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However, circulating cell-free tumor DNA (ctDNA) is highly diluted by cfDNA from non-cancer cells, complicating ctDNA detection and analysis. To identify low-frequency variants, we developed a program, eVIDENCE, which is a workflow for filtering candidate variants detected by using the ThruPLEX tag-seq (Takara Bio), a commercially-available molecular barcoding kit. We analyzed 27 cfDNA samples from hepatocellular carcinoma patients. Sequencing libraries were constructed and hybridized to our custom panel targeting about 80 genes. An initial variant calling identified 36,500 single nucleotide variants (SNVs) and 9,300 insertions and deletions (indels) across the 27 samples, but the number was much greater than expected when compared with previous cancer genome studies. eVIDENCE was applied to the candidate variants and finally 70 SNVs and 7 indels remained. Of the 77 variants, 49 (63.6%) showed VAF of\u2009&lt;\u20091% (0.20\u20130.98%). Twenty-five variants were selected in an unbiased manner and all were successfully validated, suggesting that eVIDENCE can identify variants with VAF of\u2009\u2265\u20090.2%. Additionally, this study is the first to detect hepatitis B virus integration sites and genomic rearrangements in the <jats:italic>TERT<\/jats:italic> region from cfDNA of HCC patients. We consider that our method can be applied in the examination of cfDNA from other types of malignancies using specific custom gene panels and will contribute to comprehensive ctDNA analysis.<\/jats:p>","DOI":"10.1038\/s41598-019-51459-4","type":"journal-article","created":{"date-parts":[[2019,10,23]],"date-time":"2019-10-23T04:26:13Z","timestamp":1571804773000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["eVIDENCE: a practical variant filtering for low-frequency variants detection in cell-free DNA"],"prefix":"10.1038","volume":"9","author":[{"given":"Kei","family":"Mizuno","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shusuke","family":"Akamatsu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takayuki","family":"Sumiyoshi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing Hao","family":"Wong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1457-6233","authenticated-orcid":false,"given":"Masashi","family":"Fujita","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kazuaki","family":"Maejima","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kaoru","family":"Nakano","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Atushi","family":"Ono","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroshi","family":"Aikata","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masaki","family":"Ueno","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shinya","family":"Hayami","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroki","family":"Yamaue","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kazuaki","family":"Chayama","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takahiro","family":"Inoue","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Osamu","family":"Ogawa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hidewaki","family":"Nakagawa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Akihiro","family":"Fujimoto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2019,10,22]]},"reference":[{"key":"51459_CR1","doi-asserted-by":"publisher","first-page":"579","DOI":"10.1200\/JCO.2012.45.2011","volume":"32","author":"LA Diaz","year":"2014","unstructured":"Diaz, L. 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