μCaler MRD Solution is equipped with adapters containing dual Unique Molecular Identifiers (UMI & BMI), so as to meet the analysis requirements of ultra-low frequency mutations in plasma circulating cell-free DNA for MRD detection; in addition, this solution is designed for targeted enrichment of small Panel, integrated with upgraded and optimized hybrid capture and elution processes, and equipped with μCaler Panel designed based on innovative protocols, which can complete the whole process of capture-library preparation in same day.
Analysis of Variation Consistency in Standards
Samples were derived from Pancancer Light 800 gDNA Reference Standard (Genewell, GW-OGTM800) mixed with Human Male Genomic DNA (Promega, G1471) at the ratios of 10:0 and 1:9, respectively, to artificially mimic different mutation frequencies. Theoretical mutation frequencies at different mutation sites for the Pancancer Light 800 gDNA Reference Standard are as follows:
Figure 3. Consistency between expected allele frequency and observed allele frequency in the capture data of μCaler Panel and Traditional Panel. 30 ng simulated samples were used μCaler MRD Solution (for Illumina®) coupled with μCaler Panel and NadPrep Hybrid Capture System coupled with Traditional Panel to complete hybrid capture respectively, and sequenced on Illumina Novaseq 6000 with PE150. The detection consistency of mutations (pre-library containing UMI) was analyzed by sequencing depth from DownSampling each sample data to 80,000x before removing duplication, and the analytical filtering criteria are single-strand consensus sequences (family size ≥ 3) (SSCS ≥ 3).
Customized SNP Panels to Simulate Low Frequency Mutation Limit Detection
gDNA from two healthy donors with known mutation was mixed at ratios of 99:1, 999:1, and 9999:1 to artificially mimic samples with different mutation frequencies. Category 1 contain 19 homozygous mutation with mutation frequency of 1%, 0.1% and 0.01% respectively, and category 2 contain 11 heterozygous mutation with mutation frequency of 0.5%, 0.05% and 0.005% respectively.
Figure 4. Detection of low-frequency mutations by μCaler Panel and Traditional Panel. 50 ng simulated samples were used by μCaler MRD Solution (for Illumina®) coupled with μCaler Panel (about 3 Kb target region, covering 30 allelic loci) and NadPrep Hybrid Capture System coupled with Traditional Panel (about 42 Kb target region, covering 160 allelic loci, including the above 30 loci) respectively, and sequenced on Illumina Novaseq 6000 with PE150. Each pair of samples was subjected to the same depth (pre-library containing UMI) for three downsampling, and the detection of the mutation site was analyzed by Duplex Consensus Sequences (DCS211) as well as SSCS ≥ 3. Average valid reads supporting mutations ≥ 1 are judged as positive.
Figure 5. Comparative analysis of detection and theoretical detection for low-frequency mutations by μCaler Panel and Traditional Panel.
Note:Theoretical number of mutation is 2,750x of DCS211 depth; the distribution rule of mutation detection by 10,000 random sampling for different mutation frequencies.
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