NadPrep cfDNA Library Preparation Kit (for MGI)

Catalog Number:

#1002211 #1002212
#1003811 #1003821 #1003822 #1003820
#1003911 #1003921 #1003922 #1003925 #1003926

NadPrep cfDNA Library Preparation Kit (for MGI) is designed for preparation of high-quality libraries from cell-free DNA in plasma. This A-T ligation-based kit offers a stable and efficient library preparation solution for applications including whole genome sequencing and hybridization capture based targeted sequencing on MGI platforms.

This kit includes Bi-Molecular Identifier (BMI) adapter for duplex sequencing of cfDNA, which enhances the limit of detection of low-frequency mutations from cfDNA.

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Features

Applicable to cfDNA ranging from 1 ng to 500 ng
Generate high quality libraries from low input
Bi-molecular identifiers (BMI) adapter module enables accurate calling of low-frequency variants
Provided with DNA purification magnetic beads and optimized size-selection solution;
Optional with Single Index (SI) Adapters and MGI Unique Dual Index (MDI) Adapters.

Applications

Application	Sample type	Recommended input
Whole Genome Sequencing	cfDNA (no large fragment contamination)	≥1 ng (adjust according to test requirement)
Targeted Capture Sequencing	cfDNA (no large fragment contamination)	≥1 ng (adjust according to test requirement)

Workflow

Performance

High Efficiency

Fig 1. Efficiency of NadPrep cfDNA Library Preparation Kit (for MGI). A. Library yields prepared with NadPrep BMI Adapter (MDI) Module. B. Size distribution of one cfDNA library prepared with MDI Adapters (Agilent 2100 Bioanalyzer).

BMI for Low-Frequency Variants Analysis

Figure 2. Schematic of BMI adapters analysis

1% and 0.5% variants were mimicked by spiking plasma cfDNA from one healthy donor into that of the unrelated one, at 1% proportion. 10 ng of cfDNA mixture were used for BMI library preparation. Analysis result of low frequency variants under different analysis and filtering methods are shown follows:

Table 1. Different analysis and filtering methods.

Methods	Description
No BMI	Mark reads with the same start-stop positions as duplicates.
SSCS	makes use of start-stop position coupled with single strand molecule identifiers (Call Molecular Consensus Reads) to generate Single Strand Consensus Sequence (SSCS)
DCS211/ DCS633	makes use of start-stop position coupled with double strand (top and bottom strands) molecule identifiers to generate Duplex Consensus Sequence (DCS). the top and bottom reads≥ 1(DCS211) / 3(DCS633)
DCS211(≥2)/DCS633(≥2)	Based on the data of DCS211/ DCS633, the DCS of the same Variants should be ≥ 2

Figure 3. A. Sensitivity and B. positive predictive value (PPV) for 1% and 0.5% variants.

Fig 4. Denoising effect achieved by different stringency of consensus filtering.

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

NadPrep DNA Library Preparation Module

Package	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	60 µL	230 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

Bi-molecular identifiers (BMI) with single index(SI) adapter module

Color Of Tube Cap	Component	Volume 1003811 (24 rxn)	Volume 1003821 (96 rxn)	Volume 1003822(96 rxn)
	NadPrep M-Index (SI) Primer Mix #	12×15 µL	24×25 µL	24×25 µL
	NadPrep BMI-Adapter	60 µL	230 µL	230 µL

Package	Color Of Tube Cap	Component	Volume 1003820 (480 rxn)
Box1		NadPrep M-Index (SI) Primer Mix #	96×30 µL
Box2		NadPrep BMI-Adapter	1100 µL

Bi-molecular identifiers (BMI) with Unique Dual Index (MDI) adapter module

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

Which DNA extraction kit is recommended?

Based on our experience, the kits for DNA extraction are recommended as follows.

Item	Recommendations
Extraction kit	gDNA	DNeasy Blood & Tissue Kit (Qiagen, Cat # 69504 & 69506) TIANamp Genomic DNA Kit (TIANGEN, Cat # DP304)
	cfDNA	QIAamp Circulating Nucleic Acid Kit (Qiagen, Cat # 55114)
	FFPE gDNA	QIAamp DNA FFPE Tissue Kit (Qiagen, Cat # 56404)
Dissolvent	Nuclease-free water, 0.1 x TE (low TE), 10 mM Tris-HCl (pH 8.0-8.5) or Buffer provided in extraction kit should be used to dissolve DNA.

What are the DNA input requirements for NadPrep DNA Library Preparation Module (for MGI)?

This kit is compatible with types of dsDNA inputs, including DNA extracted from blood, tissues, FFPE and body fluid. This kit can be used to construct libraries for dsDNA input ranging from 1 to 500 ng.

For samples with suboptimal quality such as FFPE tissue samples, careful quality control (QC) and stratification on DNA integrity is highly recommended.

For common usage, the QC metrics are recommended as follows.

Item	Recommendations
Quantification	NanoDrop (based on the spectrophotometer principle), Qubit® or PicoGreen® (based on double-stranded DNA fluorescent dye).
Quality	OD₂₆₀/OD₂₈₀= 1.8 - 2.0; OD₂₆₀/OD₂₃₀= 2.0 - 2.5. OD₂₆₀/OD₂₈₀> 1.9 indicates RNA contamination while < 1.6 indicates protein, phenol or other contamination; OD₂₆₀/OD₂₃₀< 2.0 indicates carbohydrates, salts or organic solvent contamination.
Fragment distribution	gDNA	A clear band > 15 kb when running on a 1% agarose gel.
	cfDNA	A peak at ~ 160 bp followed by a peak at ~ 320 bp on Bioanalyzer or Bioptic (Qsep).
	FFPE gDNA	DNA integrity assessment by running on a 1% agarose gel or Bioanalyzer.

Does NadPrep DNA Library Preparation Module (for MGI) support PCR-free library construction?

No. PCR with indexing primers is necessary to generate full-length library.

What is the recommended sample fragmentation method?

The kit is compatible with ultrasonic and enzymatic fragmentation methods. For enzymatic fragmentation, NEBNext® dsDNA Fragmentase® (NEB; Cat M0348) or Custom 5X DNA Shearing Mix (Qiagen; Cat CM0162) is recommended; For ultrasonic fragmentation, it is recommended to use Covaris (M220 Focused-ultrasonicator, 50 μL interruption tube) or similar.

How to choose adapter module?

For different applications, adapter module is recommended as follows.

Application	Recommended adapter module	Optional
Whole-genome, metagenomic and targeted capture sequencing	Universal Adapter (SI) Module	BMI Adapter (SI) Module
RNA sequencing	BMI Adapter (SI) Module	Universal Adapter (SI) Module
Low frequency mutation detection (e.g. ≤ 1% variant frequency)	BMI Adapter (SI) Module	-

How to improve cleanup efficiency?

The following tips help to improve the cleanup efficiency.

1) never overdry the beads;

2) increase incubation time;

3) increase the volume of magnetic beads (NOT suitable for size selection step);

4) use freshly prepared 80% ethanol.

When using Adapter and Primer Module, how to cope with the adapters?

Adapter: insert molar ratio is essential. Excessive adapter will result in residues of adapter or adapter dimers. Insufficient adapter will affect ligation efficiency and therefore reduce library yield. For universal adapters, please refer to the listed table to calculate the dilution fold of adapters so as to achieve the best yield.

* ~ 250 bp is commonly used for gDNA fragmentation, and ~ 160 bp is peak size observed for cfDNA in plasma. Other recommendations for handling adapters are as follows.

• Diluent: Use TE Solution in the kit to dilute adapters. Diluted adapters are not recommended for long-term storage;

• Storage: Aliquot to avoid repeated freezing and thawing.

While pipetting, there is solution remaining on the pipette tips. How to solve it?

Ligation Buffer, DNA Ligase and End Repair & A-Tailing Enzyme are relatively viscous. It is recommended to use low-absorption tips and pipetting slowly. If residue remains in tip, repeatedly pipette or use the reverse pipetting.

Is the library compatible with Agilent, Roche and other targeted capture systems?

We provided a total solution for targeted capture sequencing. The library kit is compatible with a variety of targeted capture systems, such as SureSelect (Agilent) and NimbleGen™ SeqCap™ EZ (Roche).

Product	Scale	Catalog#
NadPrep DNA Library Preparation Module (for MGI), 24 rxn	24 rxn	1002211
NadPrep DNA Library Preparation Module (for MGI), 96 rxn	96 rxn	1002212
NadPrep BMI Adapter (SI) Module Set A1 (for MGI), 24 rxn	SI 1-12	1003811
NadPrep BMI Adapter (SI) Module Set B1 (for MGI), 96 rxn	SI 1-24	1003821
NadPrep BMI Adapter (SI) Module Set B2 (for MGI), 96 rxn	SI 25-48	1003822
NadPrep BMI Adapter (SI) Module Set D1 (for MGI), 480 rxn	SI 1-96	1003820
NadPrep BMI Adapter (MDI) Module Set A1 (for MGI), 24 rxn	MDI 1-12	1003911
NadPrep BMI Adapter (MDI) Module Set B1 for (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