42 research outputs found
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Efficient In Silico Identification of a Common Insertion in the MAK Gene which Causes Retinitis Pigmentosa
Background: Next generation sequencing (NGS) offers a rapid and comprehensive method of screening for mutations associated with retinitis pigmentosa and related disorders. However, certain sequence alterations such as large insertions or deletions may remain undetected using standard NGS pipelines. One such mutation is a recently-identified Alu insertion into the Male Germ Cell-Associated Kinase (MAK) gene, which is missed by standard NGS-based variant callers. Here, we developed an in silico method of searching NGS raw sequence reads to detect this mutation, without the need to recalculate sequence alignments or to screen every sample by PCR. Methods: The Linux program grep was used to search for a 23 bp “probe” sequence containing the known junction sequence of the insert. A corresponding search was performed with the wildtype sequence. The matching reads were counted and further compared to the known sequences of the full wildtype and mutant genomic loci. (See https://github.com/MEEIBioinformaticsCenter/grepsearch.) Results: In a test sample set consisting of eleven previously published homozygous mutants, detection of the MAK-Alu insertion was validated with 100% sensitivity and specificity. As a discovery cohort, raw NGS reads from 1,847 samples (including custom and whole exome selective capture) were searched in ~1 hour on a local computer cluster, yielding an additional five samples with MAK-Alu insertions and solving two previously unsolved pedigrees. Of these, one patient was homozygous for the insertion, one compound heterozygous with a missense change on the other allele (c. 46G>A; p.Gly16Arg), and three were heterozygous carriers. Conclusions: Using the MAK-Alu grep program proved to be a rapid and effective method of finding a known, disease-causing Alu insertion in a large cohort of patients with NGS data. This simple approach avoids wet-lab assays or computationally expensive algorithms, and could also be used for other known disease-causing insertions and deletions
Identification of a Novel Large Multigene Deletion and a Frameshift Indel in PDE6B as the Underlying Cause of Early-Onset Recessive Rod-Cone Degeneration
A family, with two affected identical twins with early-onset recessive inherited retinal degeneration, was analyzed to determine the underlying genetic cause of pathology. Exome sequencing revealed a rare and previously reported causative variant (c.1923_1969delinsTCTGGG; p.Asn643Glyfs*29) in th
Mitochondrial genome sequence analysis: A custom bioinformatics pipeline substantially improves Affymetrix MitoChip v2.0 call rate and accuracy
BACKGROUND: Mitochondrial genome sequence analysis is critical to the diagnostic evaluation of mitochondrial disease. Existing methodologies differ widely in throughput, complexity, cost efficiency, and sensitivity of heteroplasmy detection. Affymetrix MitoChip v2.0, which uses a sequencing-by-genotyping technology, allows potentially accurate and high-throughput sequencing of the entire human mitochondrial genome to be completed in a cost-effective fashion. However, the relatively low call rate achieved using existing software tools has limited the wide adoption of this platform for either clinical or research applications. Here, we report the design and development of a custom bioinformatics software pipeline that achieves a much improved call rate and accuracy for the Affymetrix MitoChip v2.0 platform. We used this custom pipeline to analyze MitoChip v2.0 data from 24 DNA samples representing a broad range of tissue types (18 whole blood, 3 skeletal muscle, 3 cell lines), mutations (a 5.8 kilobase pair deletion and 6 known heteroplasmic mutations), and haplogroup origins. All results were compared to those obtained by at least one other mitochondrial DNA sequence analysis method, including Sanger sequencing, denaturing HPLC-based heteroduplex analysis, and/or the Illumina Genome Analyzer II next generation sequencing platform.
RESULTS: An average call rate of 99.75% was achieved across all samples with our custom pipeline. Comparison of calls for 15 samples characterized previously by Sanger sequencing revealed a total of 29 discordant calls, which translates to an estimated 0.012% for the base call error rate. We successfully identified 4 known heteroplasmic mutations and 24 other potential heteroplasmic mutations across 20 samples that passed quality control.
CONCLUSIONS: Affymetrix MitoChip v2.0 analysis using our optimized MitoChip Filtering Protocol (MFP) bioinformatics pipeline now offers the high sensitivity and accuracy needed for reliable, high-throughput and cost-efficient whole mitochondrial genome sequencing. This approach provides a viable alternative of potential utility for both clinical diagnostic and research applications to traditional Sanger and other emerging sequencing technologies for whole mitochondrial genome analysis
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Panel-based Genetic Diagnostic Testing for Inherited Eye Diseases is Highly Accurate and Reproducible and More Sensitive for Variant Detection Than Exome Sequencing
Purpose Next-generation sequencing (NGS) based methods are being adopted broadly for genetic diagnostic testing, but the performance characteristics of these techniques have not been fully defined with regard to test accuracy and reproducibility. Methods: We developed a targeted enrichment and NGS approach for genetic diagnostic testing of patients with inherited eye disorders, including inherited retinal degenerations, optic atrophy and glaucoma. In preparation for providing this Genetic Eye Disease (GEDi) test on a CLIA-certified basis, we performed experiments to measure the sensitivity, specificity, reproducibility as well as the clinical sensitivity of the test. Results: The GEDi test is highly reproducible and accurate, with sensitivity and specificity for single nucleotide variant detection of 97.9% and 100%, respectively. The sensitivity for variant detection was notably better than the 88.3% achieved by whole exome sequencing (WES) using the same metrics, due to better coverage of targeted genes in the GEDi test compared to commercially available exome capture sets. Prospective testing of 192 patients with IRDs indicated that the clinical sensitivity of the GEDi test is high, with a diagnostic rate of 51%. Conclusion: The data suggest that based on quantified performance metrics, selective targeted enrichment is preferable to WES for genetic diagnostic testing
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Substitution of a single non-coding nucleotide upstream of TMEM216 causes non-syndromic retinitis pigmentosa and is associated with reduced TMEM216 expression
Genome analysis of individuals affected by retinitis pigmentosa (RP) identified two rare nucleotide substitutions at the same genomic location on chromosome 11 (g.61392563 [GRCh38]), 69 base pairs upstream of the start codon of the ciliopathy gene TMEM216 (c.-69G>A, c.-69G>T [GenBank: NM_001173991.3]), in individuals of South Asian and African ancestry, respectively. Genotypes included 71 homozygotes and 3 mixed heterozygotes in trans with a predicted loss-of-function allele. Haplotype analysis showed single-nucleotide variants (SNVs) common across families, suggesting ancestral alleles within the two distinct ethnic populations. Clinical phenotype analysis of 62 available individuals from 49 families indicated a similar clinical presentation with night blindness in the first decade and progressive peripheral field loss thereafter. No evident systemic ciliopathy features were noted. Functional characterization of these variants by luciferase reporter gene assay showed reduced promotor activity. Nanopore sequencing confirmed the lower transcription of the TMEM216 c.-69G>T allele in blood-derived RNA from a heterozygous carrier, and reduced expression was further recapitulated by qPCR, using both leukocytes-derived RNA of c.-69G>T homozygotes and total RNA from genome-edited hTERT-RPE1 cells carrying homozygous TMEM216 c.-69G>A. In conclusion, these variants explain a significant proportion of unsolved cases, specifically in individuals of African ancestry, suggesting that reduced TMEM216 expression might lead to abnormal ciliogenesis and photoreceptor degeneration
Beyond Sector Retinitis Pigmentosa: Expanding the Phenotype and Natural History of the Rhodopsin Gene Codon 106 Mutation (Gly-to-Arg) in Autosomal Dominant Retinitis Pigmentosa
Sector and pericentral are two rare, regional forms of retinitis pigmentosa (RP). While usually defined as stable or only very slowly progressing, the available literature to support this claim is limited. Additionally, few studies have analyzed the spectrum of disease within a particular genotype. We identified all cases (9 patients) with an autosomal dominant Rhodopsin variant previously associated with sector RP (RHO c.316G > A, p.Gly106Arg) at our institution. Clinical histories were reviewed, and testing included visual fields, multimodal imaging, and electroretinography. Patients demonstrated a broad phenotypic spectrum that spanned regional phenotypes from sector-like to pericentral RP, as well as generalized disease. We also present evidence of significant intrafamilial variability in regional phenotypes. Finally, we present the longest-reported follow-up for a patient with RHO-associated sector-like RP, showing progression from sectoral to pericentral disease over three decades. In the absence of comorbid macular disease, the long-term prognosis for central visual acuity is good. However, we found that significant progression of RHO p.Gly106Arg disease can occur over protracted periods, with impact on peripheral vision. Longitudinal widefield imaging and periodic ERG reassessment are likely to aid in monitoring disease progression
Identification of Homozygous and Heterozygous <i>MAK</i>-Alu Insertions in a Discovery Sample Set.
<p>Identification of Homozygous and Heterozygous <i>MAK</i>-Alu Insertions in a Discovery Sample Set.</p
Characterization of the test sample set.
<p>A) Samples from previously reported patients with Alu insertion in <i>MAK</i> exon 9 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142614#pone.0142614.ref021" target="_blank">21</a>] and control samples were PCR amplified to detect homozygous alleles for Alu insertion and WT alleles. B) Sequence of the inserted element (280 bp Alu, 54 bp poly-A and 13 bp duplication of exon 9 sequence). C) Sanger sequence of the exon 9 Alu insertion breakpoints.</p
Specificity and Sensitivity of <i>In Silico</i> Method to Detect the <i>MAK</i>-Alu insertion.
<p>Specificity and Sensitivity of <i>In Silico</i> Method to Detect the <i>MAK</i>-Alu insertion.</p