Sequence Capture and Next-Generation Resequencing of Multiple Tagged Nucleic Acid Samples for Mutation Screening of Urea Cycle Disorders

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.

Performance Evaluation of the Next‐Generation Sequencing Approach for Molecular Diagnosis of Hereditary Hearing Loss

“…25 Published studies on NGS have used a different variant allele threshold, ranging from 15% to 40%. 13,14,16,17 In the current study, using a threshold of 20%, we were able to detect all true variants correctly in 5 SNHL genes sequenced. When this threshold was increased to 30%, we failed to correctly call 7 true variants (7/280) in the 5 genes evaluated, prompting us to routinely use a threshold of 20%.…”

“…13,14,[17][18][19][20][21] In this study, we have validated the efficiency of an NGS-based targeted resequencing approach in detecting the sequence variants in the genes causing SNHL. An important aspect of targeted resequencing is the capture or enrichment of the target regions with high specificity.…”

“…Recent studies evaluating its performance in the clinical diagnostic setting have shown this technology as an efficient tool for diagnostic mutation screening. [13][14][15][16][17] In the present study, we evaluated the performance of an NGSbased targeted resequencing test, called OtoSeq, to detect the sequence variations in the genes causing SNHL. …”

Accurate molecular diagnosis of phenylketonuria and tetrahydrobiopterin-deficient hyperphenylalaninemias using high-throughput targeted sequencing

“…Only SNVs and InDels of up to 30 bp and found within 150 bp of the ends of the enriched targets were considered for subsequent analysis. Functional annotation of high-quality variants was performed using Annovar, 11 providing a comparison of the predicted variants to the National Center for Biotechnology Information (NCBI) SNP Database build 132 (dbSNP132), the March 2010 pilot release of the 1000 Genomes project (1000G; www.1000genomes.org), conservation around variants based on phastCons, 12 segmental duplication filter, gene annotation (exon/intron/UTR), amino-acid substitutions and splice variants based on UCSC Genome Browser 13 tracks, as well as multiple estimates of the impact of amino-acid substitution on the structure and function of proteins (tools: Sift, 14 Polyphen2, 15 PhyloP, 16 and MutationTaster 17 ). The reference sequences used for the four genes targeted in this study were NM_000277 (PAH), NM_000320 (QDPR), NM_000161 (GCH1), and NM_000317 (PTS).…”

Novel Pathogenic Variant (c.580C>T) in the CPS1 Gene in a Newborn With Carbamoyl Phosphate Synthetase 1 Deficiency Identified by Whole Exome Sequencing

“…With the aid of the allosteric activator N-acetylglutamate, synthesized by NAGS, CPS1 catalyzes the first step in the urea cycle, converting ammonia to carbamoyl phosphate [3]. Although UCD diagnosis is primarily based on the biochemical measurement of intermediary metabolites, such measurements cannot be used to distinguish between NAGS deficiency and CPS1D; genetic pathogenic variant testing is needed to distinguish these conditions [4]. …”