Identification of a CCG-Enriched Expanded Allele in Patients with Myotonic Dystrophy Type 1 Using Amplification-Free Long-Read Sequencing
2022; Elsevier BV; Volume: 24; Issue: 11 Linguagem: Inglês
10.1016/j.jmoldx.2022.08.003
ISSN1943-7811
AutoresYu‐Chih Tsai, Laure de Pontual, Cheryl Heiner, Tanya Stojkovic, Denis Furling, Guillaume Bassez, Geneviève Gourdon, Stéphanie Tomé,
Tópico(s)Fungal and yeast genetics research
ResumoMyotonic dystrophy type 1 (DM1) exhibits highly heterogeneous clinical manifestations caused by an unstable CTG repeat expansion reaching up to 4000 CTG. The clinical variability depends on CTG repeat number, CNG repeat interruptions, and somatic mosaicism. Currently, none of these factors are simultaneously and accurately determined due to the limitations of gold standard methods used in clinical and research laboratories. An amplicon method for targeting the DMPK locus using single-molecule real-time sequencing was recently developed to accurately analyze expanded alleles. However, amplicon-based sequencing still depends on PCR, and the inherent bias toward preferential amplification of smaller repeats can be problematic in DM1. Thus, an amplification-free long-read sequencing method was developed by using CRISPR/Cas9 technology in DM1. This method was used to sequence the DMPK locus in patients with CTG repeat expansion ranging from 130 to >1000 CTG. We showed that elimination of PCR amplification improves the accuracy of measurement of inherited repeat number and somatic repeat variations, two key factors in DM1 severity and age at onset. For the first time, an expansion composed of >85% CCG repeats was identified by using this innovative method in a DM1 family with an atypical clinical profile. No-amplification targeted sequencing represents a promising method that can overcome research and diagnosis shortcomings, with translational implications for clinical and genetic counseling in DM1. Myotonic dystrophy type 1 (DM1) exhibits highly heterogeneous clinical manifestations caused by an unstable CTG repeat expansion reaching up to 4000 CTG. The clinical variability depends on CTG repeat number, CNG repeat interruptions, and somatic mosaicism. Currently, none of these factors are simultaneously and accurately determined due to the limitations of gold standard methods used in clinical and research laboratories. An amplicon method for targeting the DMPK locus using single-molecule real-time sequencing was recently developed to accurately analyze expanded alleles. However, amplicon-based sequencing still depends on PCR, and the inherent bias toward preferential amplification of smaller repeats can be problematic in DM1. Thus, an amplification-free long-read sequencing method was developed by using CRISPR/Cas9 technology in DM1. This method was used to sequence the DMPK locus in patients with CTG repeat expansion ranging from 130 to >1000 CTG. We showed that elimination of PCR amplification improves the accuracy of measurement of inherited repeat number and somatic repeat variations, two key factors in DM1 severity and age at onset. For the first time, an expansion composed of >85% CCG repeats was identified by using this innovative method in a DM1 family with an atypical clinical profile. No-amplification targeted sequencing represents a promising method that can overcome research and diagnosis shortcomings, with translational implications for clinical and genetic counseling in DM1. Short tandem repeats are an important source of genetic variation and phenotypic variability in disease and health that are not always well characterized and understood due to their complexities. Among these repeated elements, unstable repeat expansions are associated with >20 diseases, including the complex and variable myotonic dystrophy type 1 (DM1) disorder.1Khristich A.N. Mirkin S.M. On the wrong DNA track: molecular mechanisms of repeat-mediated genome instability.J Biol Chem. 2020; 295: 4134-4170Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar DM1 is caused by an unstable CTG repeat expansion in the 3′ untranslated region of the DM1 protein kinase (DMPK) gene, which usually increases across generations and over time in somatic tissues.2Harper P.S. Myotonic Dystrophy. W.B. Saunders, London, UK2001Google Scholar,3Tomé S. Gourdon G. DM1 phenotype variability and triplet repeat instability: challenges in the development of new therapies.Int J Mol Sci. 2020; 21: 457Crossref Scopus (24) Google Scholar In DM1, longer expanded alleles are usually associated with a worsening of clinical severity and an earlier age of onset.4De Antonio M. Dogan C. Hamroun D. Mati M. Zerrouki S. Eymard B. Katsahian S. Bassez G. French Myotonic Dystrophy Clinical NetworkUnravelling the myotonic dystrophy type 1 clinical spectrum: a systematic registry-based study with implications for disease classification.Revue Neurol (Paris). 2016; 172: 572-580Crossref PubMed Scopus (147) Google Scholar This anticipation phenomenon is particularly obvious in DM1.5Harper P.S. Harley H.G. Reardon W. Shaw D.J. Anticipation in myotonic dystrophy: new light on an old problem.Am J Hum Genet. 1992; 51: 10-16PubMed Google Scholar DM1 is mainly characterized by an unusually broad clinical spectrum of symptoms divided into five distinct clinical forms ranging from late onset to the congenital forms, which are often associated with the largest size of inherited disease–associated allele.4De Antonio M. Dogan C. Hamroun D. Mati M. Zerrouki S. Eymard B. Katsahian S. Bassez G. French Myotonic Dystrophy Clinical NetworkUnravelling the myotonic dystrophy type 1 clinical spectrum: a systematic registry-based study with implications for disease classification.Revue Neurol (Paris). 2016; 172: 572-580Crossref PubMed Scopus (147) Google Scholar Facial dysmorphisms, muscle weakness, and cognitive impairment are more frequent symptoms at an earlier onset, whereas cardiac defects and cataracts are more common in DM1 patients with later forms of the disease. In DM1, it is laborious, for several reasons, to diagnose patients and classify them into distinct clinical categories based exclusively on mutation status and size of CTG repeats when known. First, the long-expanded allele and the precise number of CTG repeats are complicated to identify and measure by using conventional methods, particularly for the larger repeat expansions.6Kamsteeg E.-J. Kress W. Catalli C. Hertz J.M. Witsch-Baumgartner M. Buckley M.F. van Engelen B.G.M. Schwartz M. Scheffer H. Best practice guidelines and recommendations on the molecular diagnosis of myotonic dystrophy types 1 and 2.Eur J Hum Genet. 2012; 20: 1203-1208Crossref PubMed Scopus (103) Google Scholar,7Savić Pavićević D. Miladinović J. Brkušanin M. Šviković S. Djurica S. Brajušković G. Romac S. Molecular genetics and genetic testing in myotonic dystrophy type 1.Biomed Res Int. 2013; 2013: 1391821Crossref Scopus (44) Google Scholar Second, patients with DM1 exhibit high clinical and genetic variability that cannot be exclusively explained by the size of the CTG repeats.4De Antonio M. Dogan C. Hamroun D. Mati M. Zerrouki S. Eymard B. Katsahian S. Bassez G. French Myotonic Dystrophy Clinical NetworkUnravelling the myotonic dystrophy type 1 clinical spectrum: a systematic registry-based study with implications for disease classification.Revue Neurol (Paris). 2016; 172: 572-580Crossref PubMed Scopus (147) Google Scholar Third, additional factors such as somatic mosaicism are important disease modifiers contributing to the high genotype and phenotype variability observed in DM1. Somatic mosaicism biased toward expansions contributes to the progressive nature of the various DM1 symptoms and also to the variation in the age of onset.8Morales F. Couto J.M. Higham C.F. Hogg G. Cuenca P. Braida C. Wilson R.H. Adam B. del Valle G. Brian R. Sittenfeld M. Ashizawa T. Wilcox A. Wilcox D.E. Monckton D.G. Somatic instability of the expanded CTG triplet repeat in myotonic dystrophy type 1 is a heritable quantitative trait and modifier of disease severity.Hum Mol Genet. 2012; 21: 3558-3567Crossref PubMed Scopus (131) Google Scholar, 9Morales F. Vásquez M. Corrales E. Vindas-Smith R. Santamaría-Ulloa C. Zhang B. Sirito M. Estecio M.R. Krahe R. Monckton D.G. Longitudinal increases in somatic mosaicism of the expanded CTG repeat in myotonic dystrophy type 1 are associated with variation in age-at-onset.Hum Mol Genet. 2020; 29: 2496-2507Crossref PubMed Scopus (24) Google Scholar, 10Cumming S.A. Jimenez-Moreno C. Okkersen K. Wenninger S. Daidj F. Hogarth F. Littleford R. Gorman G. Bassez G. Schoser B. Lochmüller H. van Engelen B.G.M. Monckton D.G. OPTIMISTIC ConsortiumGenetic determinants of disease severity in the myotonic dystrophy type 1 OPTIMISTIC cohort.Neurology. 2019; 93: e995-e1009Crossref PubMed Scopus (51) Google Scholar, 11Overend G. Légaré C. Mathieu J. Bouchard L. Gagnon C. Monckton D.G. Allele length of the DMPK CTG repeat is a predictor of progressive myotonic dystrophy type 1 phenotypes.Hum Mol Genet. 2019; 28: 2245-2254Crossref PubMed Scopus (32) Google Scholar Fourth, the majority of patients with DM1 inherit pure CTG repeat expansion. However, >8% of patients with known DM1 carry interruptions that vary in type (CCG, CAG, CTC, and CGG) and number between families and also among individuals of the same family.12Peric S. Pesovic J. Savic-Pavicevic D. Rakocevic Stojanovic V. Meola G. Molecular and clinical implications of variant repeats in myotonic dystrophy type 1.Int J Mol Sci. 2021; 23: 354Crossref PubMed Scopus (8) Google Scholar Interruptions are frequently associated with intergenerational contractions and stabilization of the CTG repeat as well as milder DM1 symptoms and/or additional symptoms.11Overend G. Légaré C. Mathieu J. Bouchard L. Gagnon C. Monckton D.G. Allele length of the DMPK CTG repeat is a predictor of progressive myotonic dystrophy type 1 phenotypes.Hum Mol Genet. 2019; 28: 2245-2254Crossref PubMed Scopus (32) Google Scholar,13Braida C. Stefanatos R.K.A. Adam B. Mahajan N. Smeets H.J.M. Niel F. Goizet C. Arveiler B. Koenig M. Lagier-Tourenne C. Mandel J.-L. Faber C.G. de Die-Smulders C.E.M. Spaans F. Monckton D.G. Variant CCG and GGC repeats within the CTG expansion dramatically modify mutational dynamics and likely contribute toward unusual symptoms in some myotonic dystrophy type 1 patients.Hum Mol Genet. 2010; 19: 1399-1412Crossref PubMed Scopus (117) Google Scholar, 14Musova Z. Mazanec R. Krepelova A. Ehler E. Vales J. Jaklova R. Prochazka T. Koukal P. Marikova T. Kraus J. Havlovicova M. Sedlacek Z. Highly unstable sequence interruptions of the CTG repeat in the myotonic dystrophy gene.Am J Med Genet A. 2009; 149A: 1365-1374Crossref PubMed Scopus (124) Google Scholar, 15Botta A. Rossi G. Marcaurelio M. Fontana L. D'Apice M.R. Brancati F. Massa R. Monckton D.G. Sangiuolo F. Novelli G. Identification and characterization of 5′ CCG interruptions in complex DMPK expanded alleles.Eur J Hum Genet. 2017; 25: 257-261Crossref PubMed Scopus (29) Google Scholar, 16Cumming S.A. Hamilton M.J. Robb Y. Gregory H. McWilliam C. Cooper A. Adam B. McGhie J. Hamilton G. Herzyk P. Tschannen M.R. Worthey E. Petty R. Ballantyne B. Warner J. Farrugia M.E. Longman C. Monckton D.G. Scottish Myotonic Dystrophy ConsortiumDe novo repeat interruptions are associated with reduced somatic instability and mild or absent clinical features in myotonic dystrophy type 1.Eur J Hum Genet. 2018; 26: 1635-1647Crossref PubMed Scopus (53) Google Scholar, 17Santoro M. Masciullo M. Silvestri G. Novelli G. Botta A. Myotonic dystrophy type 1: role of CCG, CTC and CGG interruptions within DMPK alleles in the pathogenesis and molecular diagnosis.Clin Genet. 2017; 92: 355-364Crossref PubMed Scopus (40) Google Scholar, 18Tomé S. Dandelot E. Dogan C. Bertrand A. Geneviève D. Péréon Y. DM Contraction Study Group Simon M. Bonnefont J.-P. Bassez G. Gourdon G. Unusual association of a unique CAG interruption in 5′ of DM1 CTG repeats with intergenerational contractions and low somatic mosaicism.Hum Mutat. 2018; 39: 970-982Crossref PubMed Scopus (29) Google Scholar, 19Wenninger S. Cumming S.A. Gutschmidt K. Okkersen K. Jimenez-Moreno A.C. Daidj F. Lochmüller H. Hogarth F. Knoop H. Bassez G. Monckton D.G. van Engelen B.G.M. Schoser B. Associations between variant repeat interruptions and clinical outcomes in myotonic dystrophy type 1.Neurol Genet. 2021; 7: e572Crossref PubMed Scopus (8) Google Scholar, 20Miller J.N. van der Plas E. Hamilton M. Koscik T.R. Gutmann L. Cumming S.A. Monckton D.G. Nopoulos P.C. Variant repeats within the DMPK CTG expansion protect function in myotonic dystrophy type 1.Neurol Genet. 2020; 6: e504Crossref PubMed Scopus (11) Google Scholar, 21Ballester-Lopez A. Koehorst E. Almendrote M. Martínez-Piñeiro A. Lucente G. Linares-Pardo I. Núñez-Manchón J. Guanyabens N. Cano A. Lucia A. Overend G. Cumming S.A. Monckton D.G. Casadevall T. Isern I. Sánchez-Ojanguren J. Planas A. Rodríguez-Palmero A. Monlleó-Neila L. Pintos-Morell G. Ramos-Fransi A. Coll-Cantí J. Nogales-Gadea G. A DM1 family with interruptions associated with atypical symptoms and late onset but not with a milder phenotype.Hum Mutat. 2020; 41: 420-431Crossref PubMed Scopus (17) Google Scholar, 22Pešović J. Perić S. Brkušanin M. Brajušković G. Rakočević-Stojanović V. Savić-Pavićević D. Repeat interruptions modify age at onset in myotonic dystrophy type 1 by stabilizing DMPK expansions in somatic cells.Front Genet. 2018; 9: 601Crossref PubMed Scopus (26) Google Scholar Fifth, single-nucleotide polymorphisms in the DNA mismatch repair gene MSH3, required for maintenance of genomic integrity, have been shown to reduce levels of somatic mosaicism and are associated with delayed onset in patients with DM1.23Morales F. Vásquez M. Santamaría C. Cuenca P. Corrales E. Monckton D.G. A polymorphism in the MSH3 mismatch repair gene is associated with the levels of somatic instability of the expanded CTG repeat in the blood DNA of myotonic dystrophy type 1 patients.DNA Repair (Amst). 2016; 40: 57-66Crossref PubMed Scopus (58) Google Scholar,24Flower M. Lomeikaite V. Ciosi M. Cumming S. Morales F. Lo K. Hensman Moss D. Jones L. Holmans P. TRACK-HD Investigators, OPTIMISTIC Consortium Monckton D.G. Tabrizi S.J. MSH3 modifies somatic instability and disease severity in Huntington's and myotonic dystrophy type 1.Brain. 2019; 142: 1876-1886Crossref Scopus (83) Google Scholar The contribution of CTG repeat length, somatic mosaicism, structural variants, and/or modifier genes on the DM1 genotype and phenotype remains poorly understood due to technical difficulties in analyzing these factors. Currently, the molecular diagnostic procedure used in DM1 genetic testing is divided into two steps.6Kamsteeg E.-J. Kress W. Catalli C. Hertz J.M. Witsch-Baumgartner M. Buckley M.F. van Engelen B.G.M. Schwartz M. Scheffer H. Best practice guidelines and recommendations on the molecular diagnosis of myotonic dystrophy types 1 and 2.Eur J Hum Genet. 2012; 20: 1203-1208Crossref PubMed Scopus (103) Google Scholar,7Savić Pavićević D. Miladinović J. Brkušanin M. Šviković S. Djurica S. Brajušković G. Romac S. Molecular genetics and genetic testing in myotonic dystrophy type 1.Biomed Res Int. 2013; 2013: 1391821Crossref Scopus (44) Google Scholar The first is to detect or rule out possible DM1 expansions using PCR and fragment-length analysis (Table 1). This method cannot differentiate between individuals homozygous for a normal allele and individuals whose CTG repeat expansion could not be amplified by PCR. A second step is therefore necessary to identify large expanded alleles and estimate the approximate size by using Southern blot. All these methods are time-consuming procedures that do not simultaneously provide information on somatic mosaicism and structural variants of expansion, two important prognostic parameters. Furthermore, triplet-repeat primed PCR (TP-PCR) is also used to directly detect the presence of an expanded allele.25Warner J.P. Barron L.H. Goudie D. Kelly K. Dow D. Fitzpatrick D.R. Brock D.J. A general method for the detection of large CAG repeat expansions by fluorescent PCR.J Med Genet. 1996; 33: 1022-1026Crossref PubMed Google Scholar However, no size repeat can be estimated by this method, which leads to a loss of information necessary for performing genetic counseling. To date, TP-PCR is the only diagnostic method, which makes it possible to reveal variant repeats in the first 100 CTG within the 5ʹ and 3′ end of the CTG array. However, this method is unable to identify interruptions at the middle of large repeat expansions of >200 to 300 CTG repeats and to provide the exact number and type of interruptions, regardless of the size of the repeats. Diagnostic laboratories generally use several methodologic approaches to detect CTG repeat expansion and roughly estimate the size of the repeat, which leads to long diagnostic delays. Unfortunately, no diagnostic method can accurately estimate somatic mosaicism, which is an important parameter in predicting disease progression.8Morales F. Couto J.M. Higham C.F. Hogg G. Cuenca P. Braida C. Wilson R.H. Adam B. del Valle G. Brian R. Sittenfeld M. Ashizawa T. Wilcox A. Wilcox D.E. Monckton D.G. Somatic instability of the expanded CTG triplet repeat in myotonic dystrophy type 1 is a heritable quantitative trait and modifier of disease severity.Hum Mol Genet. 2012; 21: 3558-3567Crossref PubMed Scopus (131) Google Scholar, 9Morales F. Vásquez M. Corrales E. Vindas-Smith R. Santamaría-Ulloa C. Zhang B. Sirito M. Estecio M.R. Krahe R. Monckton D.G. Longitudinal increases in somatic mosaicism of the expanded CTG repeat in myotonic dystrophy type 1 are associated with variation in age-at-onset.Hum Mol Genet. 2020; 29: 2496-2507Crossref PubMed Scopus (24) Google Scholar, 10Cumming S.A. Jimenez-Moreno C. Okkersen K. Wenninger S. Daidj F. Hogarth F. Littleford R. Gorman G. Bassez G. Schoser B. Lochmüller H. van Engelen B.G.M. Monckton D.G. OPTIMISTIC ConsortiumGenetic determinants of disease severity in the myotonic dystrophy type 1 OPTIMISTIC cohort.Neurology. 2019; 93: e995-e1009Crossref PubMed Scopus (51) Google Scholar, 11Overend G. Légaré C. Mathieu J. Bouchard L. Gagnon C. Monckton D.G. Allele length of the DMPK CTG repeat is a predictor of progressive myotonic dystrophy type 1 phenotypes.Hum Mol Genet. 2019; 28: 2245-2254Crossref PubMed Scopus (32) Google ScholarTable 1Genetic Testing Strategy for DM1InformationPCRSouthern blotTP-PCRMutation status✓✓✓CTG repeat size 85% of CCG were identified in patients with DM1 for whom amplification of the triplet repeat expansion failed according to PCR and TP-PCR. Participants with DM1 were recruited by the Genetics Department of Nantes Hospital, the Genetics Department of the Necker–Enfants Malades Hospital, the DM-Scope registry, and the Neuromuscular Disease Reference Center of Pitié-Salpêtrière Hospital in France. Written informed consent was obtained from all participants. Genomic DNA samples were initially genotyped for DM1 by using conventional PCR, TP-PCR, or Southern blot.6Kamsteeg E.-J. Kress W. Catalli C. Hertz J.M. Witsch-Baumgartner M. Buckley M.F. van Engelen B.G.M. Schwartz M. Scheffer H. Best practice guidelines and recommendations on the molecular diagnosis of myotonic dystrophy types 1 and 2.Eur J Hum Genet. 2012; 20: 1203-1208Crossref PubMed Scopus (103) Google Scholar,7Savić Pavićević D. Miladinović J. Brkušanin M. Šviković S. Djurica S. Brajušković G. Romac S. Molecular genetics and genetic testing in myotonic dystrophy type 1.Biomed Res Int. 2013; 2013: 1391821Crossref Scopus (44) Google Scholar DNA was extracted in diagnostic laboratories. High-molecular weight DNA was extracted from immortalized lymphoblastoid cells by using the Monarch Genomic DNA Purification Kit (catalog no. T3010S; New England Biolabs, Evry-Courcouronnes, France). Human Hg19 reference sequences surrounding the DM1 repeat region were used to design Cas9 CRISPR RNA (crRNA) sequences. Candidate target sequences were generated by using the Genetic Perturbation Platform sgRNA Designer webtool on the Broad Institute website. The latest design tool, which provides results the same as the earlier version of the tool, is available through the link (Broad Institute, https://portals.broadinstitute.org/gppx/crispick/public, last accessed July 25, 2022). Two upstream and three downstream top ranking candidate sequences, which could generate approximately 2 kb target fragments, were custom synthesized and tested in targeted sequencing experiments with HEK293 genomic DNA. The combination of upstream and downstream crRNAs that produced the highest sequencing yield was selected for the current study. The final crRNA sequences are the DM1-L1-crRNA sequence (5′-CCCCATCGGGACAACGCAGA-3′) and the DM1-R1-crRNA sequence (5′-GGGCGTGTATAGACACCTGG-3′), which generate a target capture region of 2361 bp. Preparation of the SMRTbell library was performed according to the "Procedure and Checklist-No-Amp Targeted Sequencing utilizing the CRISPR-Cas9 system" (available on the Pacific Biosciences website) per manufacturer instructions.28Tsai Y.-C. Zafar F. McEachin Z.T. McLaughlin I. Van Blitterswijk M. Ziegle J. Schüle B. Multiplex CRISPR/Cas9-guided No-Amp targeted sequencing panel for spinocerebellar Ataxia repeat expansions.in: Proukakis C. Genomic Structural Variants in Nervous System Disorders. Neuromethods. vol. 182. Humana, New York, NY2022: 95-120Crossref Scopus (3) Google Scholar Five patients were analyzed by using SMRT Cell. Approximately 3 to 5 μg of native genomic DNA was dephosphorylated with 0.05 U/μL of shrimp alkaline phosphatase for each patient (catalog no. M0371S; New England Biolabs) to reduce the amount of off-target molecules in the final SMRTbell library. Guide RNAs (gRNAs) were formed by annealing crRNAs (Integrated DNA Technologies, Leuven, Belgium) to tracrRNAs (catalog no. 1072533; Integrated DNA Technologies) at a 1:1 ratio and a 5 μM concentration. The sequences of each crRNA targeting DM1 and various control loci used in the experiments are shown in Table 2. The gRNA–Cas9 complex was prepared by incubating 400 nmol/L gRNA with 400 nmol/L Cas9 nuclease (catalog no. M0386M; New England Biolabs) for 10 minutes at 37°C. Dephosphorylated genomic DNAs were digested with the gRNA–Cas9 complex for 1 hour at 37°C. DM1 and control gRNA were multiplexed in the same digestion reaction. Cas9 digestion products were purified by using a 0.45X volume of AMPure PB Beads (catalog no. 100-265-900; Pacific Biosciences). Barcoded adapters recommended by Pacific Biosciences were ligated to purified Cas9 digestion products using 0.4 μmol/L barcode adapters and 0.9U/μL T4 DNA ligase (catalog no. EL0013; Thermo Fisher Scientific, Illkirch-Graffenstaden, France) to form a library of symmetric SMRTbell template molecules. The SMRTbell library was purified by using a 0.45X volume of AMPure PB Beads (catalog no. 100-265-900; Pacific Biosciences). Failed ligation products and gDNA fragments were removed by nuclease treatment using 1.2 U/μL Exonuclease III (catalog no. M0206L; New England Biolabs) and an Enzyme Clean Up Kit (catalog no. 101-746-400; Pacific Biosciences) for 2 hours at 37°C. After nuclease treatment, the SMRTbell library was incubated with 41 μg/mL SOLu-Trypsin (catalog no. EMS0004; Sigma-Aldrich; Saint-Quentin-Fallavier, France) for 20 minutes at 37°C. The nuclease-treated SMRTbell library was purified by using a 0.45X volume of AMPure PB beads for the first purification and a 0.42X volume of AMPure PB Beads for the second purification. Finally, the purified SMRTbell library was resuspended in 6.3 μL of elution buffer (catalog no. 101-633-500; Pacific Biosciences) for targeted SMRT sequencing.Table 2crRNA and Genomic Coordinate for DMPK and Control LociLocuscrRNA1crRNA2GRCh38Size of the fragment,∗Fragment size after CRISPR/Cas9 digestion. bp (non-expanded allele)DMPK5′-CCCCATCGGGACAACGCAGA-3′5′-GGGCGTGTATAGACACCTGG-3′Chromosome 192328(45,769,144-45,771,505)HTT5′-CTTATTAACAGCAGAGAACT-3′5′-TAAACTTTGAAGACGAGACA-3′Chromosome 43806(3,072,601-3,076,440)C9orf725′-TTGGTATTTAGAAAGGTGGT-3′5′-GGAAGAAAGAATTGCAATTA-3′Chromosome 93639(27,571,393-27,575,065)FMR15′-CGCGCGTCTGTCTTTCGACC-3′5′-CCTTTATGCAAAGTTAGCTC-3′Chromosome X3662(147,911,739-147,915,434)crRNA, CRISPR RNA; DM1, myotonic dystrophy type 1; GRCh38, Genome Reference Consortium Human Build 38.∗ Fragment size after CRISPR/Cas9 digestion. Open table in a new tab crRNA, CRISPR RNA; DM1, myotonic dystrophy type 1; GRCh38, Genome Reference Consortium Human Build 38. Following the "Procedure and Checklist-No-Amp Targeted Sequencing Utilizing the CRISPR-Cas9 System" per manufacturer instructions,28Tsai Y.-C. Zafar F. McEachin Z.T. McLaughlin I. Van Blitterswijk M. Ziegle J. Schüle B. Multiplex CRISPR/Cas9-guided No-Amp targeted sequencing panel for spinocerebellar Ataxia repeat expansions.in: Proukakis C. Genomic Structural Variants in Nervous System Disorders. Neuromethods. vol. 182. Humana, New York, NY2022: 95-120Crossref Scopus (3) Google Scholar final SMRTbell libraries were annealed with Sequencing Primer v4 from the No-Amp Accessory Kit (catalog no. 101-788-900; Pacific Biosciences) and bound with Sequel DNA Sequencing Polymerase 3.0 in the Sequel Binding Kit 3.0 (catalog no. 101-626-600; Pacific Biosciences). Polymerase-bound SMRTbell complexes were purified with 0.6X AMPure PB Beads and sequenced on the Sequel System using Sequel Sequencing Kit 3.0 (Pacific Biosciences) and a customized setting with 4 hours of complex immobilization and 20-hour movie collection. Sequencing data repeat sequence analysis was performed following the "Analysis Procedure–No-Amp Data Preparation and Repeat Analysis" (available on the Pacific Biosciences website) per manufacturer instructions.28Tsai Y.-C. Zafar F. McEachin Z.T. McLaughlin I. Van Blitterswijk M. Ziegle J. Schüle B. Multiplex CRISPR/Cas9-guided No-Amp targete
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