한빛사 논문
Seyeon Kim1*; Sara Baldassari2*; Stéphanie Baulac2+; Jeong Ho Lee1,3+
1Graduate School of Medical Science and Engineering, KAIST, Daejeon, Korea
2Sorbonne Université, Institut du Cerveau - Paris Brain Institute - ICM, Inserm, CNRS, F75013, Paris, France
3SoVarGen, Inc., Daejeon 34051, Korea.
*equally contributed.
+equally contributed.
Abstract
In our study, the brain variant allele frequency (VAF) in our cohort of 12 patients varied from 1 to 24%. Previously, it was reported that atypical meningioma patient with 45% of brain VAF in the tumor presents 4% of VAF in cfDNA of CSF by ddPCR. Thus, only a very low fraction of these variants is expected to be detected in the cfDNA from CSF samples in epilepsy patients. With our protocol, the average amount of cfDNA of 0.4ng/µL (range 0.05- 2.7) was therefore insufficient to confidently call variants with VAF below 1% (according to the Biorad guidelines for rare mutation detection (https://www.biorad.com/webroot/web/pdf/lsr/literature/Bulletin_6628.pdf, Appendix B, Table 7). We therefore opted for a protocol that includes a step of pre-amplification of the cfDNA to increase the ddPCR assay sensitivity. Because we are well aware of the possible false-positive signals induced by pre-amplification, we performed multiple replicates (3–15) and used 3 to 5 controls, followed by appropriate statistical tests. Accordingly, with these stringent filters, we confidently detected the variants in 3 out of 12 cases with an average VAF of 0.54%, and did not consider the ambiguous cases (eg, KR-6 BRAF p.Val600Glu with 0.02% of VAF in cfDNA) in which the variants were also amplified in the controls.
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