Abstract Introduction: Circulating tumor DNA (ctDNA) based MRD detection has been proven effective for predicting the relapse of various cancers after curative surgery. We developed a custom-built, tumor-informed MRD detection method named OncoWES-MRD. Somatic variants were identified and prioritized by whole-exome sequencing (WES) of tumor tissues and matched peripheral blood samples. Patient-specific panel targeting up to 50 top-ranked single-nucleotide and Indel variants was designed to analyze plasma for assessing the presence of ctDNA. The ctDNA level was also quantified by in-house software. Here, we report the analytical validation results of this assay. Methods: The analytical validation was performed on a colorectal cancer cell line SW480. The sheared SW480 DNA was titrated to normal DNA from NA12878 cell line to produce samples with a variant allele frequency (VAF) of 0.1%. The dilution accuracy was confirmed by droplet digital PCR (ddPCR). Then the 0.1% samples were serially diluted to VAF of 0.05%, 0.02%, 0.01%, 0.005%, 0.003%, and 0.001%. As for these dilution gradients below the detection limit blank of the ddPCR assay (0.1%), the changes of exogenously introduced viral DNA copies were used as indicators of the dilution ratio. To explore the relations between the number of variants and the limit of detection (LoD), a customized capture panel covering 200 prioritized variants for SW480 was designed. Each gradient was tested 42 times with an average sequencing depth above 100,000X. The analytical specificity assessment was conducted on 20 NA12878 wild-type samples and 44 blood samples from 22 healthy donors. The library prep starting DNA input was 60 ng for each reaction. ctDNA fraction was evaluated using the maximum likelihood method, and also taking allelic copy number and cancer cell fraction into consideration. Results: The WES-MRD assay by monitoring 50 variants demonstrated a sample-level sensitivity of 97.6% at VAF of 0.003%, and 76.2% sensitivity at VAF of 0.001%, along with a specificity of 98.4%. The LOD of 0.1% VAF was achieved at the single variant level. The assay performed 95.2% analytical sensitivity at 0.02% VAF, even with 6 somatic mutations, therefore defining the minimal monitoring number. Based on WES sequencing data of 514 solid tumors, 96.1% of samples met the assay requirement of variant numbers. The upper limit monitoring variants number was set to 50, as keep increasing the monitoring variants showed limited sensitivity improvement while specificity was impaired. In the quantitative aspect, a strong linear correlation between the theoretical cell line tumor fractions with the estimated ctDNA fractions (R2 = 0.98). Conclusions: The WES-based personalized MRD assay demonstrated high sensitivity and high specificity to detect tumor-derived variants down to VAF of 0.003% and accurately quantified. Citation Format: Wei Gao, Wuqiang Cao, Xiaoling Zeng, Zihan Tian, Xue Zhang, Ning Fu, Pansong Li. Analytical validation of an NGS-based, personalized and tumor-informed liquid biopsy assay for detecting minimal residual disease (MRD) with high sensitivity and specificity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5578.