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μCaler Total Solution for AML

μCaler AML MRD comprehensive solution is based on liquid-phase hybridization target enrichment technology and specifically designed for adult Acute Myeloid Leukemia (AML) MRD research. This comprehensive solution selects MRD targets that cover approximately 90% of AML cases, allowing for the analysis of various mutations, including base substitutions, insertions/deletions, and gene fusions. By combining UMI and the μCaler hybrid capture system, it achieves ultra-high detection sensitivity and can complete the entire experimental process within same day. μCaler AML Panel v1.0 is designed for detecting common mutations in adult acute myeloid leukemia. The panel covers approximately 42.5 Kb region of the genome, targeting 32 genes. It enables enrichment of various types of mutation information, including base substitutions, insertions/deletions, and gene fusions, making it suitable for MRD monitoring.

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Performance

Precision coverage

● Reference was made to multiple databases (COSMIC & TCGA) and guidelines to select regions more likely to contain mutations.

●~90% of cases have at least one mutation, and ~60% of cases have three or more mutations (AML_OHSU_2022 cohort).

● Covers approximately 42.5 Kb of the genome, allowing detection of base substitutions, insertions/deletions, and gene fusions in 32 genes associated with AML.

Gene list

ASXL1 Exon 12,13	BRINP3 Exon 3,8	CBL Exon 8,9	CEBPA* Full CDS	DNMT3A Exon 8-23	EZH2 Exon 4-6,8,13-20	FLT3 Exon 14,15,20	GATA2 Exon 3-6	HNRNPK Exon 4-6,10,12,15,16	IDH1 Exon 4	IDH2 Exon 4
JAK2 Exon 14	KIT Exon 8,17	KMT2A† Intron 8-10	KRAS Exon 2,3	MYH11† Intron 32	NPM1 Exon 10,11	NRAS Exon 2,3	PHF6* Full CDS	PTEN Exon 5,7	PTPN11 Exon 3,13	RAD21* Full CDS
RUNX1* Full CDS	SF3B1 Exon 14,15	SMC1A EXON 2,9,11,13,15-17,22		SMC3 Exon 9,10,13,19,24,25,27		SRSF2 Exon 1	STAG2 Exon 4,5,7-9,14,16,18,19,24,26-30		TET2* Full CDS	TP53* Full CDS
U2AF1 Exon 2	WT1 Exon 6-9

Note：* Indicates that the gene is covered across the entire coding sequence (CDS) region; † Indicates that the gene is covered in fusion-related intronic regions.

Low background noise

Figure 1. Analysis of background noise after DCS211 filtering using different fragmentation methods. The different methods are A. Conventional enzymatic fragmentation; B. Sonication and C. NadPrep NEM fragmentation, respectively. NadPrep NEM fragmentation significantly reduces background noise compared to the other two methods from the source.

Higher sensitivity

Figure 2. Duplex depth (DCS211) detected by μCaler AML MRD comprehensive solution and sonication-based library preparation under different input conditions. The μCaler AML MRD comprehensive solution user manual was used as a reference, and the filtering was performed based on Duplex Consensus Sequences (DCS211). The sequencing mode is Illumina Novaseq 6000, PE150.

Lower sequencing cost

Figure 3. Minimum amount of raw data required for μCaler AML MRD comprehensive solution and traditional hybridization capture method to achieve a specific depth (satisfying Duplex Consensus Sequences analysis).

Note：Traditional indicates the conventional hybridization capture method, calculated based on the on-target rate of 15%.

Stable and efficient

Figure 4. Stability performance of μCaler AML MRD comprehensive solution in different experimental batches. A.On-target rate; B. Uniformity of data yield. The captured library were prepared from gDNA samples according to the user manual of μCaler AML MRD comprehensive solution. Using BWA to alignment to the reference genome hg19 and on-target rate was calculated by the number of reads.

High-speed and convenient

Capture performance

Figure 5. Capture performance of μCaler AML MRD comprehensive solution. 300 ng of fragmented DNA were used to prepare the pre-library, and the hybridizationcapture was completed with μCaler AML Panel v1.0. A. Mappability, Ontargetand target covered; B. Sequencing depth; C. Coverage uniformity and consistency.

Note：The samples were derived from the proportionate mixing of Pancancer Light 800 gDNA Reference Standard (Genewell, GW-OGTM800) and Human Male Genomic DNA Standard (Promega, G1471) to simulate samples with different allele frequency (0.025%~0.1%).

Analysis of mutation in standard

Figure 6. Mutation analysis of the μCaler AML MRD comprehensive solution. A. Average depth (DCS211 ≥ 1); B. Reads supporting mutations; C. Theoretical and observed mutation frequencies.

Note：The sequencing mode is Illumina Novaseq 6000, PE150.

NEM Fragment Module

Color of Tube Cap	Component	Volume 1002801（24 rxn）
	NEM Fragment Buffer	60 μL
	NEM Fragment Enzyme Mix	60 μL
	Stop Solution	150 μL

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

Blocker

Color of Tube Cap

Component

Volume

1106212 (16 rxn)

Volume

1106211 (96 rxn)

μCalerNanoBlockers (for MGI, DI)

40 μL

210 μL

Hybrid Capture

Packing No.	Color of Tube Cap	Component	Volume 1105102 (16 rxn)	Volume 1105101 (96 rxn)
Packing No.	Color of Tube Cap	Component	Volume 1105102 (16 rxn)	Volume 1105101 (96 rxn)
Box 1		μHyb #1	840 μL	3×1700 μL
		μHyb #2	130 μL	790 μL
		Human Cot DNA	45 μL	260 μL
Box 2		Streptavidin Beads	480 μL	2.9 mL
	/	Wash Buffer A	2×5.3 mL	3×21 mL
	/	Wash Buffer B	2.9 mL	20 mL

Panel

Catalog	Color of Tube Cap	Component	Volume
1101412		μCaler AML Panel v1.0, 16 rxn	40 μL
1101411		μCaler AML Panel v1.0, 96 rxn	210 μL

Type	Product	Detail	Catalog#
NEM Fragment Module	NadPrep NEM Fragment Module, 24 rxn	24 rxn	1002801
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 AML Panel v1.0, 16 rxn	16 rxn	1101412
Panel	μCaler AML Panel v1.0, 96 rxn	96 rxn	1101411