Identification of Sequence Variants in Genetic Disease-Causing Genes Using Targeted Next-Generation Sequencing

Abstract

BackgroundIdentification of gene variants plays an important role in research on and diagnosis of genetic diseases. A combination of enrichment of targeted genes and next-generation sequencing (targeted DNA-HiSeq) results in both high efficiency and low cost for targeted sequencing of genes of interest.Methodology/Principal FindingsTo identify mutations associated with genetic diseases, we designed an array-based gene chip to capture all of the exons of 193 genes involved in 103 genetic diseases. To evaluate this technology, we selected 7 samples from seven patients with six different genetic diseases resulting from six disease-causing genes and 100 samples from normal human adults as controls. The data obtained showed that on average, 99.14% of 3,382 exons with more than 30-fold coverage were successfully detected using Targeted DNA-HiSeq technology, and we found six known variants in four disease-causing genes and two novel mutations in two other disease-causing genes (the STS gene for XLI and the FBN1 gene for MFS) as well as one exon deletion mutation in the DMD gene. These results were confirmed in their entirety using either the Sanger sequencing method or real-time PCR.Conclusions/SignificanceTargeted DNA-HiSeq combines next-generation sequencing with the capture of sequences from a relevant subset of high-interest genes. This method was tested by capturing sequences from a DNA library through hybridization to oligonucleotide probes specific for genetic disorder-related genes and was found to show high selectivity, improve the detection of mutations, enabling the discovery of novel variants, and provide additional indel data. Thus, targeted DNA-HiSeq can be used to analyze the gene variant profiles of monogenic diseases with high sensitivity, fidelity, throughput and speed.

Improving Mutation Screening in Familial Hematuric Nephropathies through Next Generation Sequencing

“…It has recently been shown to be efficient in diagnostic screening of human diseases, 15 including renal diseases, such as autosomal dominant polycystic kidney disease 16 and steroid-resistant nephrotic syndrome. 17 Here, we used multiplex PCR, amplicon quantification, library barcoding, and sample pooling followed by NGS for mutation analysis of three type IV collagen genes in a large series of patients affected with AS or BFH.…”

Integration of targeted sequencing and NIPT into clinical practice in a Chinese family with maple syrup urine disease

“…The sensitivity and efficiency of this array in MPS have been evaluated previously. 5 The target region includes exons and adjacent flanking parts (total: 5.5 kb) of four known MSUD-related genes, including BCKDHA, BCKDHB, DBT, and DLD.…”

“…Library pooling, hybridization, sequencing, and reads alignment were performed as previously described. [5][6][7] Single-nucleotide variants and indels (insertion-deletion sites) were detected using the SOAPsnp software and Samtools, respectively.…”

“…5 All led to a potential mutation, c.392A>G in the gene BCKDHA. Sanger sequencing validated that the suspected novel missense mutation (c.392A>G) exists in the proband and her mother Original research article (Figure 2b), consistent with the MPS results.…”

Next-generation whole-exome sequencing contribution to identification of rare autosomal recessive diseases

“…That gives enough insight to "pinpoint" the mutation responsible for a disease. At present several national projects using NGS are released in Canada, Korea, UK, USA, and other countries (6,7).…”