μCaler Total Solution for Methylation

Catalog Number:

#1105201
#1105202

μCaler Targeted Methylation Sequencing Comprehensive Solution, featuring an exclusive patented μCaler hybridization capture system, enabling the completions of the entire process within same day with simple and convenient operations. This solution captures the converted methylation libraries using a patented probe design scheme that covers targets in all methylation states. Furthermore, with a hybridization capture system optimized for methylation is not only suitable for targeted enrichment with mini-Panels but also applicable for capturing special regions like AT-rich sequences.

μCaler Hybrid Capture Reagents v2 is designed for targeted enrichment of small Panel and hybrid capture of various types of pre-libraries, 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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Workflow

fig-01

*The above schematic diagram is taking NadPrep Methyl Stubby Adapter (UDI) (with 10 nt Index) as an example.

Features

• Precise detection of large-scale CpG sites:

Simultaneously assessment of multiple genes' CpG sites associated with various cancers, achieving precise comprehensive joint testing

• Accurate quantification of methylation levels:

High-precision coverage for accurate quantification of different methylation states ranging from 0 to 100% in various samples

• Fast and convenient operation:

Avoid inconsistent amount of raw data, ensuring stability in capture sequencing results, thereby reducing the risk of rework

• Highly efficient and stable: Avoid inconsistent amount of raw data, ensuring stability in capture sequencing results, thereby reducing the risk of rework
• Cost-effective sequencing: Utilizinge a mixed hybrid mode to reduce the quantity of hybrid reaction, lowering sequencing costs and enhancing cost-effectiveness

Performance

Precise detection of large-scale CpG sites

Figure 1. Performance of μCaler targeted methylation sequencing with custom panels containing different scales of CpG sites. Human genomic DNA standards (Promega, G1471) were used to prepare pre-libraries using the NadPrep Methyl Library Preparation Module in conjunction with the NadPrep Methyl Stubby Adapter (UDI) Module (with 10 nt Index) and NadPrep DNA Methyl Bisulfite Conversion Module. Hybrid capture were completed with μCaler Methyl Cervical Cancer Panel (MCC) and μCaler Methyl Multiple Cancer Panel (MMC), followed by sequencing on Illumina Novaseq 6000, PE 150. For each sample, 1 Mb reads pair was randomly selected for data analysis. A. Capture performance of MCC; B. CpG site coverage depth of MCC; C. Capture performance of MMC; D. CpG site coverage depth of MMC.

Note:The coverage area for MCC is approximately 1.4 Kb, containing 125 CpG sites; whereas the coverage area for MMC is approximately 3.6 Kb, containing 336 CpG sites.

Accurate quantification of methylation levels

Figure 2. Methylation levels of simulated samples detected by different methods. Pre-libraries were prepared using 50 ng of simulated samples with varying methylation levels, with 500 ng input per pre-library, and hybrid capture was completed with MCC. A-E. The detection results for simulated samples at methylation levels of 0%, 2%, 10%, 50%, and 100%, respectively.
Note: The samples consist of 100% methylated DNA (zymo, D5014-2) as a positive control and 0% Methylated DNA (Zymo, D5014-1) as a negative control, mixed at different ratios to simulate different methylation levels. μCaler: μCaler targeted methylation sequencing; Pyro: Pyrosequencing.

Fast and convenient operation

Highly efficient and stable

Figure 3. Capture performance of μCaler targeted methylation sequencing for mini panels. Pre-libraries were prepared using human genomic DNA standards (Promega, G1471), with 500 ng input per pre-library, and hybrid capture was completed with MBT, MMC, and MPC, respectively. A. Mappability and on-target rate; B. Target covered of CpG sites; C. Conversion rate of cytosine bases; D. CpG methylation levels.

Note: The coverage area for MBT is approximately 1.2 Kb, containing 159 CpG sites; while the coverage area for MPC is approximately 6.2 Kb, containing 620 CpG sites.

Cost-effective sequencing

Figure 4. Capture performance of μCaler targeted methylation sequencing under different mixed hybridization modes. Pre-libraries were prepared using human genomic DNA Standards (Promega, G1471), with 500 ng input per pre-library, and hybrid capture was performed with MCC. A. Target covered of CpG sites; B. CpG Methylation levels.

Application example of clinical sample

Figure 5. Comparison of detection results from different technical approaches using clinical samples. A.Detection results for samples with high methylation levels (Tumor); B. Detection results for samples with low methylation levels (Normal); C. Methylation levels of CpG sites related to chromosome 7 (Chr 7).
Note: μCaler targeted methylation sequencing was performed with MBT for hybrid capture.

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

Methyl Library Prep Module

Package	Color of Cap Tube	Component	Volume 1002501 (24 rxn)	Volume 1002502 (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	60 µL	230 µL
		2X HiFi-Methyl PCR Master Mix	720 µL	2×1600 µL
	/	Nuclease Free Water	2.5 mL	8 mL
		TE Solution	1 mL	4 mL
Box2	/	NadPrep SP Beads	4 mL	15 mL

BS Conversion Module

Package	Color of Cap Tube	Component	Volume 1002701 (24 rxn)	Volume 1002702 (96 rxn)
Package	Color of Cap Tube	Component	Volume 1002701 (24 rxn)	Volume 1002702 (96 rxn)
Box 1	/	Bisulfite Diluent	4.5 mL	9 mL
		Bisulfite Mix	4×1 tube	8×1 tube
		Desulf Buffer	1.8 mL	7.5 mL
Box 2	/	Nuclease Free Water	2.5 mL	8 mL
	/	Bisulfite Beads	4.8 mL	3×6.2 mL
		DNA Protect Buffer	100 μL	380 μL

Methyl Adapter (SI) Module (for MGI)

Color of Cap Tube	Component	Volume 1003631 (24 rxn)	Volume 1003632 (96 rxn)	Volume 1003633 (96 rxn)
	NadPrep M-Index (SI) Primer Mix #	12×15 µL	24×25 µL	24×25 µL
	NadPrep Methyl M-Adapter	60 µL	230 µL	230 µL

Methyl Adapter (MDI) Module (for MGI)

Color of Cap Tube

Component

Volume
1003740 (24 rxn)

Volume
1003741 (96 rxn)

Volume
1003742 (96 rxn)

Volume

1003743（96 rxn）

Volume

1003744（96 rxn）

NadPrep M-Index (MDI) Primer Mix #

12×15 µL

24×25 µL

NadPrep Methyl M-Adapter

60 µL

230 µL

Methyl Stubby Adapter (UDI) Module

Color of Cap Tube	Component	Volume 1003371 (24 rxn)	Volume 1003381 (96 rxn)	Volume 1003382 (96 rxn)
	NadPrep Universal UDI-Index Primer Mix #	12×15 µL	24×25 µL	24×25 µL
	NadPrep Methyl Stubby Adapter	60 µL	230 µL	230 µL
	NadPrep Amplification Primer Mix II	20 µL	75 µL	75 µL

Blocker

Color of Cap Tube

Component

Volume

1106102 (16 rxn)

Volume

1106101 (96 rxn)

μCalerNanoBlockers (for Illumina^®)

40 μL

210 μL

Hybrid Reagents

Package	Color of Cap Tube	Component	Volume 1105202 (16 rxn)	Volume 1105201 (96 rxn)
Package	Color of Cap Tube	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

Type	Product	Detail	Catalog#
Methyl Library Prep Module	NadPrep Methyl Library Preparation Module, 24 rxn	24 rxn	1002501
Methyl Library Prep Module	NadPrep Methyl Library Preparation Module, 96 rxn	96 rxn	1002502
BS Conversion Module	NadPrep DNA Methyl Bisulfite Conversion Module, 24 rxn	24 rxn	1002701
BS Conversion Module	NadPrep DNA Methyl Bisulfite Conversion Module, 96 rxn	96 rxn	1002702
Methyl Stubby Adapter (UDI) Module	NadPrep Methyl Stubby Adpater (UDI) Module Set A1 (with 10 nt Index), 24 rxn	UDI 1-12	1003371
	NadPrep Methyl Stubby Adpater (UDI) Module Set B1 (with 10 nt Index), 96 rxn	UDI 1-24	1003381
	NadPrep Methyl Stubby Adpater (UDI) Module Set B2 (with 10 nt Index), 96 rxn	UDI 25-48	1003382
Blocker	μCaler NanoBlockers (for Illumina®), 16 rxn	16 rxn	1106102
Blocker	μCaler NanoBlockers (for Illumina®), 96 rxn	96 rxn	1106101
Hybrid Reagent	μCaler Hybrid Capture Reagents v2, 16 rxn	16 rxn	1105202
Hybrid Reagent	μCaler Hybrid Capture Reagents v2, 96 rxn	96 rxn	1105201