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NadPrep cfDNA Library Preparation Kit (for MGI)

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

workflow

Performance

High Efficiency
DNBSEQ-BMI-Fig1

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

consensus sequencing-fig2

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


cfDNALibrary Pic3

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

cfDNALibrary Pic4
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  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

/

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.


图片1


* ~ 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

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

Product Sheet

Application Note

Demo Data

NadPrep for MGI Hema Research gDNA Promega female G1521
DNA library was created from human gDNA (Promega, G1521) using Nanodigmbio NadPrep library preparation kit (for MGI). The library was enriched in a capture using the Hema Research Panel v1.0, then sequenced.
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NadPrep DNA Universal Library Preparation Kit (for MGI)

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