NxSeq AmpFREE Low DNA Library Kit

You’re doing next gen sequencing for a reason; you want big data! To get there, you know you need the best fragment libraries possible to get the most information out of each sample. So why spend your time, resources and expensive sequencing reagents on anything but the best fragment DNA libraries?

The NxSeq™ AmpFREE Low DNA Library Kit allows you to build the best fragment DNA libraries possible. We optimised each step of the protocol to ensure peak performance on Illumina sequencers. In addition, these kits require only 75 ng of sheared DNA input, and produce libraries in about 2 hours using a streamlined, easy to follow protocol.

Better Data
Higher Efficiency Libraries
More Reads from Challenging FFPE Samples
Excellent Sequencing Data Examples
Minimal GC Bias
Faster, Easier Protocol
Higher Efficiency Libraries

More Sequenceable DNA Fragments per Library = More Data

Figure 1. Percentage of library DNA with correctly ligated adaptors measured by qPCR. Duplicate libraries were prepped per kit/organism (Human, Staphylococcus aureus, Rhodobacter sphaeroides (1 library only), and E. coli) according to the manufacturer’s recommended input amounts and protocols. Adaptor ligation efficiency was measured by qPCR using the KAPA Library Quantification Kit (Complete ROX Low, Item No.KK4873) and matching amplified library as an internal standard.

Sequencing Impact of Higher Efficiency Library Construction

Better Libraries Increase the Number of Reads per Library

Library Kit DNA Input Total Number of Sequencing Reads Per Library
Staphylococcus aureus
E. coli K12
NxSeq AmpFREE Low DNA Library Kit 75 ng 5,649,946 4,305,882
Kapa Hyper Prep Kit 250 ng 4,838,726 (-15%) 1,647,452 (-62%)
Illumina TruSeq DNA PCR-Free Library Prep Kit 1 µg 38,768 (-99%) 1,543,558 (-64%)

Figure 2a. Number of sequencing reads generated per library after multiplexing and running on a MiSeq Instrument. DNA fragment libraries were prepped in parallel for each kit/organism according to the manufacturer’s recommended input amounts and protocols. Libraries were quantitated and normalized to 2 nM using the Bioanalyzer (size) and Qubit Fluorometer (amount). Equimolar amounts of each library were multiplexed and sequenced with a single MiSeq run using 2 ×150 bp chemistry. The number of sequencing reads obtained are shown as well as the percent reduction (%) in total reads compared to the appropriate NxSeq AmpFREE Kit results.

More Proof with Challenging FFPE Samples

Library Kit Sample Type Input Amount Total Reads Mapped Reads
(repeat masked)
NxSeq AmpFREE Low DNA Library Kit Normal gDNA 75 ng 2,163,636 900,338
FFPE DNA 75 ng 1,767,818 688,074
FFPE DNA 150 ng 1,706,714 656,658
Kapa Hyper Prep Kit Normal gDNA 250 ng 1,567,276 (-28%) 650,296 (-28%)
FFPE DNA 250 ng 1,270,870 (-28%) 487,872 (-29%)

Figure 2b. Number of sequencing reads generated from matching normal and FFPE gDNA sample libraries. DNA fragment libraries were prepped using the two indicated kits according to the manufacturer’s recommended input amounts and protocols. Libraries were constructed from normal gDNA (Biochain, Cat. No. D1234142-S02) and DNA extracted from a matching FFPE human kidney tissue (Biochain Cat. No. T2234142-S02) using the Qiagen AllPrep DNA/RNA FFPE Kit. The gDNA samples were sheared to ~250 bp before starting library construction. Final libraries were quantitated and normalized to 2 nM using the Bioanalyzer (size) and Qubit Fluorometer (amount). Equimolar amounts of each library were multiplexed and sequenced with a single MiSeq run using 2 × 150 bp chemistry. The number of sequencing reads obtained are shown as well as the percent reduction (%) in total and mapped reads compared to the corresponding NxSeq AmpFREE Kit results using 75 ng of input DNA.

Highly Mappable Reads (>92%) from Human, Staphylococcus and Rhodobacter gDNA Sequencing

Sequencing Stat

Human Staphylococcus

Rhodobacter

Genome size, GC percentage

~3 Gbp 45% GC

2,821,361 33% GC

4,602,977 69% GC

Raw reads

3,131,114

1,260,836

3,900,174

Mapped reads

2,979,237 (95.15%)

1,174,111 (93.12%)

3,613,165 (92.64%)

Read length

148.9 bp

148.8 bp

149.6 bp

Total bases

443,767,447

174,694,261

540,403,552

Genome fraction

0.11

0.97

1.00

Avg. coverage

0.15X

62X

117X

Figure 3. Representative gDNA sequencing stats from three different organisms. Genomic DNA fragment libraries were generated using the NxSeq AmpFREE Low DNA Library Kit using 75 ng of sheared gDNA input from three organisms (human, Staphylococcus aureus, and Rhodobacter sphaeroides). The final libraries were quantitated and normalized to 2 nM final concentrations using the Bioanalyzer and Qubit fluorometer, and 5 µL of each library was run on a MiSeq using 2 x 150 bp chemistry and analysed.

Minimal Bias Detected

Figure 4. Sequencing bias measured for three different organisms with varying GC content. DNA fragment libraries were generated from gDNA of three organisms with varying GC content ( Staphylococcus aureus, 24% GC; E. coli K12, 50% GC; and Rhodobacter sphaeroides, 68% GC) according to the manufacturer’s recommended input amounts and protocols. Samples were quantitated using the Bioanalyzer and Qubit fluorometer and normalized to 2 nM final concentrations. Five µL of each library sample was sequenced on a MiSeq using 2 x 150 bp v2 chemistry and analysed. Normalized coverage was calculated as the (average coverage of all windows with X% GC content) divided by the (overall average coverage).

Don’t spend 25-74% More per Library with Another Kit

Save Big with the NxSeq AmpFREE Low DNA Library Kit

PCR-Free Fragment Library Kit Cat. No. Size
(rxns) 2016 US
List Price Cost/Reaction Per Rxn Cost Increase
vs. NxSeq Kit
NxSeq
AmpFREE Low
DNA Library Kit 14000-1 12 $240 $20
14000-2 48 $921 $19 12 rxn 48 rxn
Kapa Hyper Prep Kit KK8501 8 $264 $33 +65% +74%
KK8503 24 $696 $29 +45% +53%
KK8505 96 $2400 $25 +25% +32%
Faster Protocol with Significantly Less Hands-on Time

Save Time and Get Your Samples on the Sequencer Sooner!

read more

Key features

• Low Input: Requires as little as 75 ng of sheared input DNA allowing use of limiting samples.
• High Efficiency: Optimised adaptor ligation produces more sequenceable fragments in each library, yielding better coverage & depth from single or multiplexed libraries.
• PCR-free: Prevents the introduction of PCR-bias, providing more uniform coverage.
• Fast: 2 hour, 10 minute protocol saves you time and gets your samples on the sequencer sooner.
• Affordable: Best priced and best performing kit available.

Product Details

The NxSeq AmpFREE Low DNA Library Kit is compatible with our NxSeq Adaptors or other dT-tailed, Illumina-compatible adaptors.

Each NxSeq AmpFREE Low DNA Library Kit contains Enzyme Mix (EM), 2X Buffer (2XB), Ligase (LIG) and Elution Buffer (EB). Adaptors must be purchased separately.

Resources

Brochures and guides

case study mcgill nxseq ampfree

Posters

NxSeq Fragment Library AmpFREE Low Input PAG 2016

FAQs

FAQs

Documents

• SDS 001 14000 14300 1 14400 1 NxSeq AmpFREE Low DNA Library Kit
• SDS 14000 NxSeq AmpFREE Low DNA Library Kit
• NxSeq AmpFREE Low DNA Library Kit
• NxSeq UltraLow DNA Library Kit Quick Protocol
• F103 US NxSeq AmpFREE Kit
• case study mcgill nxseq ampfree
• NxSeq Fragment Library AmpFREE Low Input PAG 2016