Assessment of Target Enrichment Platforms Using Massively Parallel Sequencing for the Mutation Detection for Congenital Muscular Dystrophy

Overview

Journal J Mol Diagn

Publisher Elsevier

Specialties Molecular Biology
Pathology

Date 2012 Mar 20

PMID 22426012

Citations 19

Authors

C Alexander Valencia

Devin Rhodenizer

Shruti Bhide

Ephrem Chin

Martin Robert Littlejohn

Lisa Mari Keong

Anne Rutkowski

Carsten Bonnemann

Madhuri Hegde

Affiliations

Soon will be listed here.

Abstract

Sequencing individual genes by Sanger sequencing is a time-consuming and costly approach to resolve clinically heterogeneous genetic disorders. Panel testing offers the ability to efficiently and cost-effectively screen all of the genes for a particular genetic disorder. We assessed the analytical sensitivity and specificity of two different enrichment technologies, solution-based hybridization and microdroplet-based PCR target enrichment, in conjunction with next-generation sequencing (NGS), to identify mutations in 321 exons representing 12 different genes involved with congenital muscular dystrophies. Congenital muscular dystrophies present diagnostic challenges due to phenotypic variability, lack of standard access to and inherent difficulties with muscle immunohistochemical stains, and a general lack of clinician awareness. NGS results were analyzed across several parameters, including sequencing metrics and genotype concordance with Sanger sequencing. Genotyping data showed that both enrichment technologies produced suitable calls for use in clinical laboratories. However, microdroplet-based PCR target enrichment is more appropriate for a clinical laboratory, due to excellent sequence specificity and uniformity, reproducibility, high coverage of the target exons, and the ability to distinguish the active gene versus known pseudogenes. Regardless of the method, exons with highly repetitive and high GC regions are not well enriched and require Sanger sequencing for completeness. Our study demonstrates the successful application of targeted sequencing in conjunction with NGS to screen for mutations in hundreds of exons in a genetically heterogeneous human disorder.

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References

Hu H, Wrogemann K, Kalscheuer V, Tzschach A, Richard H, Haas S . Mutation screening in 86 known X-linked mental retardation genes by droplet-based multiplex PCR and massive parallel sequencing. Hugo J. 2011; 3(1-4):41-9. PMC: 2882650. DOI: 10.1007/s11568-010-9137-y. View

Tucker T, Marra M, Friedman J . Massively parallel sequencing: the next big thing in genetic medicine. Am J Hum Genet. 2009; 85(2):142-54. PMC: 2725244. DOI: 10.1016/j.ajhg.2009.06.022. View

Askree S, Hjelm L, Pervaiz M, Adam M, Bean L, Hedge M . Allelic dropout can cause false-positive results for Prader-Willi and Angelman syndrome testing. J Mol Diagn. 2011; 13(1):108-12. PMC: 3069869. DOI: 10.1016/j.jmoldx.2010.11.006. View

Yeager M, Orr N, Hayes R, Jacobs K, Kraft P, Wacholder S . Genome-wide association study of prostate cancer identifies a second risk locus at 8q24. Nat Genet. 2007; 39(5):645-9. DOI: 10.1038/ng2022. View

Ur Rehman A, Morell R, Belyantseva I, Khan S, Boger E, Shahzad M . Targeted capture and next-generation sequencing identifies C9orf75, encoding taperin, as the mutated gene in nonsyndromic deafness DFNB79. Am J Hum Genet. 2010; 86(3):378-88. PMC: 2833391. DOI: 10.1016/j.ajhg.2010.01.030. View

Mardis E, Ding L, Dooling D, Larson D, McLellan M, Chen K . Recurring mutations found by sequencing an acute myeloid leukemia genome. N Engl J Med. 2009; 361(11):1058-66. PMC: 3201812. DOI: 10.1056/NEJMoa0903840. View

Holt R, Jones S . The new paradigm of flow cell sequencing. Genome Res. 2008; 18(6):839-46. DOI: 10.1101/gr.073262.107. View

Muntoni F, Voit T . The congenital muscular dystrophies in 2004: a century of exciting progress. Neuromuscul Disord. 2004; 14(10):635-49. DOI: 10.1016/j.nmd.2004.06.009. View

Asmann Y, Wallace M, Thompson E . Transcriptome profiling using next-generation sequencing. Gastroenterology. 2008; 135(5):1466-8. DOI: 10.1053/j.gastro.2008.09.042. View

10.

Hoppman-Chaney N, Peterson L, Klee E, Middha S, Courteau L, Ferber M . Evaluation of oligonucleotide sequence capture arrays and comparison of next-generation sequencing platforms for use in molecular diagnostics. Clin Chem. 2010; 56(8):1297-306. DOI: 10.1373/clinchem.2010.145441. View

11.

Schessl J, Zou Y, Bonnemann C . Congenital muscular dystrophies and the extracellular matrix. Semin Pediatr Neurol. 2006; 13(2):80-9. DOI: 10.1016/j.spen.2006.06.003. View

12.

Porreca G, Zhang K, Li J, Xie B, Austin D, Vassallo S . Multiplex amplification of large sets of human exons. Nat Methods. 2007; 4(11):931-6. DOI: 10.1038/nmeth1110. View

13.

Hodges E, Xuan Z, Balija V, Kramer M, Molla M, Smith S . Genome-wide in situ exon capture for selective resequencing. Nat Genet. 2007; 39(12):1522-7. DOI: 10.1038/ng.2007.42. View

14.

Marguerat S, Wilhelm B, Bahler J . Next-generation sequencing: applications beyond genomes. Biochem Soc Trans. 2008; 36(Pt 5):1091-6. PMC: 2563889. DOI: 10.1042/BST0361091. View

15.

Albert T, Molla M, Muzny D, Nazareth L, Wheeler D, Song X . Direct selection of human genomic loci by microarray hybridization. Nat Methods. 2007; 4(11):903-5. DOI: 10.1038/nmeth1111. View

16.

Ng S, Turner E, Robertson P, Flygare S, Bigham A, Lee C . Targeted capture and massively parallel sequencing of 12 human exomes. Nature. 2009; 461(7261):272-6. PMC: 2844771. DOI: 10.1038/nature08250. View

17.

Volpi L, Roversi G, Colombo E, Leijsten N, Concolino D, Calabria A . Targeted next-generation sequencing appoints c16orf57 as clericuzio-type poikiloderma with neutropenia gene. Am J Hum Genet. 2009; 86(1):72-6. PMC: 2801743. DOI: 10.1016/j.ajhg.2009.11.014. View

18.

Okou D, Steinberg K, Middle C, Cutler D, Albert T, Zwick M . Microarray-based genomic selection for high-throughput resequencing. Nat Methods. 2007; 4(11):907-9. DOI: 10.1038/nmeth1109. View

19.

Hoischen A, Gilissen C, Arts P, Wieskamp N, van der Vliet W, Vermeer S . Massively parallel sequencing of ataxia genes after array-based enrichment. Hum Mutat. 2010; 31(4):494-9. DOI: 10.1002/humu.21221. View

20.

Daiger S, Sullivan L, Bowne S, Birch D, Heckenlively J, Pierce E . Targeted high-throughput DNA sequencing for gene discovery in retinitis pigmentosa. Adv Exp Med Biol. 2010; 664:325-31. PMC: 2909649. DOI: 10.1007/978-1-4419-1399-9_37. View