New lung cancer panel for high-throughput targeted resequencing.
Ontology highlight
ABSTRACT:
SUBMITTER: Kim EH
PROVIDER: S-EPMC4099348 | biostudies-literature | 2014 Jun
REPOSITORIES: biostudies-literature
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Project description:BackgroundAlthough the sequencing landscape is rapidly evolving and sequencing costs are continuously decreasing, whole genome sequencing is still too expensive for use on a routine basis. Targeted resequencing of only the regions of interest decreases both costs and the complexity of the downstream data-analysis. Various target enrichment strategies are available, but none of them obtain the degree of coverage uniformity, flexibility and specificity of PCR-based enrichment. On the other hand, the biggest limitation of target enrichment by PCR is the need to design large numbers of partially overlapping assays to cover the target.ResultsTo overcome the aforementioned hurdles, we have developed primerXL, a state-of-the-art PCR primer design pipeline for targeted resequencing. It uses an optimized design criteria relaxation cascade and a thorough downstream in silico evaluation process to generate high quality singleplex PCR assays, reducing the need for amplicon normalization, and outperforming other target enrichment strategies and similar primer design tools when considering assay quality, coverage uniformity and target coverage. Results of four different sequencing projects with 2348 amplicons in total covering 470 kb are presented. PrimerXL can be accessed at www.primerxl.org .ConclusionPrimerXL is an state-of-the-art, easy to use primer design webtool capable of generating high-quality targeted resequencing assays. The workflow is fully customizable to suit every researchers' needs, while an innovative relaxation cascade ensures maximal target coverage.
| S-EPMC5588703 | biostudies-literature
Project description:ObjectivesWe report development of a targeted resequencing gene panel for focal epilepsy, the most prevalent phenotypic group of the epilepsies.MethodsThe targeted resequencing gene panel was designed using molecular inversion probe (MIP) capture technology and sequenced using massively parallel Illumina sequencing.ResultsWe demonstrated proof of principle that mutations can be detected in 4 previously genotyped focal epilepsy cases. We searched for both germline and somatic mutations in 251 patients with unsolved sporadic or familial focal epilepsy and identified 11 novel or very rare missense variants in 5 different genes: CHRNA4, GRIN2B, KCNT1, PCDH19, and SCN1A. Of these, 2 were predicted to be pathogenic or likely pathogenic, explaining ?0.8% of the cohort, and 8 were of uncertain significance based on available data.ConclusionsWe have developed and validated a targeted resequencing panel for focal epilepsies, the most important clinical class of epilepsies, accounting for about 60% of all cases. Our application of MIP technology is an innovative approach that will be advantageous in the clinical setting because it is highly sensitive, efficient, and cost-effective for screening large patient cohorts. Our findings indicate that mutations in known genes likely explain only a small proportion of focal epilepsy cases. This is not surprising given the established clinical and genetic heterogeneity of these disorders and underscores the importance of further gene discovery studies in this complex syndrome.
| S-EPMC4844234 | biostudies-literature
Project description:Although genomewide association studies have successfully identified associations of many common single-nucleotide polymorphisms (SNPs) with common diseases, the SNPs implicated so far account for only a small proportion of the genetic variability of tested diseases. It has been suggested that common diseases may often be caused by rare alleles missed by genomewide association studies. To identify these rare alleles we need high-throughput, high-accuracy resequencing technologies. Although array-based genotyping has allowed genomewide association studies of common SNPs in tens of thousands of samples, array-based resequencing has been limited for 2 main reasons: the lack of a fully multiplexed pipeline for high-throughput sample processing, and failure to achieve sufficient performance. We have recently solved both of these problems and created a fully multiplexed high-throughput pipeline that results in high-quality data. The pipeline consists of target amplification from genomic DNA, followed by allele enrichment to generate pools of purified variant (or nonvariant) DNA and ends with interrogation of purified DNA on resequencing arrays. We have used this pipeline to resequence approximately 5 Mb of DNA (on 3 arrays) corresponding to the exons of 1,500 genes in >473 samples; in total >2,350 Mb were sequenced. In the context of this large-scale study we obtained a false positive rate of approximately 1 in 500,000 bp and a false negative rate of approximately 10%.
| S-EPMC2672536 | biostudies-literature
Project description:BackgroundSmall cell lung cancer (SCLC) is an aggressive and invasive malignancy that presents at advanced clinical stage with no more effective treatments. Development of a method for its early detection would be useful, also new therapeutic target need to be discovered; however, there is a lack of information about its oncogenic driver gene mutations.ObjectivesWe aim to identify the SCLC-related genomic variants that associate with clinical staging and serum protein biomarkers observed in other types of lung cancer.MethodsWe screened formalin-fixed paraffin-embedded (FFPE) biopsy tissues of 32 Chinese SCLC patients using the 303 oncogenic driver gene panel generated by Tiling PCR amplification sequencing (tPAS) and analyzed the patients' corresponding serum protein levels of CYFRA21-1 CEA, NSE, and SCCA.ResultsIn total, we found 147 SCLC-related mutant genes, among these, three important genes (TP53, RB1, KMT2D) as well as five novel genes LRRK2, BRCA1, PTCH1, ARID2, and APC that altogether occurred in 90% of patients. Furthermore, increased mutations to 6 genes (WT1, NOTCH1, EPHA3, KDM6A, SETD2, ACVR1B) significantly associated with higher serum NSE levels (P = 0.0016) and higher clinical stages II + III compared to stage I (P = 0.06).ConclusionsOur panel is relatively reliable in detecting the oncogenic mutations of Chinese SCLC patients. Based on our findings, it may be possible to combine SCLC-related mutations and serum NSE for a simple detection of clinical staging.
| S-EPMC8046547 | biostudies-literature
Project description:The next-generation sequencing technology coupled with the growing number of genome sequences opens the opportunity to redesign genotyping strategies for more effective genetic mapping and genome analysis. We have developed a high-throughput method for genotyping recombinant populations utilizing whole-genome resequencing data generated by the Illumina Genome Analyzer. A sliding window approach is designed to collectively examine genome-wide single nucleotide polymorphisms for genotype calling and recombination breakpoint determination. Using this method, we constructed a genetic map for 150 rice recombinant inbred lines with an expected genotype calling accuracy of 99.94% and a resolution of recombination breakpoints within an average of 40 kb. In comparison to the genetic map constructed with 287 PCR-based markers for the rice population, the sequencing-based method was approximately 20x faster in data collection and 35x more precise in recombination breakpoint determination. Using the sequencing-based genetic map, we located a quantitative trait locus of large effect on plant height in a 100-kb region containing the rice "green revolution" gene. Through computer simulation, we demonstrate that the method is robust for different types of mapping populations derived from organisms with variable quality of genome sequences and is feasible for organisms with large genome sizes and low polymorphisms. With continuous advances in sequencing technologies, this genome-based method may replace the conventional marker-based genotyping approach to provide a powerful tool for large-scale gene discovery and for addressing a wide range of biological questions.
| S-EPMC2694477 | biostudies-literature
Project description:Targeted resequencing of genomic DNA from organisms such as humans is an important tool enabling experimental access to variation within the species and between similar species. Taking full advantage of the reference genome sequences in designing robust, specific PCR assays and using stringent conditions, resequencing can be done efficiently without purification of the PCR product. By using a 10-fold greater amount of one primer when setting up the PCR initially in a new version of asymmetric PCR, one simply adds the rest of the sequencing reagents at the end of PCR and allows the sequencing reaction to proceed, with the excess PCR primer serving as the sequencing primer. We demonstrated that this streamlined protocol can be used with PCR products up to 1300 bp and had up to a 97% success rate in high-throughput analysis of allele frequencies for >30,000 single-nucleotide polymorphisms (SNPs). SNP primers and characterization results are provided at http://snp.wustl.edu.
| S-EPMC430165 | biostudies-literature
Project description:BACKGROUND: Although the throughput of next generation sequencing is increasing and at the same time the cost is substantially reduced, for the majority of laboratories whole genome sequencing of large cohorts of cancer samples is still not feasible. In addition, the low number of genomes that are being sequenced is often problematic for the downstream interpretation of the significance of the variants. Targeted resequencing can partially circumvent this problem; by focusing on a limited number of candidate cancer genes to sequence, more samples can be included in the screening, hence resulting in substantial improvement of the statistical power. In this study, a successful strategy for prioritizing candidate genes for targeted resequencing of cancer genomes is presented. RESULTS: Four prioritization strategies were evaluated on six different cancer types: genes were ranked using these strategies, and the positive predictive value (PPV) or mutation rate within the top-ranked genes was compared to the baseline mutation rate in each tumor type. Successful strategies generate gene lists in which the top is enriched for known mutated genes, as evidenced by an increase in PPV. A clear example of such an improvement is seen in colon cancer, where the PPV is increased by 2.3 fold compared to the baseline level when 100 top fitSNP genes are sequenced. CONCLUSIONS: A gene prioritization strategy based on the fitSNP scores appears to be most successful in identifying mutated cancer genes across different tumor entities, with variance of gene expression levels as a good second best.
| S-EPMC3291573 | biostudies-other
Project description:Background: Recent advanced technologies, such as high-throughput sequencing, have enabled the identification of a broad spectrum of variants. Using targeted-gene-panel resequencing for Parkinson's disease (PD)-associated genes, we have occasionally found several single-nucleotide variants (SNVs), which are thought to be disease-associated, in PD patients. To confirm the significance of these potentially disease-associated variants, we performed genome association analyses, using next-generation target resequencing, to evaluate the associations between the identified SNVs and PD. Methods: We obtained genomic DNA from 766 patients, who were clinically diagnosed with PD, and 336 healthy controls, all of Japanese origin. All data were analyzed using Ion AmpliSeq panel sequences, with 29 PD- or dementia-associated genes in a single panel. We excluded any variants that did not comply with the Hardy-Weinberg equilibrium in the control group. Variant frequencies in the PD and control groups were compared using PLINK. The identified variants were confirmed to a frequency difference of P < 0.05, after applying the Benjamini-Hochberg procedure using Fisher's exact test. The pathogenicity and prevalence of each variant were estimated based on a public gene database. Results: We identified three rare variants that were significantly associated with PD: rs201012663/rs150500694 in SYNJ1 and rs372754391 in DJ-1, which are intronic variants, and rs7412 in ApoE, which is an exonic variant. The variants in SYNJ1 and ApoE were frequently identified in the control group, and rs201012663/rs150500694 in SYNJ1 may play a protective role against PD. The DJ-1 variant was frequently identified in the PD group, with a high odds ratio of 2.2. Conclusion: The detected variants may represent genetic modifiers or disease-related variants in PD. Targeted-gene-panel resequencing may represent a useful method for detecting disease-causing variants and genetic association studies in PD.
| S-EPMC7550729 | biostudies-literature
Project description:During the past three decades, we have witnessed remarkable advances in our understanding of the molecular etiologies of hereditary neurodegenerative diseases, which have been accomplished by 'positional cloning' strategies. The discoveries of the causative genes for hereditary neurodegenerative diseases accelerated not only the studies on the pathophysiologic mechanisms of diseases, but also the studies for the development of disease-modifying therapies. Genome-wide association studies (GWAS) based on the 'common disease-common variants hypothesis' are currently undertaken to elucidate disease-relevant alleles. Although GWAS have successfully revealed numerous susceptibility genes for neurodegenerative diseases, odds ratios associated with risk alleles are generally low and account for only a small proportion of estimated heritability. Recent studies have revealed that the effect sizes of the disease-relevant alleles that are identified based on comprehensive resequencing of large data sets of Parkinson disease are substantially larger than those identified by GWAS. These findings strongly argue for the role of the 'common disease-multiple rare variants hypothesis' in sporadic neurodegenerative diseases. Given the rapidly improving technologies of next-generation sequencing next-generation sequencing (NGS), we expect that NGS will eventually enable us to identify all the variants in an individual's personal genome, in particular, clinically relevant alleles. Beyond this, whole genome resequencing is expected to bring a paradigm shift in clinical practice, where clinical practice including diagnosis and decision-making for appropriate therapeutic procedures is based on the 'personal genome'. The personal genome era is expected to be realized in the near future, and society needs to prepare for this new era.
| S-EPMC2875051 | biostudies-literature
Project description:Knowledge of the origins, distribution, and inheritance of variation in the malaria parasite (Plasmodium falciparum) genome is crucial for understanding its evolution; however the 81% (A+T) genome poses challenges to high-throughput sequencing technologies. We explore the viability of the Roche 454 Genome Sequencer FLX (GS FLX) high throughput sequencing technology for both whole genome sequencing and fine-resolution characterization of genetic exchange in malaria parasites.We present a scheme to survey recombination in the haploid stage genomes of two sibling parasite clones, using whole genome pyrosequencing that includes a sliding window approach to predict recombination breakpoints. Whole genome shotgun (WGS) sequencing generated approximately 2 million reads, with an average read length of approximately 300 bp. De novo assembly using a combination of WGS and 3 kb paired end libraries resulted in contigs ? 34 kb. More than 8,000 of the 24,599 SNP markers identified between parents were genotyped in the progeny, resulting in a marker density of approximately 1 marker/3.3 kb and allowing for the detection of previously unrecognized crossovers (COs) and many non crossover (NCO) gene conversions throughout the genome.By sequencing the 23 Mb genomes of two haploid progeny clones derived from a genetic cross at more than 30× coverage, we captured high resolution information on COs, NCOs and genetic variation within the progeny genomes. This study is the first to resequence progeny clones to examine fine structure of COs and NCOs in malaria parasites.
| S-EPMC3055840 | biostudies-other