{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,9,28]],"date-time":"2025-09-28T11:14:15Z","timestamp":1759058055749,"version":"3.41.2"},"reference-count":29,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2020,3,16]],"date-time":"2020-03-16T00:00:00Z","timestamp":1584316800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"DOI":"10.13039\/100000050","name":"National Heart, Lung, and Blood Institute","doi-asserted-by":"publisher","award":["R01HD087292"],"award-info":[{"award-number":["R01HD087292"]}],"id":[{"id":"10.13039\/100000050","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000051","name":"National Human Genome Research Institute","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000051","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000065","name":"National Institute of Neurological Disorders and Stroke","doi-asserted-by":"publisher","award":["NS105078"],"award-info":[{"award-number":["NS105078"]}],"id":[{"id":"10.13039\/100000065","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["GM106373"],"award-info":[{"award-number":["GM106373"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["currentprotocols.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["CP Human Genetics"],"published-print":{"date-parts":[[2020,6]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>As genome sequencing methodologies have become more sensitive in detecting low\u2010frequency rare\u2010variant events, the link between post\u2010zygotic mutagenesis and somatic mosaicism in the etiology of several human genetic conditions other than cancers has become more clear. Given that current clinical\u2010genomics diagnostic methods have limited detection sensitivity for mosaic events, a copy\u2010number variant (CNV) deletion inherited from a parent with low\u2010level (&lt;10%) mosaicism can be erroneously interpreted in the proband to represent a <jats:italic>de novo<\/jats:italic> germline event. Here, we describe three sensitive, precise, and cost\u2010efficient methods that can quantitatively assess the potential degree of parental somatic mosaicism levels for CNV deletions: droplet digital PCR (ddPCR), PCR amplicon\u2013based next\u2010generation sequencing (NGS), and quantitative PCR. ddPCR using the EvaGreen fluorescent dye protocol can specifically quantify the deleted or non\u2010deleted alleles by analyzing the number of droplets positive for a fluorescent signal for each event. PCR amplicon\u2013based NGS assesses the allele frequencies of a heterozygous single\u2010nucleotide polymorphism within a deletion region. The difference in number of reads between the two genotypes indicates the level of somatic mosaicism for the CNV deletion. Quantitative PCR can be applied where the relative quantity of the deletion junction\u2013specific product represents the level of mosaicism. Clinical implementation of these quantitative variant\u2010detection methods enables potentially more accurate assessment of disease recurrence risk in family\u2010based genetic counseling, allowing couples to engage in more informed family planning. \u00a9 2020 by John Wiley &amp; Sons, Inc.<\/jats:p><jats:p><jats:bold>Basic Protocol<\/jats:bold>: Droplet digital PCR (ddPCR)<\/jats:p><jats:p><jats:bold>Alternate Protocol 1<\/jats:bold>: PCR amplicon\u2013based next\u2010generation sequencing<\/jats:p><jats:p><jats:bold>Alternate Protocol 2<\/jats:bold>: Quantitative real\u2010time PCR (qPCR)<\/jats:p>","DOI":"10.1002\/cphg.99","type":"journal-article","created":{"date-parts":[[2020,3,16]],"date-time":"2020-03-16T18:33:43Z","timestamp":1584383623000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Quantitative Assessment of Parental Somatic Mosaicism for Copy\u2010Number Variant (CNV) Deletions"],"prefix":"10.1002","volume":"106","author":[{"given":"Qian","family":"Liu","sequence":"first","affiliation":[{"name":"Department of Molecular and Human Genetics Baylor College of Medicine  Houston Texas"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Christopher M.","family":"Grochowski","sequence":"additional","affiliation":[{"name":"Department of Molecular and Human Genetics Baylor College of Medicine  Houston Texas"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Weimin","family":"Bi","sequence":"additional","affiliation":[{"name":"Department of Molecular and Human Genetics Baylor College of Medicine  Houston Texas"},{"name":"Baylor Genetics  Houston Texas"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"James R.","family":"Lupski","sequence":"additional","affiliation":[{"name":"Department of Molecular and Human Genetics Baylor College of Medicine  Houston Texas"},{"name":"Human Genome Sequencing Center Baylor College of Medicine  Houston Texas"},{"name":"Texas Children's Hospital  Houston Texas"},{"name":"Department of Pediatrics Baylor College of Medicine  Houston Texas"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pawe\u0142","family":"Stankiewicz","sequence":"additional","affiliation":[{"name":"Department of Molecular and Human Genetics Baylor College of Medicine  Houston Texas"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2020,3,16]]},"reference":[{"key":"e_1_2_6_2_1_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrg3424"},{"key":"e_1_2_6_2_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41591-019-0711-0"},{"key":"e_1_2_6_2_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ajhg.2014.08.010"},{"key":"e_1_2_6_2_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ajhg.2014.07.003"},{"key":"e_1_2_6_2_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mrrev.2010.04.002"},{"key":"e_1_2_6_2_6_1","doi-asserted-by":"publisher","DOI":"10.1021\/ac202028g"},{"key":"e_1_2_6_2_7_1","doi-asserted-by":"publisher","DOI":"10.2174\/138920210793176065"},{"key":"e_1_2_6_2_8_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41588-018-0259-9"},{"key":"e_1_2_6_2_9_1","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/bty036"},{"key":"e_1_2_6_2_10_1","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btm091"},{"key":"e_1_2_6_2_11_1","doi-asserted-by":"publisher","DOI":"10.1038\/nn.4598"},{"key":"e_1_2_6_2_12_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1239503"},{"key":"e_1_2_6_2_13_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrg2626"},{"key":"e_1_2_6_2_14_1","doi-asserted-by":"publisher","DOI":"10.1159\/000184717"},{"key":"e_1_2_6_2_15_1","doi-asserted-by":"publisher","DOI":"10.1021\/ac202578x"},{"key":"e_1_2_6_2_16_1","doi-asserted-by":"publisher","DOI":"10.1038\/ng.3469"},{"key":"e_1_2_6_2_17_1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.1754"},{"key":"e_1_2_6_2_18_1","first-page":"7.4A.1","article-title":"DNA sequencing by the dideoxy method","volume":"47","author":"Slatko B. 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