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 optimized 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.
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, cat #KK4873) and matching amplified library as an internal standard.Back to top
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 |
|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.
Genome size, GC percentage
~3 Gbp 45% GC
2,821,361 33% GC
4,602,977 69% GC
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 analyzed.
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 analyzed. Normalized coverage was calculated as the (average coverage of all windows with X% GC content) divided by the (overall average coverage).
Save Big with the NxSeq® AmpFREE Low DNA Library Kit
|PCR-Free Fragment Library Kit||Cat. No.||Size |
|2016 US |
|Cost/Reaction||Per Rxn Cost Increase |
vs. NxSeq® Kit
|Lucigen NxSeq® |
DNA Library Kit
|14000-2||48||$921||$19||12 rxn||48 rxn|
|Kapa Hyper Prep Kit||KK8501||8||$264||$33||+65%||+74%|
Save Time and Get Your Samples on the Sequencer Sooner!Back to top