μCaler MRD Solution

Catalog Number:

#1100100
#1103111 #1103112 #1103113 #1103114

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.

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Schematic Diagram of Working Principle in μCaler Hybrid System

Find target region by probe to form stable binding

Rapid binding of multiple probe conjugation effects for further stabilization

principle

Feature

Simplified process for easier operation

More Faster for hybridization capture,Complete the whole process from samples to capture-library preparation in same day

Small Panel has stable and excellent capture performance

Compatible with automated workstations

Performance

More Convenient

rapid

More Stable

Different types of samples and inputs

Figure 1. Capture performance of μCaler MRD Solution on cfDNA and gDNA libraries with different inputs. A. Mappability and On-target rate; B. Target covered; C. Fold 80 base penalty.Hybrid capture was accomplished using μCaler MRD Solution (for Illumina®) and M71 (8.5 Kb, GC range 25-70%) for Healthy Human-derived Pooled cfDNA Standards and Human Male Genomic DNA (Promega, G1471), and sequenced on Illumina Novaseq 6000 with PE150. The BWA was used for alignment of raw reads to the reference genome (hg38) and On-target rate was calculated by the number of reads.

Different types of panels

Figure 2. Capture performance of μCaler^®MRD Solution for different types of panels. A. Mappability and On-target rate; B. Target covered; C. Fold 80 base penalty.Hybrid capture was accomplished using μCaler MRD Solution (for Illumina®) for Human Male Genomic DNA (Promega, G1471) as well as M27 (3.2 Kb, GC range 25-55%), M44 (5.28 Kb, GC range 25-70%), M50 (5 Kb, GC range 37-90%), and M580 (58 Kb, GC range 19-99%).

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.

For research use only. Not for use in diagnostic procedures.

Lib Prep Module

Packing No.	Color of Tube Cap	Component	Volume 1002101 (24 rxn)	Volume 1002103 (96 rxn)
Box1		EndRepair & A-Tailing Buffer	180 µL	690 µL
		End Repair & A-Tailing Enzyme	120 µL	460 µL
		Ligation Buffer	750 µL	2×1500 µL
		DNA Ligase	65 µL	250 µL
		2X HiFi PCR Master Mix	720 µL	2×1600 µL
		NadPrep Amplification Primer Mix II	145 µL	650 µL
	/	Nuclease Free Water	2.5 mL	8 mL
		TE Solution	1 mL	4 mL
Box2	/	NadPrep SP Beads	4 mL	15 mL

Packing No.	Color of Tube Cap	Component	Volume 1002211(24 rxn)	Volume 1002212 (96 rxn)
Box1		EndRepair & A-Tailing Buffer	180 µL	690 µL
		End Repair & A-Tailing Enzyme	120 µL	460µL
		Ligation Buffer	750 µL	2×1500 µL
		DNA Ligase	65 µL	250 µL
		2X HiFi PCR Master Mix	720 µL	2×1600 µL
		NadPrep M-Amplification Primer Mix (for MGI, SI)	20 µL	75 µL
		NadPrep M-Amplification Primer Mix (for MGI, DI)	20 µL	75 µL
		NadPrep M-Amplification Primer Mix (for MGI, DI)	20 µL	75 µL
	/	Nuclease Free Water	2.5 mL	8 mL
		TE Solution	1 mL	4 mL
Box2	/	NadPrep SP Beads	4 mL	15 mL

Adapter Module

Color of Tube Cap	Component	Volume 1103111 (24 rxn)	Volume 1103121 (96 rxn)	Volume 1103122 (96 rxn)
	NadPrep Universal UDI-Index Primer Mix #	12×15 µL	24×25 µL	24×25 µL
	NadPrep UMI Adapter	60 µL	230 µL	230 µL
	NadPrep Amplification Primer Mix II	20 μL	75 μL	75 μL

Color of Tube Cap	Component	Volume 1003911 (24 rxn)	Volume 1003921 (96 rxn)	Volume 1003922(96 rxn)	Volume 1003925 (96 rxn)	Volume 1003926 (96 rxn)
	NadPrep M-Index (MDI) Primer Mix #	12×15 µL	24×25 µL	24×25 µL	24×25 µL	24×25 µL
	NadPrep BMI-Adapter	60 µL	230 µL	230 µL	230 µL	230 µL

Blocker

Color of Tube Cap

Component

Volume

1106102 (16 rxn)

Volume

1106101 (96 rxn)

μCalerNanoBlockers (for Illumina^®)

40 μL

210 μL

Color of Tube Cap

Component

Volume

1106212 (16 rxn)

Volume

1106211 (96 rxn)

μCaler NanoBlockers (for MGI, DI)

40 μL

210 μL

Hybrid Capture

Packing No.	Color of Tube Cap	Component	Volume 1105202 (16 rxn)	Volume 1105201 (96 rxn)
Packing No.	Color of Tube Cap	Component	Volume 1105202 (16 rxn)	Volume 1105201 (96 rxn)
Box 1		μHyb #1	840 μL	3×1700 μL
		μHyb #2	130 μL	790 μL
		Human Cot DNA	45 μL	260 μL
		μEnhancer	1300 μL	5×1600 μL
Box 2		Streptavidin Beads	480 μL	2.9 mL
	/	Wash Buffer A Pro	2×5.8 mL	3×23 mL
	/	Wash Buffer B	2.9 mL	20 mL

Panel

Catalog	/Color of Tube Cap	Component	Volume
1100100		μCaler Custom Panel, 24rxn	60 μL

Type	Product	Detail	Catalog#
Lib Prep Module	NadPrep DNA Library Preparation Kit (for Illumina®) G24	24 rxn	1002101
Lib Prep Module	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
	NadPrep BMI Adapter (MDI) Module Set A1 (for MGI), 24 rxn	MDI 1-12	1003911
	NadPrep BMI Adapter (MDI) Module Set B1 (for MGI), 96 rxn	MDI 1-24	1003921
	NadPrep BMI Adapter (MDI) Module Set B2 (for MGI), 96 rxn	MDI 25-48	1003922
	NadPrep BMI Adapter (MDI) Module Set B3 (for MGI), 96 rxn	MDI 49-72	1003925
	NadPrep BMI Adapter (MDI) Module Set B4 (for MGI), 96 rxn	MDI 73-96	1003926
Blocker	μCaler NanoBlockers (for Illumina®), 16 rxn	16 rxn	1106102
	μCaler NanoBlockers (for Illumina®), 96 rxn	96 rxn	1106101
	μCaler NanoBlockers (for MGI, DI), 16 rxn	16 rxn	1106212
	μCaler NanoBlockers (for MGI, DI), 96 rxn	96 rxn	1106211
Hybrid Capture	μCaler Hybrid Capture Reagents, 16 rxn	16 rxn	1105102
Hybrid Capture	μCaler Hybrid Capture Reagents, 96 rxn	96 rxn	1105101
Panel	μCaler Custom Panel, 16 rxn	16 rxn	1101201