Next-generation sequencing (NGS) is widely used in biomedical research, but its adoption has been limited in molecular diagnostics. One application of NGS is the targeted resequencing of genes whose ...mutations lead to an overlapping clinical phenotype. This study evaluated the comparative performance of the Illumina Genome Analyzer and Roche 454 GS FLX for the resequencing of 16 genes associated with hypertrophic cardiomyopathy (HCM). Using a single human genomic DNA sample enriched by long-range PCR (LR-PCR), 40 GS FLX and 31 Genome Analyzer exon variants were identified using >or=30-fold read-coverage and >or=20% read-percentage selection criteria. Twenty-seven platform concordant variants were Sanger-confirmed. The discordant variants segregated into two categories: variants with read coverages >or=30 on one platform but <30-fold on the alternate platform and variants with read percentages >or=20% on one platform but <20% on the alternate platform. All variants with <30-fold coverage were Sanger-confirmed, suggesting that the coverage criterion of >or=30-fold is too stringent for variant discovery. The variants with <20% read percentage were identified as reference sequence based on Sanger sequencing. These variants were found in homopolymer tracts and short-read misalignments, specifically in genes with high identity. The results of the current study demonstrate the feasibility of combining LR-PCR with the Genome Analyzer or GS FLX for targeted resequencing of HCM-associated genes.
In the relatively short time frame since the introduction of next generation sequencing, it has become a method of choice for complex genomic research studies. As a paradigm shifting technology, we ...are now witnessing its translation into clinical diagnostic laboratories for patient care. Multi-gene panels for a variety of disorders are now available in several clinical laboratories based on targeted gene enrichment followed by next generation sequencing. Genome wide interrogation of protein coding regions, or exome sequencing, has been successfully and increasingly applied in the research setting for the elucidation of candidate genes and causal variants in individuals and families with a diversity of rare and complex genetic disorders. Based on this progress, exome sequencing is also beginning a translational process into clinical practice. However, introducing exome sequencing as a diagnostic modality poses new technical and bioinformatics challenges for clinical laboratories. In this review, we present technical and bioinformatics aspects of exome sequencing, describe representative examples from the literature of how exome sequencing has been used for candidate gene discovery, and discuss considerations for its clinical translation.
One limitation of small amplicon melting is the inability to genotype certain nearest-neighbor symmetric variations without manipulating the sample. We have developed a method for these exceptions: a ...high-resolution melting single nucleotide extension assay. Single nucleotide extension was performed in a new instrument, the LightScanner 32 (LS32), which uses capillary reaction tubes and is capable of real-time PCR and sequential high-resolution melting of 32 samples. Asymmetric PCR used Platinum Taq and LC Green Plus in the master mix for target amplification. Dideoxynucleotides and extension oligonucleotides were sequestered in the tube cap and added post-PCR, maintaining a closed system. One dideoxynucleotides was used per capillary tube. Samples were cycled five times to incorporate dideoxynucleotides into the extension products using ThermoSequenase, followed by high-resolution melting. Single nucleotide polymorphisms from the RET proto-oncogene ( n = 7), hemochromatosis ( HFE , n = 30), coagulation factor 2 ( F2 , n = 29), coagulation factor 5 ( F5 , n = 30), and methylenetetrahydrofolate reductase ( MTHFR , n = 60) genes were genotyped. The DNA melting profiles identified the target single nucleotide polymorphisms by the lowest melting temperature transition. All genotypes had a distinctive melting pattern. The method was 100% concordant with samples previously genotyped at HFE , MTHFR , and F2 and 90% concordant with F5. F5 discordants were genotyped correctly by redesigning the assay. Our results demonstrate that although single nucleotide polymorphisms can be successfully differentiated using this methodology, the method requires careful optimization.
Amplicon melting is a closed-tube method for genotyping that does not require probes, real-time analysis, or allele-specific polymerase chain reaction. However, correct differentiation of homozygous ...mutant and wild-type samples by melting temperature (Tm ) requires high-resolution melting and closely controlled reaction conditions. When three different DNA extraction methods were used to isolate DNA from whole blood, amplicon Tm differences of 0.03 to 0.39°C attributable to the extractions were observed. To correct for solution chemistry differences between samples, complementary unlabeled oligonucleotides were included as internal temperature controls to shift and scale the temperature axis of derivative melting plots. This adjustment was applied to a duplex amplicon melting assay for the methylenetetrahydrofolate reductase variants 1298A>C and 677C>T. High- and low-temperature controls bracketing the amplicon melting region decreased the Tm SD within homozygous genotypes by 47 to 82%. The amplicon melting assay was 100% concordant to an adjacent hybridization probe (HybProbe) melting assay when temperature controls were included, whereas a 3% error rate was observed without temperature correction. In conclusion, internal temperature controls increase the accuracy of genotyping by high-resolution amplicon melting and should also improve results on lower resolution instruments.
Real-time PCR data analysis for quantification has been the subject of many studies aimed at the identification of new and improved quantification methods. Several analysis methods have been proposed ...as superior alternatives to the common variations of the threshold crossing method. Notably, sigmoidal and exponential curve fit methods have been proposed. However, these studies have primarily analyzed real-time PCR with intercalating dyes such as SYBR Green. Clinical real-time PCR assays, in contrast, often employ fluorescent probes whose real-time amplification fluorescence curves differ from those of intercalating dyes. In the current study, we compared four analysis methods related to recent literature: two versions of the threshold crossing method, a second derivative maximum method, and a sigmoidal curve fit method. These methods were applied to a clinically relevant real-time human herpes virus type 6 (HHV6) PCR assay that used a minor groove binding (MGB) Eclipse hybridization probe as well as an Epstein–Barr virus (EBV) PCR assay that used an MGB Pleiades hybridization probe. We found that the crossing threshold method yielded more precise results when analyzing the HHV6 assay, which was characterized by lower signal/noise and less developed amplification curve plateaus. In contrast, the EBV assay, characterized by greater signal/noise and amplification curves with plateau regions similar to those observed with intercalating dyes, gave results with statistically similar precision by all four analysis methods.
Unlabeled probe detection with a double-stranded DNA (dsDNA) binding dye is one method to detect and confirm target amplification after PCR. Unlabeled probes and amplicon melting have been used to ...detect small deletions and single-nucleotide polymorphisms in assays where template is in abundance. Unlabeled probes have not been applied to low-level target detection, however.
Herpes simplex virus (HSV) was chosen as a model to compare the unlabeled probe method to an in-house reference assay using dual-labeled, minor groove binding probes. A saturating dsDNA dye (LCGreen Plus) was used for real-time PCR. HSV-1, HSV-2, and an internal control were differentiated by PCR amplicon and unlabeled probe melting analysis after PCR.
The unlabeled probe technique displayed 98% concordance with the reference assay for the detection of HSV from a variety of archived clinical samples (n = 182). HSV typing using unlabeled probes was 99% concordant (n = 104) to sequenced clinical samples and allowed for the detection of sequence polymorphisms in the amplicon and under the probe.
Unlabeled probes and amplicon melting can be used to detect and genotype as few as 10 copies of target per reaction, restricted only by stochastic limitations. The use of unlabeled probes provides an attractive alternative to conventional fluorescence-labeled, probe-based assays for genotyping and detection of HSV and might be useful for other low-copy targets where typing is informative.
High-resolution amplicon melting is a simple method for genotyping that uses only generic PCR primers and a saturating DNA dye. Multiplex amplicon genotyping has previously been reported in a single ...color, but two instruments were required: a carousel-based rapid cycler and a high-resolution melting instrument for capillaries. Manual transfer of capillaries between instruments and sequential melting of each capillary at 0.1 degrees C/s seriously limited the throughput. In this report, a single instrument that combines rapid-cycle real-time PCR with high-resolution melting LightScanner-32 (LS-32), Idaho Technology, Salt Lake City, UT was used for multiplex amplicon genotyping. The four most common mutations associated with thrombophilia, F5 (factor V Leiden 1691G>A), F2 (prothrombin 20210G>A), and methylenetetrahydrofolate reductase (MTHFR; 1298A>C and 677C>T) were genotyped in a single homogeneous assay with internal controls to adjust for minor chemistry and instrument variation. Forty temperature cycles required 9.2 min, and each capillary required 2.2 min by melting at 0.3 degrees C/s, 3x the prior rate. Sample volume was reduced from 20 microl to 10 microl. In a blinded study of 109 samples (436 genotypes), complete concordance with standard assays was obtained. In addition, the rare variant MTHFR 1317T>C was genotyped correctly when present. The LS-32 simplifies more complex high-resolution melting assays by reducing hands-on manipulation, total time of analysis, and reagent cost while maintaining the resolution necessary for multiplex amplicon genotyping.
Next-generation sequencing was performed for 2 families with an undiagnosed neurologic disease. Analysis revealed X-linked mutations in the proteolipid protein 1 (PLP1) gene, which is associated with ...X-linked Pelizaeus-Merzbacher disease and Spastic Paraplegia type 2. In family A, the novel PLP1 missense mutation c.617T>A (p.M206K) was hemizygous in the 2 affected male children and heterozygous in the mother. In family B, the novel de novo PLP1 frameshift mutation c.359_369del (p.G120fs) was hemizygous in the affected male child. Although PLP1 mutations have been reported to cause an increasingly wide range of phenotypes inclusive of the dystonia, spastic paraparesis, motor neuronopathy, and leukodystrophy observed in our patients, atypical features included the cerebrospinal fluid deficiency of neurotransmitter and pterin metabolites and the delayed appearance of myelin abnormalities on neuroimaging studies. Next-generation sequencing studies provided a diagnosis for these families with complex leukodystrophy disease phenotypes, which expanded the spectrum of PLP1-associated leukodystrophy clinical phenotypes.
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