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μCaler Total Solution for Methylation

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

图1

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

图2 A
图2 B
图2 C
图2 D
图2 E
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

流程图

Highly efficient and stable

图3

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

图4

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

图5-A
图5-B
图5-C
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)

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 24×25 µL 24×25 µL
 24×25 µL

NadPrep Methyl M-Adapter 60 µL 230 µL 230 µL 230 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)

μCaler NanoBlockers (for Illumina®) 40 μL 210 μL

Hybrid Reagents

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
NadPrep Methyl Library Preparation Module, 96 rxn 96 rxn 1002502
BS Conversion Module NadPrep DNA Methyl Bisulfite Conversion Module, 24 rxn 24 rxn 1002701
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
μCaler NanoBlockers (for Illumina®), 96 rxn 96 rxn 1106101
Hybrid Reagent μCaler Hybrid Capture Reagents v2, 16 rxn 16 rxn 1105202
μCaler Hybrid Capture Reagents v2, 96 rxn 96 rxn 1105201

Product Sheet

Application Note

Demo Data

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