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xGen™ DNA Library Preparation Kits

Comprehensive coverage with fewer PCR duplicates

Streamline preparation of comprehensive NGS libraries from dsDNA. These kits utilize enzymatic fragmentation (EZ) or mechanical shearing (MC) methods to generate libraries suitable for PCR-free, PCR-amplified, and targeted sequencing research applications.

xGen NGS—made for DNA library preparation.

xGen DNA Library Prep Kit EZ and EZ UNI

  • Low input, high complexity—up to 3x fewer duplicates from 1 ng of DNA.
  • Improved yield for enrichment—specially formulated PCR master mix boosts yield from a minimal number of PCR cycles.
  • High multiplex capability—pre-plated UDI primers enable multiplexing of up to 1536 samples.
  • Compatible with xGen Normalase™ Module—streamlines normalization of sequencing libraries from multiplexing
  • Workflow design for easy automation

Ordering

Product details

Low input and PCR-free workflows

The xGen DNA Library EZ Kits are available in two configurations (Figure 1): xGen DNA Library EZ kit (Standard kit) and xGen DNA Library EZ UNI kit (Universal kit).

  • The xGen DNA Library EZ kit supports indexing by PCR workflow, it includes xGen Stubby Adapters. Indexing primers are supplied separately.
  • The xGen DNA Library EZ UNI kit supports an indexing by ligation workflow for optional PCR amplification or PCR-free workflow, amplification primers are included in the kit. Indexed adapters are supplied separately.

Both kits include an enzymatic fragmentation module, a high efficiency ligase, and a PCR Master Mix that can generate library yields sufficient for hybridization-based enrichment or direct sequencing. Both kits have workflows compatible with high throughput applications and have been automated on multiple liquid handlers.

Figure 1. Workflow for the xGen DNA Library Prep EZ Kits. xGen DNA Library Prep EZ Kits are compatible with either an indexing by PCR workflow using xGen Stubby Adapters included in the kit and indexing primers supplied separately (left) or an indexing by ligation workflow using full length, indexed Y adapters (right). Both kits include amplification reagents for Indexing PCR or optional PCR amplification.

Table 1. Supported applications

Research applicationsxGen DNA Library EZ Kit and EZ UNI Kit
Whole genome sequencing
Whole exome sequencing
Variant detection Germline + somatic
Genotyping
CNV detection

Table 2. Specifications

FeaturesxGen DNA Library EZ KitxGen DNA Library EZ UNI Kit
Sample typesFresh frozen tissue, genomic DNA, PCR amplicons, high quality FFPE*
Range of input concentrations 0.1–1000 ng0.1–1000 ng
Indexing compatibilityCDI primers up to 96-plex, compatible with xGen Normalase Module UDI primers up to 1536-plex, compatible with xGen Normalase ModulexGen indexing primers for custom, full-length and truncated adapters, compatible with xGen Normalase Module
System compatibility and multiplexing formatIllumina® sequencing instruments compatibilityManual and automated. Please inquire for a list of compatible liquid handling robots and scripts.

*Optimization of the enzymatic fragmentation step may be required.

xGen Deceleration Module enables additional control over fragment length with the xGen DNA Library EZ Kits.

The xGen Deceleration Module

  • generates an average library insert size of 550 bp
  • offers flexible fragmentation times on automation platforms

Comprehensive coverage

The xGen DNA Library EZ Kit was benchmarked against another supplier of DNA library prep kit that uses enzymatic fragmentation and a widely used HiFi DNA polymerase. Two different libraries for each concentration of input DNA (Coriell NA 12878; 100 ng, 10 ng, and 1 ng) with both kits were generated and evaluated for enrichment using the xGen Pan-Cancer Panel to assess library quality metrics (Table 3). For comparative analysis, identical SPRI®-based size selections (Beckman Coulter) were used for both library prep kits. Libraries were sequenced 2x101 bp on an Illumina® MiSeq® System using v2 chemistry, and reads were normalized to 460k per sample.

The xGen DNA Library EZ Kit required a lower number of PCR cycles to generate yields equivalent to the kit from the alternate supplier. At lower inputs, particularly at 1 ng, the xGen DNA Library EZ Kit provided higher target coverage, a significantly lower percentage of PCR duplicates, and better coverage uniformity compared to the other supplier’s kit. The xGen DNA Library EZ Kit also produced a more uniform insert size across the input quantities evaluated, when compared to the other kit.

Table 3. Targeted sequencing metrics.

Library prepInput (ng)Yield ng/µl (PCR cycles)Aligned insert (bp)Duplicates (%)Mean coverage (%)20X Coverage (%)30X Coverage (%)40X Coverage (%)50X Coverage (%)
xGen DNA Library EZ-sample 110097 (5)2350.7849.197.389.470.945.9
xGen DNA Library EZ- sample 295 (5)2330.5142.796.083.657.629.1
Other supplier, sample 178 (5)2110.3548.597.289.570.044.1
Other supplier, sample 284 (5)2120.2841.595.982.354.225.4
xGen DNA Library EZ-sample 11068 (8)2261.5048.897.489.469.944.6
xGen DNA Library EZ-sample 273 (8)2231.1742.596.083.056.228.7
Other supplier, sample 198 (11)2501.6147.896.386.566.742.2
Other supplier, sample 291 (11)2531.2341.994.579.053.128.3
xGen DNA Library EZ-sample 1178 (11)2298.842.196.282.554.727.3
xGen DNA Library EZ-sample 277 (11)2276.837.193.973.739.713.9
Other supplier, sample 1100 (17)31646.613.914.30.980.110.08
Other supplier, sample 2103 (17)31541.512.58.890.420.090.06

The xGen DNA Library EZ Kit was also benchmarked against another supplier of a DNA library kit using enzymatic fragmentation and 1 ng of DNA harvested from a mock bacterial community (ATCC® MSA-1000®). The DNA was converted into libraries using either the xGen DNA Library EZ Kit or the other enzymatic fragmentation library prep kit. The xGen DNA Library EZ Kit provided a greater library yield from fewer PCR cycles (Table 4), better normalized coverage across distinct GC compositions (Figure 2), improved coverage uniformity (Figure 2), and fewer PCR duplicates (Table 4) compared to the other supplier’s kit.

Figure 2. Higher normalized coverage observed using xGen DNA Library Prep EZ Kit versus another supplier of an enzymatic fragmentation-based library prep kit. To generate the two libraries, the xGen DNA Library EZ Kit (A) and a comparable, enzymatic fragmentation-based library prep kit from another supplier (B) was used on a mock bacterial community (ATCC, MSA-1000) with 1 ng DNA input. For comparative analysis, a 0.65x SPRI-based, DNA cleanup was used after PCR for both kits. The two libraries were sequenced 2x151 bp on an Illumina® MiniSeq® System in High Output mode. Reads were normalized to 5 M per sample. The mock bacterial community contained the following strains: B. cereus (GC% = 35.5), B. adolescentis (GC% = 59.4), C. beijerinckii (GC% = 29.9), D. radiodurans (GC% = 66.7), E. faecalis (GC% = 37.8), E. coli (GC% = 50.8), L. gasseri (GC% = 35.3), R. sphaeroides(GC% = 68.9), S. epidermidis (GC% = 32.0), and S. mutans (GC% = 36.8).

Table 4. Targeted sequencing metrics.

Library prep kitLibrary yield ng/µ L (PCR cycles)Aligned insert (bp)Duplicates (%)Mean coverage (%)10X coverage (%)20X coverage (%)30X coverage (%)
xGen DNA Library EZ-MSA-sample 126 (11)313.70.6933.498.195.164.7
xGen DNA Library EZ-MSA-sample 226 (11)319.50.7133.798.195.365.9
Other supplier, sample-MSA-sample 14.4 (13)321.32.0932.698.086.748.5
Other supplier, sample-MSA-sample 24.7 (13)3282.2932.898.087.048.6

xGen DNA Library Prep MC Kit and MC UNI

  • The xGen DNA Library Prep MC Kit is compatible with Covaris® sheared DNA—this kit supports 1 ng to 1 µg, PCR-free libraries from 50 ng input DNA.
  • High multiplex capability—pre-plated UDI primers enable multiplexing of up to 1536 samples.
  • Compatible with xGen Normalase™ Module—streamlines normalization of sequencing libraries from multiplexing
  • Workflow design for easy automation

Product details

The xGen DNA Library Prep MC Kits offer a versatile solution to streamline dsDNA, NGS sample preparation for Illumina® sequencing platforms. This workflow processes fragmented DNA, such as Covaris®-sheared DNA, for rapid and efficient end repair, adenylation, and adapter ligation. It includes an optional library amplification step, thus enabling both manual and automated workflows. These kits can also be used for targeted hybridization capture. The HiFi Polymerase Master Mix, which is supplied in the kit, is suitable for pre-hyb PCR to produce yields of 500 ng or more from as little as 1 ng DNA input. These kits are also compatible with any of the predesigned xGen Hyb Panels or a xGen Custom Hyb Panel.

Table 5. Specifications

FeaturexGen DNA Library MC KitxGen DNA Library MC UNI Kit
Sample typesGenomic DNA extracted from tissue, blood, microbial isolates, and environmental samples
Input concentration and type1 ng–1 µg input DNA in 50 µL volume
Indexing compatibilityCDI primers up to 96-plex and UDI primers up to 1536-plexxGen indexing primers for custom full-length and truncated adapters
System compatibility and multiplexing formatIllumina® sequencing instruments
Workflow compatibilityManual and automated. Please inquire for a list of compatible liquid handling robots and scripts.

Rapid, flexible workflow

The xGen DNA Library Prep MC protocol (Figure 4) contains two enzymatic incubations, an indexing or optional PCR, and bead-purification steps. The protocol minimizes sample handling, shortening the overall, library preparation time to two hours with PCR.

Figure 3. Workflow for the xGen DNA Library Prep MC Kits. These kits are available in two configurations to support two indexing workflows. The incubation steps consist of end repair, polishing of dsDNA, and A-tailing, all performed in a single End Prep reaction followed by ligation of either a stubby Y adapter (xGen DNA Library Prep MC kit, left) or full-length indexed Y adapter (xGen DNA Library Prep MC UNI Kit, right). The xGen DNA Library Prep MC workflow incorporates an indexing PCR step after adapter ligation to complete the adapter sequences. PCR is an optional step for the MC UNI Kit. xGen DNA Library Prep MC Kits are compatible with various indexing primers and full-length indexed Y adapters, including UDI and UMI adapters and up to 1536 UDI primer pairs. Both kits are compatible with the Normalase module. The xGen DNA Library Prep MC workflow is comparable to other kit suppliers, where a 60-minute end repair/A-tailing step (ER/A) is followed by a 15-minute adapter ligation step (L), a bead-based purification (B), an optional library amplification step (PCR), and a second, bead-based purification (B).

Product data

Figure 4. Balanced genome coverage. PCR-free libraries were constructed with the xGen DNA Library Prep MC UNI and two other suppliers’ kits, using 100 ng Coriell NA12878 DNA that was Covaris-sheared to 350 bp, in duplicate. All libraries were constructed using IDT for Illumina® TruSeq® UDI DNA Indexes (Cat. No. 20020590) at supplier-specified concentrations. A consistent bead ratio was used to generate equivalent insert sizes across supplier kits for direct comparison. Libraries were quantified by qPCR and sequenced on an Illumina® NovaSeq ® instrument, and data were normalized to 270 M reads per sample. Right Panel: PCR-free library yields were comparable across all three kits. Left Panel: xGen DNA Library Prep MC UNI showed a more balanced coverage of 971 high GC regions, relative to the mean coverage of other suppliers’ kits that included high GC regions greater than the mean coverage. A deviation from a relative coverage of 1 represents a reduction in coverage uniformity (GC rich bed file from Ross et al, Characterizing and Measuring Bias in Sequence Data; Genome Biol. 2013).

xGen Exome and Pan-Cancer Panels

The xGen DNA Library Prep MC and the two other suppliers’ kits were evaluated with the xGen Pan-Cancer and Exome enrichment panels using duplicate samples of Coriell NA12878 genomic DNA that was Covaris-sheared to 200 bp. A total of 10 ng sheared DNA was used for the xGen Pan-Cancer Panel, and 100 ng sheared DNA was used for the xGen Exome Panel v2. For Pan-Cancer, xGen DNA Library Prep MC and competitor libraries were prepared with truncated Y adapters at supplier-specified concentration and an xGen CDI Primer kit. The Exome libraries were prepared with the xGen DNA Library Prep MC UNI and other suppliers’ kits using IDT for Illumina® TruSeq® UD DNA Indexes (Cat. No. 20020590) at supplier-specified concentration. For both panels, a consistent bead ratio was used to generate equivalent insert sizes across supplier kits for direct comparison. Table 6 lists the experimental results.

The upper section of Table 6 lists data obtained in the following experiment: xGen Pan-Cancer captured libraries were sequenced on a MiSeq® with 100 bp, paired-end reads and were normalized to 1.5 M reads. xGen DNA Library Prep MC Kit yields using the HiFi Master Mix were equivalent to other supplier kits that used Kapa HiFi Hot Start Ready Mix, thus showing library amplification for pre-hyb PCR. Due to MiSeq chemistry, PCR duplicates directly reflect library complexity. All three kits demonstrated similar target coverage, estimated library size, and uniformity of target coverage.

The lower section of Table 6 lists data obtained in the following experiment: Exome capture libraries were sequenced on a NovaSeq® instrument (Illumina) with 150 bp, paired end reads and were normalized to 80 M reads. The xGen DNA Library Prep MC yields using the HiFi Master Mix were equivalent to other supplier kits that used Kapa HiFi Hot Start Ready Mix, thus showing comprehensive library amplification for pre-hyb PCR. Due to NovaSeq chemistry, PCR duplicates include cluster duplicates and do not directly reflect library complexity. However, all three kits showed similar target coverage and uniformity of target coverage.

Table 6. Data from targeted sequencing libraries created with xGen DNA Library Prep Kit MC followed by xGen Pan-Cancer Hyb Panel (upper) or xGen Exome Hyb Panel (lower) capture reactions.

PrepCapture runInput (ng)Pre-Hyb yield (ng/ul)Total readsMean insert (bp)Mean coverageDuplicates (%)Estimated libraryFold 80 base penalty*50X Coverage (%)100X Coverage (%)On-Target (%)
xGen DNA Library Prep MCxGen Pan-Cancer Hyb Panel MiSeq®1097.21.5 M201205X9.410 X 106099.19980.3
110201206X8.811 X 10699.198.980.3
Supplier A101201203X9.610 X 10699.29980.2
96201200X10.69 X 10699.29980
Supplier B99.6196203X10.19 X 10699.19980.3
105196203X9.99 X 10699.198.980.2
XGen DNA Library Prep MCxGen Exome Hyb Panel NovaSeq®10010080 M212146X9.1N/A1.5385.67789.1
110211146X8.21.4885.97888.6
Supplier A96.5211143X9.11.5885.67688.7
101213141X91.5485.87688.2
Supplier B96201145X8.21.5785.67689.1
98.4207141X91.5585.87688.7

* Fold 80 Base Penalty is the fold over-coverage necessary to raise 80% of bases in targets to the mean coverage.

Figure 5. Normalase technology. 384 xGen DNA MC libraries were generated with 1 ng Coriell NA12878 gDNA and were each uniquely indexed with xGen Normalase Unique Dual Indexing primers during library amplification. Libraries were pooled with equal volumes, following PCR. The pool was quantified by Qubit™ (Thermo Fisher) and loaded on an Illumina MiSeq® system to obtain the percent reads Identified from each index. The equal volume pools coefficient of variation (CV) was 21%, showing amplification with the xGen Normalase indexing primers. The same libraries were then enzymatically normalized using xGen Normalase Module to generate an equimolar library pool, at a 4 nM concentration, and were then loaded on an Illumina MiSeq system. The Normalase pool CV as reduced to 7.4%, showing normalization of multiplexed libraries using xGen Normalase technology. Lines represent the median and 95% confidence interval.

Comprehensive metagenomics sequencing

NGS libraries were constructed from 1 ng of mock metagenome DNA (ATCC MSA-1000) that was Covaris-sheared to 350 bp, in duplicate, using xGen DNA Library Prep MC UNI and two other suppliers’ kits. Libraries were prepared using IDT for Illumina TruSeq UDI DNA Indexes (Cat. No. 20020590) at supplier-specified adapter concentrations and were amplified with the polymerase supplied in each kit. Libraries were quantified by Qubit™ (Thermo Fisher) and Agilent Bioanalyzer™ 2100 and sequenced on an Illumina MiSeq system with 150 bp, paired end reads. Data (Figure 7) for each library were normalized to 1 M reads and achieved a metagenome mean coverage of 20X.

Figure 6. Comprehensive metagenomics sequencing. Top panel: Size selecting libraries using the same SPRI ratio resulted in a balanced comparison of the sequencing results. Comparable mean insert sizes and duplication rates were obtained with xGen DNA Library Prep MC UNI and two kits from other suppliers. Bottom panel: Despite significant variation in base composition, the xGen DNA Library Prep MC UNI and the other suppliers’ kits enabled identification of each strain’s genome at the expected frequency, with minimal bias. Variability in GC composition and genome size in this experiment supported that it did not influence results from the xGen™ DNA Library Prep MC UNI kit. The mock bacterial community contained the following strains: B. cereus (GC% = 35.5), B. adolescentis (GC% = 59.4), C. beijerinckii (GC% = 29.9), D. radiodurans (GC% = 66.7), E. faecalis (GC% = 37.8), E. coli (GC% = 50.8), L. gasseri (GC% = 35.3), R. sphaeroides (GC% = 68.9), S. epidermidis (GC% = 32.0), S. mutants (GC% = 36.8).

*RUO—For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations.

TruSeq® and NovaSeq® are registered trademarks of Illumina, Inc., used with permission. All rights reserved.
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