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Sequence capture and next-generation resequencing of multiple tagged nucleic acid samples for mutation screening of urea cycle disorders


Amstutz, U; Andrey-Zürcher, G; Suciu, D; Jaggi, R; Häberle, J; Largiadèr, C R (2011). Sequence capture and next-generation resequencing of multiple tagged nucleic acid samples for mutation screening of urea cycle disorders. Clinical Chemistry, 57(1):102-111.

Abstract

BACKGROUND: Molecular genetic testing is commonly used to confirm clinical diagnoses of inherited urea cycle disorders (UCDs); however, conventional mutation screenings encompassing only the coding regions of genes may not detect disease-causing mutations occurring in regulatory elements and introns. Microarray-based target enrichment and next-generation sequencing now allow more-comprehensive genetic screening. We applied this approach to UCDs and combined it with the use of DNA bar codes for more cost-effective, parallel analyses of multiple samples.
METHODS: We used sectored 2240-feature medium-density oligonucleotide arrays to capture and enrich a 199-kb genomic target encompassing the complete genomic regions of 3 urea cycle genes, OTC (ornithine carbamoyltransferase), CPS1 (carbamoyl-phosphate synthetase 1, mitochondrial), and NAGS (N-acetylglutamate synthase). We used the Genome Sequencer FLX System (454 Life Sciences) to jointly analyze 4 samples individually tagged with a 6-bp DNA bar code and compared the results with those for an individually sequenced sample.
RESULTS: Using a low tiling density of only 1 probe per 91 bp, we obtained strong enrichment of the targeted loci to achieve ≥90% coverage with up to 64% of the sequences covered at a sequencing depth ≥10-fold. We observed a very homogeneous sequence representation of the bar-coded samples, which yielded a >30% increase in the sequence data generated per sample, compared with an individually processed sample. Heterozygous and homozygous disease-associated mutations were correctly detected in all samples.
CONCLUSIONS: The use of DNA bar codes and the use of sectored oligonucleotide arrays for target enrichment enable parallel, large-scale analysis of complete genomic regions for multiple genes of a disease pathway and for multiple samples simultaneously. This approach thus may provide an efficient tool for comprehensive diagnostic screening of mutations.

Abstract

BACKGROUND: Molecular genetic testing is commonly used to confirm clinical diagnoses of inherited urea cycle disorders (UCDs); however, conventional mutation screenings encompassing only the coding regions of genes may not detect disease-causing mutations occurring in regulatory elements and introns. Microarray-based target enrichment and next-generation sequencing now allow more-comprehensive genetic screening. We applied this approach to UCDs and combined it with the use of DNA bar codes for more cost-effective, parallel analyses of multiple samples.
METHODS: We used sectored 2240-feature medium-density oligonucleotide arrays to capture and enrich a 199-kb genomic target encompassing the complete genomic regions of 3 urea cycle genes, OTC (ornithine carbamoyltransferase), CPS1 (carbamoyl-phosphate synthetase 1, mitochondrial), and NAGS (N-acetylglutamate synthase). We used the Genome Sequencer FLX System (454 Life Sciences) to jointly analyze 4 samples individually tagged with a 6-bp DNA bar code and compared the results with those for an individually sequenced sample.
RESULTS: Using a low tiling density of only 1 probe per 91 bp, we obtained strong enrichment of the targeted loci to achieve ≥90% coverage with up to 64% of the sequences covered at a sequencing depth ≥10-fold. We observed a very homogeneous sequence representation of the bar-coded samples, which yielded a >30% increase in the sequence data generated per sample, compared with an individually processed sample. Heterozygous and homozygous disease-associated mutations were correctly detected in all samples.
CONCLUSIONS: The use of DNA bar codes and the use of sectored oligonucleotide arrays for target enrichment enable parallel, large-scale analysis of complete genomic regions for multiple genes of a disease pathway and for multiple samples simultaneously. This approach thus may provide an efficient tool for comprehensive diagnostic screening of mutations.

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Additional indexing

Item Type:Journal Article, not refereed, original work
Communities & Collections:04 Faculty of Medicine > University Children's Hospital Zurich > Medical Clinic
Dewey Decimal Classification:610 Medicine & health
Language:English
Date:January 2011
Deposited On:26 Feb 2012 10:12
Last Modified:06 Sep 2017 11:52
Publisher:American Association for Clinical Chemistry
ISSN:0009-9147
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.1373/clinchem.2010.150706
PubMed ID:21068339

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