NadPrep DNA Universal Library Preparation Kit (for Illumina®)

Catalog Number:

#1002101 #1002103
#1003211 #1003212 #1003213 #1003221
#1003224 #1003225 #1003226 #1003227

NadPrep DNA Universal Library Preparation Kit (for Illumina®) is designed for the preparation of high-quality libraries from double-stranded DNA (dsDNA) on Illumina® platforms. This A-T ligation based kit offers a stable and efficient library preparation solution for a variety of applications.

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Features

Suitable for various sample types including FFPE DNA
High complexity libraries generated from a wide range of input amounts
Adapters with unique dual indexes (UDIs) to reduce impact of index hopping on Illumina®️ platforms
Compatible with unique molecular identifiers (UMI) adapters to enable confident calling of low-frequency variants
Purification beads and optimized size selection methods included

Applications

Application	Sample type	Input recommended
Whole genome sequencing	Plant or animal gDNA (without serious degradation)	50-500 ng
	Cell-free DNA	≥1 ng (analytical sensitivity dependent)
	Microbial DNA (without serious degradation)	1-500 ng
Targeted sequencing	gDNA (without serious degradation)	≥ 10 ng (quality dependent)^*
	FFPE DNA (with average size＞500 bp)	≥ 100 ng (quality dependent)^*
	Cell-free DNA	≥ 1 ng (analytical sensitivity dependent)

*For gDNA, input recommended here indicates the amount quantified after DNA fragmentation.

Workflow

workflow

Performance

High Efficiency

Fig 1. Library yield with different types of adapters. The yields of the libraries prepared with different gDNA initial amount by using NadPrep DNA Library Preparation Kit (for Illumina®) coupled with NadPrep Universal Stubby Adapter (UDI) Module or NadPrep UDI Adapter Module (for Illumina®) Note: The sample is human female genomic DNA (Promega-Female, G1521).

Unbiased Coverage

Fig 2. Uniform coverage of regions with various GC content. Coverage distribution for libraries generated from 10 ng DNA of A. Staphylococcus aureus (GC%=33), B. Salmonella enterica LT2 (GC%=52) and C. Pseudomonas aeruginosa (GC%=67). GC% distribution per 100 bp bin is indicated by histograms and normalized coverage is represented by lines.

Accurate Variant Analysis

Fig 3. Variant calling in reference standard DNA. The DNA libraries were prepared from Quantitative Multiplex Formalin Compromised (Mild) DNA (Horizon Discovery, HD798). The enriched libraries were sequenced on HiSeq X Ten (PE150) and subsampled to 5 Gb for analysis. Vardict was used to call variants.

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

NadPrep DNA Library Preparation Module (for Illumina)

Package	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	60 µL	230 µL
		2X HiFi PCR Master Mix	720 µL	2×1600 µL
		NadPrep Amplification Primer Mix	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

NadPrep UDI Adapter Module

Color Of Tube Cap	Component	Volume 1003211 (24 rxn)	Volume 1003222 (24 rxn)	Volume 1003211(96 rxn)	Volume 1003212(96 rxn)	Volume 1003213(192 rxn)	Volume 1003224(480 rxn)
	NadPrep UDI Adapter #	12×10 µL	12×10 µL	12×22 µL	12×22 µL	24×22 µL	96×3.5 µL (Plate)×5

Universal Stubby Adapter (UDI) Module

Color Of Tube Cap	Component	Volume 1003240 (24 rxn)	Volume 1003241 (96 rxn)	Volume 1003242 (96 rxn)
	NadPrep Universal UDI-index Primer Mix #	12×15 µL	24×25 µL	24×25 µL
	NadPrep Universal Stubby Adapter	60 µL	230 µL	230 µL
	NadPrep Amplification Primer Mix II	20 µL	75 µL	75 µL

Color of Tube Cap	Component	Volume	Volume	Volume	Volume
Color of Tube Cap	Component	1003251 (96 rxn)	1003252 (96 rxn)	1003253 (96 rxn)	1003254 (96 rxn)
	NadPrep Universal UDI-index Primer Mix #	96×8 µL (Plate)	96×8 µL (Plate)	96×8 µL (Plate)	96×8 µL (Plate)
	NadPrep Universal Stubby Adapter	260 µL (Tube)	260 µL (Tube)	260 µL (Tube)	260 µL (Tube)
	NadPrep Amplification Primer Mix II	75 µL (Tube)	75 µL (Tube)	75 µL (Tube)	75 µL (Tube)

What is the possible reason for the less library yield or longer size distribution of library than expected after digestion？How to solve it ?

Possible Reason	Solution
The DNA sample contains more than 1 mM of EDTA	Purify the DNA by using magnetic beads or spin columns,and dissolve the purified DNA with TE Solution.
The DNA sample contains impurities	High quality DNA input, with OD₂₆₀ /OD₂₈₀ =1.8-2.0 and OD₂₆₀ /OD₂₃₀ =2.0-2.5. The residual guanidine salt and proteins may suppress the fragmentase activity. Purify the DNA by using magnetic beads or spin columns,and dissolve the purified DNA with TE Solution.
Fail to thoroughly mix the reaction mix	Mix thoroughly and centrifuge to collect the contents to the bottom of the tube before initiate the reaction.
Fragments self-ligation	Thoroughly read the manual before operation.

What is the possible reason for the shorter size distribution of library than expected after digestion? How to solve it ?

Possible Reason	Solution
The DNA is seriously degraded	Degraded DNA, such as FFPE C DNA, may result in short fragments (150-200 bp) without change of the same fragmentation condition.
The fragmentation process lasts too long	Avoid operating multiple samples at one time.
The operation under room temperature lasts too long	Ensure all the processes operated on ice beforeloading into the thermo cycler.

How to explain the appearance of the adapter peak in size distribution?

Possible Reason：Inappropriate dilution of adapters

Solution：For low DNA input, adapters should be diluted as recommended prior to use.

Type	Product	Scale	Catalog
Library Preparation Module	NadPrep DNA Library Preparation Kit (for Illumina^®️) G24	24 rxn	1002101
Library Preparation Module	NadPrep DNA Library Preparation Kit (for Illumina^®️) E96	96 rxn	1002103
Universal Stubby Adapter (UDI) Module	NadPrep Universal Stubby Adapter (UDI) Module Set A1, 24 rxn	Index 1-12, 24 rxn	1003240
	NadPrep Universal Stubby Adapter (UDI) Module Set B1, 96 rxn	Index 1-24, 96 rxn	1003241
	NadPrep Universal Stubby Adapter (UDI) Module Set B2, 96 rxn	Index 25-48, 96 rxn	1003242
	NadPrep Universal Stubby Adapter (UDI) Module Set E1, 96 rxn	Index 1-96, 96 rxn	1003251
	NadPrep Universal Stubby Adapter (UDI) Module Set E2, 96 rxn	Index 97-192, 96 rxn	1003252
	NadPrep Universal Stubby Adapter (UDI) Module Set E3, 96 rxn	Index 193-288, 96 rxn	1003253
	NadPrep Universal Stubby Adapter (UDI) Module Set E4, 96 rxn	Index 289-384, 96 rxn	1003254
UDI Adapter Module	NadPrep UDI Adapter Kit, Set A (for Illumina^®️)	Index 1-12， 300 pmol / Adapter	1003211
	NadPrepUDI Adapter Kit, Set B (for Illumina^®️)	Index 13-24， 300 pmol / Adapter	1003212
	NadPrep UDI Adapter Kit, Set A&B (for Illumina^®️)	Index 1-24， 300 pmol / Adapter	1003213
	NadPrep UDI Adapter Kit Set C1 (for Illumina^®️)	24 rxn	1003221
	NadPrep UDI Adapter Kit Set D1 (for Illumina^®️)	480 rxn	1003224