MPD: multiplex primer design for next-generation targeted sequencing

Wingo, Thomas S.; Kotlar, Alex V.; Cutler, David J.

doi:10.1186/s12859-016-1453-3

Cited by 19 publications

(14 citation statements)

References 10 publications

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“…To capture sequence with acceptable range of accuracy and uniform representation across all the regions in multiplexed reactions, oligonucleotides must meet certain specifications in terms of sequence specificity, efficient oligo design with minimal interaction between the probes and optimal process time[14, 16, 17, 26]. AnthOligo was implemented to satisfy these requirements with the RSE method.…”

Section: Resultsmentioning

confidence: 99%

“…Probe design for targeted enrichment is a requirement for any NGS test development. Although there exist many stand-alone tools and web-applications to help address requirements for varied target enrichment approaches, none can be implemented directly for the RSE method[8-17]. The advantage of this specific oligonucleotide design method is the ability to “space” the oligos evenly at a certain distance (thousands of bases) and thus achieve equivalent target specificity with fewer probes required as compared to the tiling approach (1X or 2X tiling density) by many custom “kit” provisions.…”

Section: Introductionmentioning

confidence: 99%

“…Step 3: Design of optimal collection of oligos for target capture using multiplex PCR required combinatorial optimization solutions[11, 17] (Figure 1). The number of heterodimer combinations C for n oligos for each input region could be calculated as: …”

Section: Introductionmentioning

confidence: 99%

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AnthOligo: Automating the design of oligonucleotides for capture/enrichment technologies

Jayaraman

Mosbruger

et al. 2019

Preprint

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1 2 Summary 1 3A number of methods have been devised to address the need for targeted genomic resequencing. 4One of these methods, Region-specific extraction (RSE) of DNA is characterized by the capture 1 5 of long DNA fragments (15-20 kb) by magnetic beads, after enzymatic extension of 1 6 oligonucleotides hybridized to selected genomic regions. Facilitating the selection of the most 1 7optimal capture oligos targeting a region of interest, satisfying the properties of temperature 1 8 (Tm) and entropy (ΔG), while minimizing the formation of primer dimers in a pooled experiment 1 9 3 1 format and customize parameters for each task. A Redis-like MapReduce framework is 3 2implemented to run sub-tasks in parallel to optimize time and system resources alongside a 'task-3 3queuing' system that runs submitted jobs as a server-side background daemon. The task of probe 3 4 design in AnthOligo commences when a user uploads an input file and concludes with the 3 5 generation of a result-set containing an optimal set of region-specific oligos. 6AnthOligo is currently available as a public web application with URL: 3 7

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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AnthOligo: Automating the design of oligonucleotides for capture/enrichment technologies

Jayaraman

Mosbruger

et al. 2019

Preprint

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“…Multiplex bar-coded polymerase chain reaction amplification was carried out using next-generation sequencing libraries prepared with the Fluidigm Access Array (Fluidigm Corporation, South San Francisco, CA, USA). 17 DNA sequencing was carried out using the Illumina HiSeq platform. Galaxy workflow was used to identify somatic mutations in genes, including the top five genes mutated in CCRCC, as reported in Cosmic (https://cancer.sanger.ac.uk/cosmic) VHL, PBRM1, SETD2, BAP1 and KDM5C, as well as NFE2L2, which is mutated in papillary RCC, and TP53 (associated with carcinogenesis in general) by comparing the tumor samples with sequencing obtained from cells in the blood of the same patients.…”

Section: Polymerase Chain Reaction and Sequencingmentioning

confidence: 99%

Molecular characteristics and markers of advanced clear cell renal cell carcinoma: Pitfalls due to intratumoral heterogeneity and identification of genetic alterations associated with metastasis

et al. 2020

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Objectives To identify clear cell renal cell carcinoma‐related gene mutations potentially associated with aggressive disease, sarcomatoid differentiation or poor prognosis. Methods We carried out genomic analysis of 217 tumor foci from 25 patients with conventional clear cell renal cell carcinoma (14 patients), clear cell renal cell carcinoma with sarcomatoid differentiation (six patients) and non‐clear cell renal cell carcinoma (five patients). Each tumor nodule on the tissue block that corresponded to the same focus on the slide was separated from the normal parenchyma and other histologically distinct areas of tumor. The isolated tumor foci were used for subsequent analyses and sequencing. Deoxyribonucleic acid from the formalin‐fixed paraffin‐embedded tissues was extracted. Multiplex bar‐coded polymerase chain reaction amplification was carried out using next‐generation sequencing libraries. Results Overall, 67 protein alterations, including amino acid alterations, frame shifts and splice site mutations in seven genes were identified in the cohort of renal cell carcinoma tumors included in this study. Fewer patients with clear cell renal cell carcinoma with sarcomatoid differentiation had clear cell renal cell carcinoma‐related mutations in comparison with patients with conventional clear cell renal cell carcinoma. Additionally, the average number of unique clear cell renal cell carcinoma‐related protein alterations per patient was significantly lower in clear cell renal cell carcinoma with sarcomatoid differentiation than in conventional clear cell renal cell carcinoma. Mutations in PBRM1 were identified in a higher proportion of patients with high‐grade tumors (World Health Organization/International Society of Urological Pathology grade 4) and in the primary tumors of six of 10 (60%) patients with metastatic disease. Conclusions Although there are pitfalls due to intratumoral heterogeneity and sampling bias, mutations in PBRM1 may be associated with metastasis and aggressive disease in clear cell renal cell carcinoma.

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“…Genomic DNA was extracted from white blood cells of 310 ALS patients and 266 non-ALS subjects using the Gentra Puregene kit (Qiagen, Hilden, Germany) according to the manufacturer's protocols. For targeted resequencing, two sets of primers were designed by using the multiplex primer design software with >90% coverage for each gene (46) and optimal multiplex design for the Access Array System (Fluidigm, South San Francisco, CA, USA). The first set was designed to capture 14 candidate genes including DLG2, MYH15, KIF27 and ABCC2, and 5 known ALS genes (GRN, SOD1, FUS, TARDBP and TBK1) (Table 1).…”

Section: Targeted Resequencingmentioning

confidence: 99%

Rare variants in MYH15 modify amyotrophic lateral sclerosis risk

Kim

Bao

Jiao

et al. 2019

Human Molecular Genetics

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Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by progressive muscular atrophy and respiratory failure. The G4C2 repeat expansion in the C9orf72 gene is the most prevalent genetic risk for ALS. Mutation carriers (C9ALS) display variability in phenotypes such as age-at-onset and duration, suggesting the existence of additional genetic factors. Here we introduce a three-step gene discovery strategy to identify genetic factors modifying the risk of both C9ALS and sporadic ALS (sALS) using limited samples. We first identified 135 candidate genetic modifiers of C9ALS using whole-genome sequencing (WGS) of extreme C9ALS cases diagnosed ~30 years apart. We then performed an unbiased genetic screen using a Drosophila model of the G4C2 repeat expansion with the genes identified from WGS analysis. This genetic screen identified the novel genetic interaction between G4C2 repeat-associated toxicity and 18 genetic factors, suggesting their potential association with C9ALS risk. We went on to test if 14 out of the 18 genes, those which were not known to be risk factors for ALS previously, are also associated with ALS risk in sALS cases. Gene-based-statistical analyses of targeted resequencing and WGS were performed. These analyses together reveal that rare variants in MYH15 represent a likely genetic risk factor for ALS. Furthermore, we show that MYH15 could modulate the toxicity of dipeptides produced from expanded G4C2 repeat. Our study presented here demonstrates the power of combining WGS with fly genetics to facilitate the discovery of fundamental genetic components of complex traits with a limited number of samples.

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MPD: multiplex primer design for next-generation targeted sequencing

Cited by 19 publications

References 10 publications

AnthOligo: Automating the design of oligonucleotides for capture/enrichment technologies

AnthOligo: Automating the design of oligonucleotides for capture/enrichment technologies

Molecular characteristics and markers of advanced clear cell renal cell carcinoma: Pitfalls due to intratumoral heterogeneity and identification of genetic alterations associated with metastasis

Rare variants in MYH15 modify amyotrophic lateral sclerosis risk

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