NxSeq® UltraLow DNA Library Kit

Lowest input, highest efficiency, Illumina-compatible DNA fragment library prep kit

• High Quality Data: High efficiency adaptor ligation produces complex libraries that yield improved sequencing depth uniformity and better coverage with fewer zero coverage regions
• Sensitive: Construct DNA fragment libraries from as little as 50 pg to as much 75 ng of sheared/fragmented DNA
• Minimal Bias: Robust, uniform PCR amplification improves coverage uniformity when working with low input amounts of genomic DNA
• Flexible:Extensively tested in de novo whole genome sequencing or resequencing, but compatible with other applications such as exome-seq, ChIP-seq and FFPE DNA samples.
• Fast: 3 hour protocol gets your samples on the sequencer quicker
• High Value: Cost-effective library and indexing kits which produce excellent sequencing results

Many DNA next generation sequencing applications or sample types require the construction of PCR-amplified DNA fragment libraries. (e.g. whole genome sequencing or resequencing from limiting genomic DNA amounts or FFPE and cell-free DNA samples, exome sequencing, ChIP-seq, etc.) To get the highest quality data for these applications and sample types, you need a DNA library preparation kit with the highest efficiency adaptor ligation followed by unbiased PCR amplification to produce the most complex libraries possible. With these complex libraries, you will produce sequencing data with uniform coverage depth and minimal zero coverage regions.   

The NxSeq® UltraLow DNA Library Kit and NxSeq® Single Indexing Kits allow you to build high quality DNA fragment libraries from extremely low DNA input amounts – as low as 50 pg depending on sample type and Illumina sequencer used. If you have more DNA, no problem; you can use as much as 75 ng of input DNA with this system.

To generate these high quality libraries, we optimized each step of the protocol to ensure peak performance on Illumina sequencers. To help with the initial steps of DNA fragmentation, we’ve included guidance for mechanical shearing on an instrument like a Covaris LE220 and an optimized protocol for enzymatic fragmentation using dsDNA Shearase Plus from Zymo Research. Not only will you produce high quality libraries, but once you have your fragmented DNA, library prep is quick and easy - about 3 hours.

This library and single indexing kits have been optimized for whole genome sequencing (WGS) and resequencing applications for de novo sequencing or SNV/mutation or copy number variation (CNV) identification. The system can, however, be used in other NGS applications such as ChIP-seq, exome-seq, and with other sample types such as metagenomic and FFPE DNA samples which require PCR-amplified DNA fragment libraries.

 

Single Index Library Construction

Figure 1. Mechanics of NxSeq® UltraLow Library Construction Using Single Indexing Primers. This workflow figure illustrates how DNA fragment libraries are constructed using the NxSeq® UltraLow Library Prep Kit and NxSeq Single Indexing Kits. Note that some PCR fragments and products are not shown to simplify the illustration.

Highest Efficiency Adaptor Ligation

Figure 2: Percentages of DNA fragments with correctly ligated adaptors measured by qPCR. Two independent sets of libraries were prepped per kit/organism (Staphylococcus aureus, Rhodobacter sphaeroides, and E. coli) according to the manufacturer’s recommended protocols using 1 ng of sheared genomic DNA. Prior to PCR amplification, adaptor ligation efficiency for each library was measured by triplicate qPCR assays using the KAPA Library Quantification Kit (Complete ROx Low, cat #KK4873) with a Lucigen-designed PCR primer set that binds to and amplifies all adaptor-ligated DNA fragments independent of the kit used. Standard P5 and P7 primers cannot be used because the Lucigen Universal Adaptor and the NEB adaptor do not contain P5 and P7 sequences prior to amplification. Efficiency data was normalized to the NxSeq UltraLow Library Kit data set (1.0) for each set of libraries and then the data libraries was averaged and plotted.

High Quality Whole Genome Sequencing Data from ≥50 pg of gDNA

Sheared gDNA Input	50 pg	250 pg	500 pg	75 ng
PCR Cycles Used	16	15	13	4
Reads Sampled per Library	312,500	312,500	312,500	312,500
No. Mapped Reads	303,442	304,135	304,894	304,935
Percent Mapped Reads	97.10%	97.32%	97.57%	97.58%
CLC Mapped Duplicates	0.84%	0.15%	0.08%	0.02%
Ave. Coverage Depth	9.62	9.72	9.74	9.84
Coverage Depth Std. Dev.	3.75	3.71	3.71	3.6
No. of Zero Locations	109	91	91	56
Ave. Zero Region Length (bp)	24	24	25	27
Total Zero Length (bp)	2651	2214	2175	1491

Table 1. Generation and Sequencing of DNA Fragment Libraries from 50 pg to 75 ng of E. coli gDNA. Mechanically sheared (Covaris LE220, peak size 300 bp) E. coli K12 genomic DNA was serially diluted and used to make duplicate libraries starting with the indicated gDNA input amounts and number of PCR amplification cycles using the NxSeq® UltraLow DNA Library Kit and a NxSeq® Single Indexing Kit. The final libraries were quantitated and diluted to 2 nM based on Bioanalyzer (size) and Qubit fluorometer (DNA quantity) analyses. The diluted libraries were then pooled and sequenced on a MiSeq using 2 × 150 bp chemistry, and the average data from the duplicate libraries are presented.

Minimal GC- or AT-bias with the NxSeq® UltraLow DNA Library Kit

Figure 3. Sequencing bias analysis for three different organisms with varying GC content.Tripilcate genomic DNA fragment libraries were generated from gDNA of three organisms with varying GC content (A - Staphylococcus aureus, 24% GC; B - E. coli K12, 50% GC; and C - Rhodobacter sphaeroides, 68% GC) using the Lucigen NxSeq® UltraLow DNA Library Kit, Kapa Hyper Prep Kit or the NEB NEBNext Ultra II Kit according to the manufacturer’s recommended protocols using 1 ng aliquots of the same mechanically sheared genomic DNA samples. The final libraries were quantitated and diluted to 2 nM based on Bioanalyzer (size) and Qubit fluorometer (DNA quantity) analyses. The diluted libraries were then pooled and sequenced on a MiSeq using 2 × 150 bp chemistry. Normalized coverage was calculated as the (average coverage of all windows with x% GC content) divided by the (overall average coverage) and the data from each set of replicates was averaged and presented. The blue area represents the percent of each genome with the indicated GC content.

 

Better Performance by All Metrics with the NxSeq® UltraLow DNA Library Kit on the HiSeq 2500 Instrument

	NxSeq®  UltraLow Kit	Kapa Hyper Prep Kit	Benefits of the NxSeq UltraLow Kit
Total PF Reads	160,630,020	150,642,156
R1 Mapped Reads R2 Mapped Reads	94.46% 94.32%	93.17% 93.13%	1.24% more mapped reads
Fragment Length (bp)	278 +/- 102	190 +/- 57	Sequenced fragments only 22 bp smaller than 300 bp input
Duplicates (Proper Read Pairs)	2.13% +/- 0.07%	2.37% +/- 0.06%	Significantly lower duplication rate
Coverage Depth	4.79X	4.07X	18% deeper coverage
Autosomal Coverage	97.25%	96.38%	0.87% better coverage of the autosomal genome or  ~24 million more basepairs covered

Table 2.  HiSeq 2500 Sequencing Results with Fragment Libraries Made with 10 ng of Human Genomic DNA. Triplicate DNA fragment libraries were constructed using either the Lucigen NxSeq® UltraLow DNA Library Kit or the Kapa Hyper Prep Kit according to each manufacturer’s recommended protocol. Briefly, human genomic DNA (NA15510, Coriell) was fragmented to a median size of 300 bp, and 10 ng aliquots of the same sheared DNA sample were used to make triplicate libraries with each kit. Eight cycles of PCR were used to amplify each library, and the final libraries were cleaned/size selected as recommended. The final libraries were sent to Hudson Alpha for sequencing where they were sized, quantitated by qPCR and Qubit, and equimolar amounts of each library were pooled and sequenced on a HiSeq 2500 using 2 × 100 bp chemistry. The average data from the triplicate libraries are shown.

 

Improved Performance with the NxSeq® UltraLow DNA Library Kit on the HiSeq X Ten Instrument

	NxSeq UltraLow Kit	Kapa Hyper Prep Kit	NEB NEBNext Ultra II Kit
Total PF Sampled Reads	200,000,000	200,000,000	200,000,000
R1 Mapped Reads	96.52%	96.02%	96.10%
R2 Mapped Reads	95.51%	95.36%	94.97%
Fragment Length (bp)	289 +/- 110	191 +/- 61	225 +/- 109
Duplicates (Proper Read Pairs)	2.15%	3.73%	3.54%
Coverage Depth	8.32	6.15	6.96
Autosomal Coverage	98.64%	98.40%	98.73%

Table 3. HiSeq X Ten Sequencing Results with Fragment Libraries Made with 10 ng of Human Genomic DNA. DNA fragment libraries were constructed using either the Lucigen NxSeq® UltraLow DNA Library Kit, Kapa Hyper Prep Kit or NEB NEBNext Ultra II Kit according to each manufacturer’s recommended protocol. Briefly, human genomic DNA (NA15510, Coriell) was fragmented to a median size of 300 bp, and 10 ng aliquots of the sheared DNA were used to make duplicate libraries with each kit. The libraries were PCR amplified for the following number of cycles: Lucigen, 8 cycles; Kapa, 8 cycles; and NEB, 10 cycles. The amplified libraries were cleaned/size selected as recommended. The final libraries were sent to Hudson Alpha for sequencing where they were sized, quantitated by qPCR and Qubit, and equimolar amounts of each library within a test set were pooled. Each set of pooled duplicate libraries was sequenced on its own lane of a HiSeq X Ten using 2 × 150 bp chemistry. During analysis, the optical duplicates generated by the HiSeq X Ten were removed using the Clumpify program, and then the indicated number of reads were analyzed. The averaged data from the replicate libraries are shown.

Fast, Streamlined Library Prep

Figure 4. Comparison of multiple library prep workflows and timing. The workflows illustrated correspond to the following kits: Lucigen, NxSeq® UltraLow DNA Library Kit; Illumina, TruSeq Nano DNA LT Library Preparation Kit; Kapa, Hyper Prep Kit with KAPA Library Amplification Primer Mix; NEB, NEBNext Ultra II DNA Library Prep Kit for Illumina.

Save on Every Library with the NxSeq® UltraLow and Single Indexing Kits

DNA Library Prep  and Single Indexing Kits	Cat. No.	Size (rxn)	2017 U.S. List Price	Cost per Library	Total Cost per Library
Lucigen NxSeq® UltraLow DNA Library Kit	15012-1	12	--	--	--
Lucigen NxSeq® Single Indexing Kit, Set A	15100-1	48	--	--
Kapa Hyper Prep Kit with KAPA Library Amplification Primer Mix (10X)	KK8500	8	--	--	--
Kapa Single-Indexed Adapter kit, Set A	KK8701	192	--	--
NEB NEBNext Ultra II DNA Libary Prep Kit for Illumina	E7645S	24	--	--	--
NEB NEBNexNEBNext Multiplex Oligos for Illumina (Index Primer Set 1)	E7335S	24	--	--
Swift Biosciences Accel-NGS 2S Plus DNA Library Kit	21024	24	--	--	--
Swift Biosciences 2S Set A Indexing Kit	26148	48	--	--

Table 4. Cost Comparison of Fragment DNA Library Prep and Single Indexing Kits. All prices were obtained from each company’s website in the U.S., 2017.