnjBra0ZMM5ZySSZxwm4Bf29ubNN35us5FtYD58WOJNhY1tf3tP2gN8WqAA4hImHhCDmSeNCB1QwPgDYvMx4rWMglEwCkbBSAAAmb48i6+F/54AAAAASUVORK5CYII=","orcid":"","institution":"Graduate School of Medicine, The University of Tokyo","correspondingAuthor":true,"prefix":"","firstName":"Tatsushi","middleName":"","lastName":"Toda","suffix":""},{"id":280048748,"identity":"0085b7ad-1ba4-4994-aad5-b248432a2264","order_by":1,"name":"Akihiko Mitsutake","email":"","orcid":"","institution":"International University of Health and Welfare","correspondingAuthor":false,"prefix":"","firstName":"Akihiko","middleName":"","lastName":"Mitsutake","suffix":""},{"id":280048749,"identity":"b5070126-cb9d-4fdf-af64-09286cdbd6bf","order_by":2,"name":"Hiroyuki Ishiura","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hiroyuki","middleName":"","lastName":"Ishiura","suffix":""},{"id":280048750,"identity":"e27023d8-793c-4932-a7ce-edbdb61a3c3c","order_by":3,"name":"Takashi Matsukawa","email":"","orcid":"","institution":"Graduate School of Medicine, The University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Takashi","middleName":"","lastName":"Matsukawa","suffix":""},{"id":280048751,"identity":"6a94c7d3-b9e7-4457-9283-3300e49ea61c","order_by":4,"name":"Jun Mitsui","email":"","orcid":"","institution":"Graduate School of Medicine, The University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Mitsui","suffix":""},{"id":280048752,"identity":"fb7a3985-eb79-41a6-bffc-cb7d4b421b88","order_by":5,"name":"Shoji Tsuji","email":"","orcid":"","institution":"Graduate School of Medicine, The University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Shoji","middleName":"","lastName":"Tsuji","suffix":""}],"badges":[],"createdAt":"2024-03-07 13:56:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4025036/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4025036/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53009967,"identity":"11a225a3-7040-4a87-be62-ffa1321fc53e","added_by":"auto","created_at":"2024-03-19 15:24:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":583439,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of RCR amplification and gel electrophoresis of RCR products\u003c/p\u003e\n\u003cp\u003ea. Overview of RCR amplification of excised genomic DNA fragment\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ein trans\u003c/em\u003e: gDNAs were cleaved using CRISPR/Cas9, generating cleaved segments with two heterozygous variants. The cleaved gDNA fragments were ligated to the \u003cem\u003eAmpR\u003c/em\u003e–\u003cem\u003eoriC\u003c/em\u003e cassette, forming circular DNA molecules. Two distinct types of circular DNA molecule, each containing one of the variants, were produced. Subsequently, the circular DNA molecules were amplified by RCR.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ein cis\u003c/em\u003e: gDNAs were cleaved using CRISPR/Cas9, generating cleaved segments with two heterozygous variants. The cleaved gDNA fragments were ligated to the \u003cem\u003eAmpR-oriC\u003c/em\u003ecassette, forming circular DNA molecules. Two types of circular DNA molecule, one containing both variants and the other without either variant, were produced. Subsequently, the circular DNA molecules were amplified by RCR.\u003c/p\u003e\n\u003cp\u003eb. Agarose gel electrophoresis of RCR products targeting 104 kb on chromosome 15\u003c/p\u003e\n\u003cp\u003eEight independent RCR products of 104 kb were loaded onto a 1% agarose gel. Undigested RCR products and those digested with SacI were assessed. Successful RCR amplification was observed in products 1, 2, 3, 4, 5, and 7 through the examination of digested RCR products sizes (16 kb, 14 kb x2, 9.2 kb x2, 6.1 kb, 4.8 kb, and 3.5 kb, respectively).\u003c/p\u003e\n\u003cp\u003eLanes 1, size standard marker (1 kb DNA ladder); 2, undigested RCR product 1; 3, digested RCR product 1; 4, undigested RCR product 2; 5, digested RCR product 2; 6, undigested RCR product 3; 7, digested RCR product 3; 8, undigested RCR product 4; 9, digested RCR product 4; 10, undigested RCR product 5; 11, digested RCR product 5; 12, undigested RCR product 6; 13, digested RCR product 6; 14, undigested RCR product 7; 15, digested RCR product 7; 16, undigested RCR product 8; 17, digested RCR product 8; 18, supercoiled DNA ladder.\u003c/p\u003e\n\u003cp\u003ec. Monoallelic and biallelic amplifications by RCR (chr10:79,309,011–79,336,568)\u003c/p\u003e\n\u003cp\u003eThe region chr10:79,309,011–79,336,568 is amplified by RCR and subsequently sequenced. One allele is amplified in products 1 and 2, whereas two alleles are amplified in product 3.\u003c/p\u003e","description":"","filename":"oriciroFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4025036/v1/f7505ad0171fa66840f5b76b.png"},{"id":53009968,"identity":"20ba06c4-c225-4434-91a7-9eb2bc2cb866","added_by":"auto","created_at":"2024-03-19 15:24:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":444535,"visible":true,"origin":"","legend":"\u003cp\u003eRCR amplification and determination of the phase\u003c/p\u003e\n\u003cp\u003ea. RCR amplification of CYP27A1 locus and subsequent transformation in Patient 1\u003c/p\u003e\n\u003cp\u003eTwo heterozygous missense variants [c.410G\u0026gt;A (p.R137Q) and c.1421G\u0026gt;A (p.R474Q)] in \u003cem\u003eCYP27A1\u003c/em\u003e were detected by whole-exome sequence analysis. A previous analysis by PCR amplification-based subcloning yielded inconsistent phase results. RCR was utilized to determine the phase of this patient. Six RCR products were prepared, and direct nucleotide sequence analysis was performed. Only one allele was amplified in products 1–4. Two alleles were amplified in products 5 and 6, for which Sanger sequencing suggested some skewed amplification. Product 6 was transformed in \u003cem\u003eE. coli\u003c/em\u003e and resultant colonies were picked. The successful transformation was confirmed by the electrophoresis of plasmid DNA, and direct nucleotide sequence analysis showed consistent phasing results.\u003c/p\u003e\n\u003cp\u003eb. Reconstruction of the haplotype by utilizing a heterozygous SNV between the variants in Patient 5\u003c/p\u003e\n\u003cp\u003eWe were unable to amplify the entire region containing two heterozygous variants (152 kb) probably owing to its large size. Whole-exome sequence analysis showed that there was a heterozygous SNV located 78 kb away from the variant (c.11576A\u0026gt;C), and we utilized this SNV to reconstruct the haplotype.\u003c/p\u003e","description":"","filename":"oriciroFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4025036/v1/6367a1a2ef12c8910a2c2fcf.png"},{"id":58670396,"identity":"550eec92-da3a-4d6c-93f5-1cae1f9839bc","added_by":"auto","created_at":"2024-06-19 14:33:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1560280,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4025036/v1/22d16c5d-bf56-42dd-9cf5-527bb5714c90.pdf"},{"id":53009970,"identity":"498a64d2-4d35-4c85-a87b-c6ab4ad2e584","added_by":"auto","created_at":"2024-03-19 15:24:51","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":168094,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"oriCsupple.docx","url":"https://assets-eu.researchsquare.com/files/rs-4025036/v1/c081619be957abbdc6db08d2.docx"},{"id":53009972,"identity":"24c2fe97-605f-4604-9515-52ec7513495d","added_by":"auto","created_at":"2024-03-19 15:24:51","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":600459,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Fig. 1. RCR amplification and determination of the phase in Patients 2 –7\u003c/p\u003e\n\u003cp\u003eb. Patient 2. In these five RCR products, only one allele is amplified.\u003c/p\u003e\n\u003cp\u003ec. Patient 3. In these eight RCR products, only one allele is amplified.\u003c/p\u003e\n\u003cp\u003ed. Patient 4. In these six RCR products, only one allele is amplified.\u003c/p\u003e\n\u003cp\u003ee. Patient 5. The region containing variant 1 (c.18325C\u0026gt;T, p.Q6109*) and heterozygous SNP (c.11576A\u0026gt;C) is amplified. In these six RCR products, only one allele is amplified. Similarly, the region containing heterozygous SNP (c.11576A\u0026gt;C) and variant 2 (c.4640_4643dup, p.Q1548Hfs*19) is amplified. In these five RCR products, only one allele is amplified.\u003c/p\u003e\n\u003cp\u003ef. Patient 6. In these eight RCR products, two alleles are amplified. Then, the molar ratio of gDNA to \u003cem\u003eoriC\u003c/em\u003e–\u003cem\u003eAmpR\u003c/em\u003e cassette is changed from 1:100 to 1:10. In these six RCR products, only one allele is amplified in five products.\u003c/p\u003e\n\u003cp\u003eg. Patient 7. In these five RCR products, only one allele is amplified.\u003c/p\u003e","description":"","filename":"oriCSupplementaryFigure.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4025036/v1/05cd2b6b884b729422db93c5.pptx"}],"financialInterests":"There is no duality of interest","formattedTitle":"Efficient variant phasing utilizing a replication cycle reaction system","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWhen two heterozygous variants are detected in genes for diseases with autosomal recessive inheritance, it is necessary to determine whether the two variants are located \u003cem\u003ein cis\u003c/em\u003e or \u003cem\u003ein trans\u003c/em\u003e. Although the genomic deoxyribonucleic acids (gDNAs) of the parents are usually analyzed, we face difficulties when parental DNAs are unavailable. Subcloning of long-range polymerase chain reaction (PCR) products obtained from gDNAs is useful, but long-range PCR\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e fails to amplify segments usually exceeding 10 kb. Subcloning of complementary DNA (cDNA) is another option, but its success largely depends on the availability of cells expressing the relevant messenger ribonucleic acid (mRNA). In addition, PCR amplification-based subcloning can occasionally yield inconsistent phase results potentially attributed to incomplete elongation and mispriming on the heterologous allele producing PCR chimera.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Droplet digital PCR is useful when these methods cannot be applied.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e This method isolates gDNA into 30,000\u0026ndash;50,000 droplets in such a way that a single DNA molecule in each droplet is amplified with allele-specific fluorescence-labeled probes. In principle, an appropriate combination of allele-specific fluorescent probes will produce double-positive droplets when DNA fragments containing physically linked variants are present in the same droplets.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e This method can be applied when the distance between variants is up to 100 kb,\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e although designing and optimizing allele-specific probes sometimes remain challenging.\u003c/p\u003e \u003cp\u003eRecently, Su\u0026rsquo;etsugu \u003cem\u003eet al\u003c/em\u003e. have successfully reconstituted the entire replication cycle of \u003cem\u003eE. coli in vitro\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e This replication cycle reaction (RCR) propagates amplified circular DNA in an isothermal reaction.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e This method can amplify a very large circular DNA carrying the \u003cem\u003eoriC\u003c/em\u003e sequence up to 0.2 Mb. Furthermore, it is characterized by a high accuracy of replication, approximately \u0026sim;1.2 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e per base per replication cycle. By combining the \u003cem\u003eoriC\u003c/em\u003e sequence and the excised gDNA fragment, we considered that we could amplify a genomic region too large to be amplified by PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). To accomplish the amplification of a large genomic DNA fragment carrying two heterozygous variants, we employed RCR and tried to determine whether the heterozygous variants are located \u003cem\u003ein cis\u003c/em\u003e or \u003cem\u003ein trans\u003c/em\u003e. First, we determined the optimal conditions for the RCR amplification of the excised gDNA fragment. We then investigated haplotyping through RCR amplification using DNA samples already analyzed by long-read sequencing, demonstrating successful haplotyping. Finally, we examined clinical samples in actual clinical practice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSamples\u003c/h2\u003e \u003cp\u003eIn this study, we utilized eight gDNA samples that had already been confirmed to carry two heterozygous variants on the basis of whole-exome or whole-genome sequence analyses. This study was approved by the Institutional Review Board of the University of Tokyo Hospital (G1396). Written informed consent was obtained from all the patients.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDigestion of gDNA\u003c/h2\u003e \u003cp\u003egDNAs were extracted from peripheral blood leukocytes following a standard procedure. Extracted gDNAs were digested using a CRISPR/Cas9 system. CHOPCHOP (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://chopchop.cbu.uib.no/\u003c/span\u003e\u003cspan address=\"https://chopchop.cbu.uib.no/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to design the CRISPR RNA (crRNA) sequence, a 20-nucleotide sequence upstream of the PAM sequence (5\u0026rsquo;-NGG-3\u0026rsquo;). Then, we synthesized a single guide RNA (sgRNA) from target-specific oligonucleotides with an EnGen sgRNA Synthesis Kit (New England Biolabs, Ipswich, MA. #E2233S) following the manufacturer\u0026rsquo;s instructions. After the purification of sgRNA using an RNeasy Plus Mini Kit (QIAGEN Venlo, The Netherlands), 10 \u0026micro;g of gDNA was cleaved with Cas9 Nuclease, \u003cem\u003eS. Pyogenes\u003c/em\u003e (New England Biolabs. #M0386S). 5 \u0026micro;L of NEBuffer r3.1, 5 \u0026micro;L of sgRNA (5 \u0026micro;M), 1 \u0026micro;L of Cas9 nuclease (20 \u0026micro;M), and nuclease-free water were mixed in a total volume of 50 \u0026micro;L. The mixture was incubated for 1 h at 37\u0026deg;C and then for 20 min at 65\u0026deg;C. The cleaved gDNA was purified with AmPure XP (Agencourt Bioscience Corp., Beverly, MA). The quality of gDNA was evaluated using a 4150 TapeStation (Agilent Technologies, Santa Clara, CA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRCR amplification of gDNA fragment\u003c/h2\u003e \u003cp\u003eAn \u003cem\u003eoriC\u003c/em\u003e cassette contained in the OriCiro Cell-Free Cloning System (OriCiro Genomics, Tokyo, Japan) was subcloned into pBR322 downstream of the ampicillin resistance gene using an In-Fusion\u0026reg; HD Cloning Kit (Takara Bio, Otsu, Japan). The nucleotide sequence of the plasmid is shown in Supplementary Methods 1.\u003c/p\u003e \u003cp\u003eWith the vector as a template, the region containing the cassette with \u003cem\u003erop\u003c/em\u003e, \u003cem\u003eori\u003c/em\u003e, \u003cem\u003eAmpR\u003c/em\u003e, and \u003cem\u003eoriC\u003c/em\u003e cassettes (\u003cem\u003eoriC\u003c/em\u003e\u0026ndash;\u003cem\u003eAmpR\u003c/em\u003e cassette) was amplified by PCR using primer pairs containing 60 nt overlapping sequences with the target sequence located on the 5\u0026rsquo;-side, followed by PCR product purification. This procedure was conducted for the subsequent transformation of RCR products. The detailed PCR procedure and the primer sequences are described in Supplementary Methods 2. Then, we ligated the cleaved gDNA fragment and PCR-amplified \u003cem\u003eoriC\u003c/em\u003e\u0026ndash;\u003cem\u003eAmpR\u003c/em\u003e cassette using the 2xRA mix contained in the OriCiro Cell-Free Cloning System to form a circular DNA molecule. The reaction conditions are as follows. The cleaved gDNA fragment and \u003cem\u003eoriC-AmpR\u003c/em\u003e cassette were mixed in a total of 2.4 \u0026micro;L at various molar ratios of gDNA to the \u003cem\u003eoriC-AmpR\u003c/em\u003e cassette (1:10, 1:100, 1:1000, and 1:10000). The amount of gDNA input was fixed at 100 ng (1.4 \u0026micro;L), and various amounts of \u003cem\u003eoriC\u003c/em\u003e\u0026ndash;\u003cem\u003eAmpR\u003c/em\u003e cassette (1.6 pg, 16 pg, 160 pg, and 1.6 ng) were added to a total volume of 1.1 \u0026micro;L. After adding 2.5 \u0026micro;L of 2x RA Mix, the mixture was incubated for 1 hour at 42\u0026deg;C to form circular DNA molecules by an enzyme-based annealing mechanism using 2xRA Mix.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Afterward, we mixed 0.5 \u0026micro;L of the product with 1 \u0026micro;L of 5x Buffer I, 1 \u0026micro;L of 5x Buffer II, 0.5 \u0026micro;L of 10x RE mix, and 2 \u0026micro;L of nuclease-free water. The resulting mixture was then incubated for 16 hours at 33\u0026deg;C for the amplification of circular DNA molecules by RCR. The successful amplification by RCR was confirmed by agarose gel electrophoresis (0.6%) of RCR products (100 V, 1 h) after digestion with specific restriction enzymes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetermination of the optimal molar ratio of gDNA to\u003c/b\u003e \u003cb\u003eoriC\u003c/b\u003e \u003cb\u003ecassette\u003c/b\u003e\u003c/p\u003e \u003cp\u003eVarious molar ratios of gDNA to the \u003cem\u003eoriC-AmpR\u003c/em\u003e cassette were evaluated to determine the optimal conditions for the ligation step. In this experiment, we used a gDNA sample (#9565) already analyzed by long-read sequencing. Whole-genome sequence analysis was conducted using a long-read sequencer, PacBio RSII (Pacific Biosciences), utilizing gDNAs extracted from lymphoblastoid cells.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Circular consensus sequence (CCS) reads were generated from the subreads using the ccs application SMRT Link version 6.0.0.47841 provided by PacBio (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ccs.how/\u003c/span\u003e\u003cspan address=\"https://ccs.how/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The reads were aligned to GRCh38/hg38 using Minimap2.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e The result was visualized using Integrated Genome Viewer (IGV) (version 2.8.6).\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThree regions (chr5:149,503,216\u0026ndash;149,513,393; chr10:79,309,011\u0026ndash;79,336,568; chr14:88,450,095\u0026ndash;88,455,957) containing heterozygous single nucleotide variants (SNVs), which were phased by the long-read sequencing, were selected, digested with sgRNA, and then amplified by RCR. Heterozygous SNVs located in the regions are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Each sample was amplified by RCR in 8 independent tubes for each molar ratio. The successful amplification by RCR was confirmed by electrophoresis after digestion with specific restriction enzymes. After confirming successful amplification, the RCR products were then subjected to direct nucleotide sequence analysis to examine whether one or two alleles were amplified in each tube after RCR amplification. The phase of the variants was determined using RCR products in which one allele was amplified.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAmplified genomic region and heterozygous SNPs in each region\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eregion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003esize (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSNP1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSNP2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003echr5:149,503,216\u0026ndash;149,513,393\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e10,178\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003echr5:149,511,792\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003echr5:149,512,042\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA/G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA/G\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003echr10:79,309,011\u0026ndash;79,336,568\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e27,558\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003echr10:79,316,010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC/G\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003echr14:88,450,095\u0026ndash;88,455,957\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e5,863\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003echr14:88,453,186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003echr14:88,453,278\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA/G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eG/T\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDirect nucleotide sequence analysis of RCR products\u003c/h2\u003e \u003cp\u003eThe obtained RCR products were purified employing ExoSAP-IT for PCR Product Clean-Up (Affymetrix, Santa Clara, CA), with one cycle of 30 min at 37\u0026deg;C followed by another cycle of 15 min at 80\u0026deg;C. The purified RCR products were subjected to nucleotide sequence analysis employing the ABI PRISM BigDye 3.1 terminator method (Applied Biosystems, Foster City, CA) and the ABI PRISM\u0026reg; 3100 Genetic Analyzer (Applied Biosystems). The sequences of the primers are shown in Supplementary Methods 3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of gDNAs from patients carrying two heterozygous variants in a clinical setting\u003c/h2\u003e \u003cp\u003eIn this experiment, we analyzed seven gDNA samples already analyzed by whole-exome sequence analysis. gDNA samples were from seven patients carrying two heterozygous variants in \u003cem\u003eSYNE1\u003c/em\u003e, \u003cem\u003eCYP27A1\u003c/em\u003e, \u003cem\u003eATP7B\u003c/em\u003e, \u003cem\u003eCOQ4\u003c/em\u003e, or \u003cem\u003eCLCN2\u003c/em\u003e. The variants are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. All the variants were classified as pathogenic or likely pathogenic according to the American College of Medical Genetics and Genomics (ACMG) guidelines.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e The distances between the two variants ranged from 4.3 to 152 kb (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of identified heterozygous variants, distance between variants in each patient, and results of RCR amplification\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e(#10614)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCYP27A1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ec.410G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ec.1421G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e5 kb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003echr2: 219,674,341\u0026ndash;219,680,561\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4/2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(NM_000784.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e(6.2 kb)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003cp\u003e(#3946)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSYNE1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ec.21250C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ec.22622_22623delAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e18 kb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003echr6: 152,527,031\u0026ndash;152,546,879\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5/0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(NM_033071.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e(20 kb)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003cp\u003e(JCAT0069)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSYNE1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ec.19943_19944delAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ec.21100C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e11 kb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003echr6: 152,545,700\u0026ndash;152,558,998\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8/0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(NM_033071.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e(13 kb)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003cp\u003e(#11528)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eATP7B\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ec.2810delT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ec.2975C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e3.3 kb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003echr13: 52,506,042\u0026ndash;52,586,194\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6/0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(NM_000053.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e(80 kb)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003cp\u003e(C0438)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eSYNE1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ec.4640_4643dup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ec.11576A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e73 kb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003echr6: 152,599,149\u0026ndash;152,671,977\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6/0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e(73 kb)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e(NM_033071.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ec.11576A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ec.18325C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e79 kb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003echr6: 152,670,346\u0026ndash;152,754,123\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5/0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e(84 kb)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003cp\u003e(#12162)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCOQ4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ec.238C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ec.718C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e8.4 kb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003echr9:131,084,924\u0026thinsp;\u0026minus;\u0026thinsp;131,096,154\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0/8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(NM_016035.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e(11 kb)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003cp\u003e(#12302)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCLCN2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ec.61dupC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ec.983\u0026thinsp;+\u0026thinsp;2T\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e4.3 kb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003echr3:184,074,010\u0026ndash;184,081,131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5/0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(NM_004366.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e(7.1 kb)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFor Patient 1, RCR products in which two alleles were amplified in a single tube were transformed in HST08 Premium Competent Cells (Takara Bio). The resultant colonies were picked, and plasmid DNA was extracted using a QIAprep Spin Miniprep Kit (QIAGEN). Direct nucleotide sequence analysis of plasmid DNA was performed.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eOptimization of molar ratio of gDNA to\u003c/b\u003e \u003cb\u003eoriC-AmpR\u003c/b\u003e \u003cb\u003ecassette and RCR success rate\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe used a gDNA sample (#9565) already analyzed by a long-read sequencer. First, we tried to amplify the genomic region of approximately 100 kb. We fixed the molar ratio of gDNA to the \u003cem\u003eoriC\u003c/em\u003e\u0026ndash;\u003cem\u003eAmpR\u003c/em\u003e cassette at 1:100. We successfully amplified the region chr15:44852987\u0026ndash;44956565 (104 kb) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eWe assumed that two alleles were amplified in each tube (biallelic amplification). However, we observed that the amplification of only one allele (monoallelic amplification) sometimes occurred (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). We examined whether the molar ratios of gDNA to the \u003cem\u003eoriC\u003c/em\u003e\u0026ndash;\u003cem\u003eAmpR\u003c/em\u003e cassette at the ligation step affected the rate of successful RCR amplification and biallelic/monoallelic amplification. Thus, three regions on chromosomes 5, 10, and 14 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were amplified at various molar ratios from 1:10 to 1:10000. As a result, we found that the molar ratio of gDNA to the \u003cem\u003eoriC\u003c/em\u003e\u0026ndash;\u003cem\u003eAmpR\u003c/em\u003e cassette considerably affected the rate of successful RCR amplification (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). When the input \u003cem\u003eoriC-AmpR\u003c/em\u003e cassette amount was low (molar ratio of 1:10, 1.6 pg), the rate of successful RCR amplification was low (4/24), and only one allele was amplified (4/4). In contrast, when the input \u003cem\u003eoriC\u003c/em\u003e\u0026ndash;\u003cem\u003eAmpR\u003c/em\u003e cassette amount was high (molar ratio of 1:1000, 1.6 ng), the rate of successful RCR amplification was high (20/24), and two alleles were amplified (18/20). Using the RCR product with monoallelic amplification, we were able to determine the phases of the variants in each region, which were consistent with those obtained by long-read sequencing.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMolar ratio of gDNA to OriC-AmpR cassette and its effect on success rate of RCR amplification and amplified alleles\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003echr5:149,503,216\u0026ndash;149,513,393\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003echr10:79,309,011\u0026ndash;79,336,568\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003echr14:88,450,095\u0026ndash;88,455,957\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003egDNA:\u003cem\u003eoriC\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003esuccess rate of\u003c/p\u003e \u003cp\u003eRCR amplification\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eamplified alleles\u003c/p\u003e \u003cp\u003e1 allele/2 alleles\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003esuccess rate of\u003c/p\u003e \u003cp\u003eRCR amplification\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eamplified alleles\u003c/p\u003e \u003cp\u003e1 allele/2 alleles\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003esuccess rate of\u003c/p\u003e \u003cp\u003eRCR amplification\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eamplified alleles\u003c/p\u003e \u003cp\u003e1 allele/2 alleles\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1:10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1:100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3/4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1/0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0/7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2/1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1:10000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2/2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of the phase of pathogenic variants by RCR amplification in a clinical setting\u003c/h2\u003e \u003cp\u003eWe used gDNA samples from patients already analyzed by whole-exome sequence analysis. In these patients, multiple heterozygous variants were detected in genes for diseases with autosomal recessive inheritance. The variants are listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. All the variants were classified as pathogenic or likely pathogenic. Nucleotide sequence analysis of the RCR products was conducted to determine the phase in seven cases that require the confirmation of compound heterozygosity. We observed the monoallelic amplification of one of two variants in six of the seven cases (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), for which the phasing of the two variants was immediately accomplished except for Patient 6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor Patient 1, four RCR products showed monoallelic amplification (only one allele was amplified in a tube), and in the other two PCR products (products 5 and 6), DNA molecules originating from both alleles were amplified (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The phase was determined using four RCR products with monoallelic amplification, but we further examined whether alleles could be separated by transforming the RCR products with biallelic amplification. Both alleles were almost equally amplified in product 6, and this product was transformed. Direct nucleotide sequence analysis of the plasmid DNA confirmed the phasing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Thus, the transformation of the RCR products enabled us to separate the alleles and determine the phase.\u003c/p\u003e \u003cp\u003eFor Patient 5, the distance between two variants was 152 kb (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Although we first tried to amplify the region containing both variants (152 kb), it was impossible to amplify the entire region. WES showed that there was a heterozygous SNV located 78 kb away from the variant (c.11576A\u0026thinsp;\u0026gt;\u0026thinsp;C). We were also able to reconstruct the haplotype by utilizing this SNV (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). We were able to amplify the region spanning variant 1 and the SNP, and the region spanning the SNP and variant 2, separately, which eventually enabled us to determine the phase.\u003c/p\u003e \u003cp\u003eFor Patient 6, two alleles were simultaneously amplified in each tube by RCR. The molar ratio of the gDNA:\u003cem\u003eoriC\u003c/em\u003e\u0026ndash;\u003cem\u003eAmpR\u003c/em\u003e cassette was changed to 1:10 to facilitate monoallelic amplification. We achieved monoallelic amplification at this molar ratio, resulting in the confirmation of the compound heterozygosity (Supplementary Fig.\u0026nbsp;1e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we successfully amplified excised gDNA fragments up to 104 kb using RCR. The analysis of RCR products proved to be valuable for phasing multiple heterozygous variants. In fact, we successfully determined the phase of the variants in seven patients with various diseases with autosomal recessive inheritance.\u003c/p\u003e \u003cp\u003eIncreasing the amount of the \u003cem\u003eoriC\u003c/em\u003e-\u003cem\u003eAmpR\u003c/em\u003e cassette increases the rate of successful RCR amplification; however, this also increases the possibility of two alleles being amplified in a single tube. Conversely, reducing the amount of the \u003cem\u003eoriC\u003c/em\u003e\u0026ndash;\u003cem\u003eAmpR\u003c/em\u003e cassette decreases the rate of successful RCR amplification but lowers the possibility of both alleles being amplified in a single tube, which means there is an increased possibility of monoallelic amplification. Thus, even when amplifying the same region, the number of amplified alleles varied depending on the conditions of the ligation step. Of note, achieving a monoallelic or skewed amplification was useful for the prompt phasing of the two variants. Maintaining a low concentration of the \u003cem\u003eoriC\u003c/em\u003e\u0026ndash;\u003cem\u003eAmpR\u003c/em\u003e cassette is preferable for monoallelic amplification. However, if this proves challenging, increasing its concentration may improve the rate of successful RCR amplification. Furthermore, our method enables the transformation of RCR products in \u003cem\u003eE. coli\u003c/em\u003e, which is also useful for phasing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eIn Patient 1, a previous study of PCR amplification-based subcloning yielded inconsistent phasing results, presumably owing to incomplete elongation and mispriming on the heterologous allele. On the other hand, RCR products with monoallelic amplification showed consistent phase results in 16 individual RCR amplification processes.\u003c/p\u003e \u003cp\u003eThe design of sgRNA necessary for cleaving gDNA varies depending on the region of interest. However, when the entire gene can be amplified by RCR, the same sgRNA can be used for that gene, regardless of the variants of interest in the gene. In this study, we were able to design the sgRNA for the RCR amplification of the entire \u003cem\u003eATP7B\u003c/em\u003e as done for Patient 4. Thus, the phasing of any variants in this gene can be performed using the same sgRNA and \u003cem\u003eoriC\u003c/em\u003e\u0026ndash;\u003cem\u003eAmpR\u003c/em\u003e cassette for patients harboring multiple heterozygous variants in \u003cem\u003eATP7B\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAs compared with other methods for phasing, the study reveals that the RCR method is simple and effective. First, no chimeric sequences were observed as in PCR-based methods. Second, this method has an advantage over ddPCR in that it does not require specialized devices. Third, designing sgRNA suitable for RCR is far more flexible than designing allele-specific probes and optimizing conditions in ddPCR. Fourth, although long-read sequencing presents another option for phasing, it is still costly. Lastly, the RCR method provides actual DNA fragments, which can be used for downstream functional analyses such as splicing assays.\u003c/p\u003e \u003cp\u003eIn summary, we established an RCR method using OriCiro technology, enabling long-range amplification. Depending on the molar ratio of the \u003cem\u003eoriC-AmpR\u003c/em\u003e cassette to gDNA, we found that the success rate varied, and monoallelic or biallelic amplification occurred. Determining the phase is one of the burdens in the diagnosis of autosomal recessively inherited diseases, particularly when DNA samples of parents are unavailable. Thus, we propose a new method for phase determination in this study.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are available on request from the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank OriCiro Genomics for helpful comments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was supported by the Japan Agency for Medical Research and Development (grant numbers JP22ek0109491 and JP23ek0109673).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Institutional Review Board of the University of Tokyo Hospital (G1396). Written information about the study was obtained from all the patients.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJia H, Guo Y, Zhao W, Wang K. Long-range PCR in next-generation sequencing: comparison of six enzymes and evaluation on the MiSeq sequencer. Sci Rep. 2014;4:5737.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJansen R, Ledley FD. Disruption of phase during PCR amplification and cloning of heterozygous target sequences. Nucleic Acids Res. 1990;18:5153\u0026ndash;5156.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRegan JF, Kamitaki N, Legler T, Cooper S, Klitgord N, Karlin-Neumann G,et al. A rapid molecular approach for chromosomal phasing. PLoS One. 2015;10:e0118270.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu\u0026rsquo;etsugu M, Takada H, Katayama T, Tsujimoto H. Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle. Nucleic Acids Res. 2017;45:11525\u0026ndash;11534.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKubota A, Ishiura H, Porto KJL, Tanaka M, Mitsui J, Unuma A, et al. DMD exon 2 duplication due to a complex genomic rearrangement is associated with a somatic mosaicism. Neuromuscul Disord. 2022;32:263\u0026ndash;269.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018; 34:3094\u0026ndash;3100.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRobinson JT, Thorvaldsd\u0026oacute;ttir H, Winckler W, Guttman M, Lander ES, Getz G, et al. Integrative genomics viewer. Nat Biotechnol. 2011;29:24\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRichards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17:405\u0026ndash;424.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"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":"","lastPublishedDoi":"10.21203/rs.3.rs-4025036/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4025036/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWhen two heterozygous variants are detected in genes for diseases with autosomal recessive inheritance, determining whether the two variants are located \u003cem\u003ein cis\u003c/em\u003e or \u003cem\u003ein trans\u003c/em\u003e is crucial. Subcloning long-range PCR products or cDNA is limited by factors such as the distance between variants (up to 10 kb) and cDNA availability. Droplet digital PCR, effective up to distances of 100 kb, faces challenges in specific probe design. We utilized replication cycle reaction (RCR) for amplifying large genomic DNA segments with multiple heterozygous variants. RCR is an \u003cem\u003ein vitro\u003c/em\u003e replication cycle based on chromosome replication in \u003cem\u003eEscherichia coli\u003c/em\u003e. Circular DNA molecules were generated by combining CRISPR/Cas9-cleaved genomic DNA fragments with an \u003cem\u003eoriC\u003c/em\u003e\u0026ndash;\u003cem\u003eAmpR\u003c/em\u003e cassette, followed by amplification through RCR. Various molar ratios of gDNA to the \u003cem\u003eoriC\u003c/em\u003e\u0026ndash;\u003cem\u003eAmpR\u003c/em\u003e cassette were evaluated to optimize the ligation step. We analyzed gDNAs from seven patients carrying two heterozygous pathogenic variants with distances ranging from 4.3 to 152 kb. A genomic region up to 104 kb could be amplified by RCR. A higher input of the \u003cem\u003eoriC\u003c/em\u003e\u0026ndash;\u003cem\u003eAmpR\u003c/em\u003e cassette resulted in a higher rate of successful RCR amplification and a lower rate of successful monoallelic amplification. Monoallelic clonal amplification occurred in six patients, facilitating a rapid determination of variant phases. A haplotype was successfully reconstructed using an SNP located 78 kb away from the variant in one patient with two variants separated by a 152 kb distance. Our method proves particularly valuable for phasing multiple heterozygous variants separated over long genomic distances.\u003c/p\u003e","manuscriptTitle":"Efficient variant phasing utilizing a replication cycle reaction system","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-19 15:24:46","doi":"10.21203/rs.3.rs-4025036/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":"1e87db62-df6e-4ca2-babd-3dbf6e40634a","owner":[],"postedDate":"March 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":29495514,"name":"Biological sciences/Genetics/Sequencing/DNA sequencing"},{"id":29495515,"name":"Health sciences/Diseases/Neurological disorders/Spinocerebellar ataxia"}],"tags":[],"updatedAt":"2024-06-19T14:25:11+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-19 15:24:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4025036","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4025036","identity":"rs-4025036","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}