28 research outputs found

    Evaluating the Clinical Validity of Gene-Disease Associations: An Evidence-Based Framework Developed by the Clinical Genome Resource

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    Supplemental Data Supplemental Data include 65 figures and can be found with this article online at http://dx.doi.org/10.1016/j.ajhg.2017.04.015. Supplemental Data Document S1. Figures S1–S65 Download Document S2. Article plus Supplemental Data Download Web Resources ClinGen, https://www.clinicalgenome.org/ ClinGen Gene Curation, https://www.clinicalgenome.org/working-groups/gene-curation/ ClinGen Gene Curation SOP, https://www.clinicalgenome.org/working-groups/gene-curation/projects-initiatives/gene-disease-clinical-validity-sop/ ClinGen Knowledge Base, https://search.clinicalgenome.org/kb/agents/sign_up OMIM, http://www.omim.org/ Orphanet, http://www.orpha.net/consor/cgi-bin/index.php With advances in genomic sequencing technology, the number of reported gene-disease relationships has rapidly expanded. However, the evidence supporting these claims varies widely, confounding accurate evaluation of genomic variation in a clinical setting. Despite the critical need to differentiate clinically valid relationships from less well-substantiated relationships, standard guidelines for such evaluation do not currently exist. The NIH-funded Clinical Genome Resource (ClinGen) has developed a framework to define and evaluate the clinical validity of gene-disease pairs across a variety of Mendelian disorders. In this manuscript we describe a proposed framework to evaluate relevant genetic and experimental evidence supporting or contradicting a gene-disease relationship and the subsequent validation of this framework using a set of representative gene-disease pairs. The framework provides a semiquantitative measurement for the strength of evidence of a gene-disease relationship that correlates to a qualitative classification: “Definitive,” “Strong,” “Moderate,” “Limited,” “No Reported Evidence,” or “Conflicting Evidence.” Within the ClinGen structure, classifications derived with this framework are reviewed and confirmed or adjusted based on clinical expertise of appropriate disease experts. Detailed guidance for utilizing this framework and access to the curation interface is available on our website. This evidence-based, systematic method to assess the strength of gene-disease relationships will facilitate more knowledgeable utilization of genomic variants in clinical and research settings

    On-target efficiency for libraries prepared from samples either treated or untreated with the mung bean nuclease.

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    <p>On-target efficiency for libraries prepared from samples either treated or untreated with the mung bean nuclease.</p

    Mung Bean Nuclease Treatment Increases Capture Specificity of Microdroplet-PCR Based Targeted DNA Enrichment

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    <div><p>Targeted DNA enrichment coupled with next generation sequencing has been increasingly used for interrogation of select sub-genomic regions at high depth of coverage in a cost effective manner. Specificity measured by on-target efficiency is a key performance metric for target enrichment. Non-specific capture leads to off-target reads, resulting in waste of sequencing throughput on irrelevant regions. Microdroplet-PCR allows simultaneous amplification of up to thousands of regions in the genome and is among the most commonly used strategies for target enrichment. Here we show that carryover of single-stranded template genomic DNA from microdroplet-PCR constitutes a major contributing factor for off-target reads in the resultant libraries. Moreover, treatment of microdroplet-PCR enrichment products with a nuclease specific to single-stranded DNA alleviates off-target load and improves enrichment specificity. We propose that nuclease treatment of enrichment products should be incorporated in the workflow of targeted sequencing using microdroplet-PCR for target capture. These findings may have a broad impact on other PCR based applications for which removal of template DNA is beneficial.</p></div

    Mung bean nuclease treatment significantly increases on-target efficiency for DNA enriched through RainDance microdroplet-PCR.

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    <p>Aliquots of RainDance enriched DNA for the same sample were either treated or not treated with mung bean nuclease, processed into TruSeq libraries, and sequenced on MiSeq as illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103491#pone-0103491-g001" target="_blank">Figure 1A</a>. Plotted is the mean value of on-target efficiency of 3 samples that went through parallel treatments (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103491#pone-0103491-t002" target="_blank">Table 2</a>). Nuclease treatment leads to significantly higher on-target efficiency (*<i>p</i> = 0.018, one-tail paired <i>t</i> test; error bar, SEM).</p

    On-target efficiency for libraries prepared from untreated RainDance captured DNA.

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    <p>On-target efficiency for libraries prepared from untreated RainDance captured DNA.</p

    Mung bean nuclease treatment abolished the high molecular DNA smear in RainDance captured DNA after end repair.

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    <p><b>A.</b> An overview of the workflow. Aliquots of 100ng of DNA enriched through microdroplet-PCR were either treated with mung-bean nuclease or untreated as a control. The differentially treated aliquots were end-repaired, concatenated, processed into TruSeq libraries and sequenced on MiSeq in parallel to rule out any batch effect. <b>B.</b> Electrophoresis analysis of DNA samples on a high-sensitivity DNA chip using the Agilent 2100 Bioanalyzer. DNA samples in all 4 lanes were derived from 200pg of the same batch of captured DNA. From left to right, the lanes are, “L” lane-the DNA size ladder, lane 1-untreated DNA enriched by RainDance microdroplet-PCR prior to end repair, lane 2- untreated DNA enriched by RainDance microdroplet-PCR post end repair, lane 3- mung bean nuclease treated DNA enriched by RainDance microdroplet-PCR prior to end repair, lane 4- mung bean nuclease treated DNA enriched by RainDance microdroplet-PCR post end repair.</p
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