μCaler MRD Solution (for Illumina®) is equipped with adapters containing dual Unique Molecular Identifiers (UMI), 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.
Package | Sub-package name | Color of Tube Cap | Component | Volume |
Box 1 | Library Prep & hybrid |
|
EndRepair & A-Tailing Buffer | 100 μL |
|
EndRepair & A-Tailing Enzyme | 65 μL | ||
|
Ligation Buffer | 380 μL | ||
|
DNA ligase | 65 μL | ||
|
2X HiFi PCR Master Mix | 1450 μL | ||
/ |
Nuclease Free Water | 4 mL | ||
|
TE Sloution | 1000 μL | ||
|
μCaler NanoBlockers (for Illumina) | 60 μL | ||
|
Human Cot DNA | 60 μL | ||
|
μHyb #1 | 1100 μL | ||
|
μHyb #2 | 180 μL | ||
|
NadPrep Amplification Primer Mix II | 75 μL | ||
UMI Adapter |
|
NadPrep UMI Adapter | 60 μL | |
|
NadPrep Universal UDI-Index Primer Mix | 12×15 μL | ||
Custom Panel |
|
μCaler Custom Panel | 60 μL | |
Box 2 | Elution |
/ |
NadPrep SP beads | 4 mL |
|
Streptavidin Beads | 750 μL | ||
/ |
Wash Buffer A | 2×8 mL | ||
/ |
Wash Buffer B | 4.7 mL |
Library conversion rate |
Sample Input |
|||||||||
1 ng |
10 ng |
20 ng |
30 ng |
50 ng |
100 ng |
200 ng |
300 ng |
500 ng |
1000 ng |
|
20% |
5.000% |
0.500% |
0.250% |
0.168% |
0.100% |
0.050% |
0.025% |
0.017% |
0.010% |
0.005% |
30% |
3.400% |
0.340% |
0.170% |
0.113% |
0.068% |
0.034% |
0.017% |
0.011% |
0.007% |
0.003% |
50% |
2.000% |
0.200% |
0.100% |
0.067% |
0.040% |
0.020% |
0.010% |
0.007% |
0.004% |
0.002% |
100% |
1.000% |
0.100% |
0.050% |
0.033% |
0.020% |
0.010% |
0.005% |
0.003% |
0.002% |
0.001% |
① Increase the sample input:The more sample input , the copies of support low-frequency mutation detection will be more;
② Increase the number of tracked mutations: the higher the number of mutation sites, the probability of detection will be higher.
|
Possible Reason |
Solution |
Relatively high starting input |
Quantification by Qubit™ 3.0 is recommended. |
Relatively high amplification cycles |
Explore the optimal amplification cycles according to the recommended protocol in the operating guideline. |
The elution is not performed according to operational guideline, leading to serious off-target. |
The elution is performed in strict accordance with the requirements of operating guideline. |
Possible Reason |
Solution |
Inaccurate starting input |
In case of lower actual starting input, precise quantification by qPCR is recommended. |
Reaction mixture is not completely transferred out when pipetting. |
Transfer all reaction mixture from each step to a new tube when pipetting. |
Some magnetic beads are discarded during hybridization and bead purification |
Avoid too intense operation during the elution, and discard supernatant without losing any magnetic beads. |
Possible Reason |
Solution |
A lot of air bubbles are generated during elution |
When using a pipette, it is necessary to gently pipette up and down to mix well to prevent the air from entering; If air bubbles are generated, first use the pipette to remove the air bubbles above the solution and then discard the supernatant. |
PCR tube cap is not replaced |
Replace the PCR tube and cap in strict accordance with the instruction for use. |
Reagent is not fully mixed |
Mix the reagents thoroughly before use according to the operation guideline and allow to warm up at the corresponding temperature. |
Type | Product | Detail | Catalog# |
Lib Prep Module | NadPrep DNA Library Preparation Kit (for Illumina®) G24 | 24 rxn | 1002101 |
NadPrep DNA Library Preparation Kit (for Illumina®) E96 | 96 rxn | 1002103 | |
Adapter Module | NadPrep UMI Adapter Kit Set A1 (with 10 nt Index), 24 rxn | 1-12 | 1103111 |
NadPrep UMI Adapter Kit Set B1 (with 10 nt Index), 96 rxn | 1-24 | 1103121 | |
NadPrep UMI Adapter Kit Set B2 (with 10 nt Index), 96 rxn | 25-48 | 1103122 | |
Blocker | μCaler NanoBlockers (for Illumina®), 16 rxn | 16 rxn | 1106102 |
μCaler NanoBlockers (for Illumina®), 96 rxn | 96 rxn | 1106101 | |
Hybrid Capture | μCaler Hybrid Capture Reagents, 16 rxn | 16 rxn | 1105102 |
μCaler Hybrid Capture Reagents, 96 rxn | 96 rxn | 1105101 | |
Panel | μCaler Custom Panel, 16 rxn | 16 rxn | 1101201 |
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