Comparing ThruPLEX DNA-Seq FLEX EF to KAPA HyperPlus and NEBNext Ultra II FS
Note: The protocols and QC procedures ThruPLEX DNA-Seq HV PLUS have been updated to accommodate lower inputs and compatibility with the Unique Dual Index Kit sets. While product naming has been revised accordingly (ThruPLEX DNA-Seq FLEX EF), reagent formulations remain unchanged.
ThruPLEX DNA-Seq FLEX is a complete, fast, and accurate system that enables reproducible sequencing readouts from challenging sample types. With the addition of an enzymatic fragmentation module, ThruPLEX DNA-Seq FLEX EF performs size-tunable enzymatic fragmentation in tandem with template repair to generate NGS-ready libraries from intact DNA with only 15 min of hands-on time (Figure 1).
When combined with Unique Dual Index Kit sets, ThruPLEX DNA-Seq FLEX EF allows for multiplexing of up to 384 libraries on Illumina® sequencing platforms. Both ThruPLEX DNA-Seq FLEX and ThruPLEX DNA-Seq FLEX EF can accommodate double-stranded DNA samples up to 200 ng with input volumes up to 30 µl. This industry-leading, three-step, single-tube workflow prevents sample loss and eliminates the need for additional time-consuming bead purifications.
Results
Familiar workflow, improved with fragmentation
Figure 1. ThruPLEX DNA-Seq FLEX EF single-tube library preparation workflow. The ThruPLEX DNA-Seq FLEX EF workflow consists of three simple steps that take place in the same well or PCR tube, eliminating the need to purify or transfer the sample material. With this latest version of ThruPLEX technology, an enzymatic fragmentation step at the start of the protocol streamlines the generation of high-complexity libraries from up to 200 ng of intact double-stranded DNA at input volumes of up to 30 µl.
ThruPLEX DNA-Seq FLEX EF
KAPA HyperPlus
NEBNext Ultra II FS
Hands-on time
15 min
20 min
20 min
Total time
2.4–2.6 hr
2.5–2.7 hr
3.1–3.2 hr
Single-tube workflow
Yes
No
No
Adapter dilution
No
Yes
Yes
Intermediate cleanup
No
Yes
Yes
Post-ligation size selection
No
No
Yes (>100 ng)
Table 1. Comparison of three leading NGS library preparation chemistries. Total time is representative of the range of time required to amplify inputs of 5 ng and 200 ng with each chemistry to yield sufficient Illumina-compatible, dual-indexed library for target enrichment. ThruPLEX FLEX is the only single-tube workflow and chemistry which does not require adapter dilution, intermediate cleanup, or post-ligation size selection. The culmination of these features is the quickest protocol with the least amount of hands-on time of any NGS library prep kit on the market.
ThruPLEX DNA-Seq FLEX EF builds upon the coveted single-tube workflow of ThruPLEX DNA-Seq FLEX with a modified template preparation step that includes an enzymatic fragmentation module in parallel with (and under the same conditions as) template repair (Figure 1, Table 1). Included with ThruPLEX DNA-Seq FLEX EF are optimized protocols for generating DNA fragments of 300 or 450 bp (Figure 2). Fragment size can be modulated by simply varying the concentration of fragmentation enzyme, thereby eliminating the need for input-specific reaction times or extra time for mechanical fragmentation. Furthermore, the kit decreases hands-on time by eliminating the need for adapter dilution and protocol optimization (Table 1). No matter what size fragment or sample input type, the total workflow time remains consistent.
Figure 2. Bioanalyzer analysis of libraries prepared using ThruPLEX DNA-Seq FLEX EF. Libraries were prepared from 5 ng of Control Human gDNA using ThruPLEX DNA-Seq FLEX EF. Post library amplification, libraries were purified following the AMPure XP protocol. An aliquot of purified library was diluted to 5 ng/μl in TE buffer, and 1 μl of this diluted sample was loaded onto a Bioanalyzer High Sensitivity DNA Analysis chip (Agilent Technologies). The blue trace is a library generated from the 300-bp protocol, and the red trace is a library generated from the 450-bp protocol.
Improved library preparation
Preparing NGS libraries from input material of a low starting concentration can lead to a library pool of poor complexity. This can be further diminished by a low input volume and template damage introduced during mechanical shearing. To combat this, ThruPLEX DNA-Seq FLEX EF accommodates a large input volume of 30 µl and utilizes enzymatic fragmentation.
Starting with samples of low concentration requires PCR amplification to obtain sufficient quantities of library for sequencing. Regions of high GC content within template molecules form strong secondary structures and can introduce PCR bias by resisting denaturation and amplification. This bias can lead to low yields, uneven representation of coverage, and low coverage depth in regions of interest. Through reformulation of ThruPLEX FLEX chemistry and workflow optimization, ThruPLEX DNA-Seq FLEX EF ensures accurate representation of the original material by minimizing bias and enabling improved coverage of high-GC regions.
Uniform library coverage across input levels
Sequencing libraries must accurately and proportionally cover a given sample’s complete sequence to represent the input material faithfully. This becomes increasingly more challenging as input concentrations are reduced as the chances of uniformly covering the sample decrease. Additionally, at lower input concentrations, reproducibility can be compromised. The ThruPLEX DNA-Seq FLEX EF kit demonstrates coverage uniformity across a wide input range, with excellent reproducibility at the lower end (Figure 3).
Figure 3. Reproducibility and uniform coverage across input levels. Correlation plots are shown for replicate library preparations generated with ThruPLEX DNA-Seq FLEX EF from 5 and 200 ng of NA12878 DNA and downsampled to 5 million total reads. Coverage of each 100-kb region of hg19 was compared across inputs. Comparison of two independent 5-ng library preps (right), and two different starting inputs of 5 and 200 ng (left), demonstrate the high reproducibility of the system.
Competitive library coverage uniformity
Generation of high-complexity libraries is critical for achieving even coverage throughout the genome for whole-genome sequencing. When compared to KAPA HyperPlus and NEBNext Ultra II FS, libraries generated using ThruPLEX DNA-Seq FLEX EF show coverage much closer to ideal normalized coverage (Figure 4). For intermediate GC compositions, all three kits perform similarly. As the GC content increases, the ThruPLEX DNA-Seq FLEX EF data remain true to the ideal normalized coverage, while results for the KAPA and NEB kits diverge dramatically. This holds true for both 5-ng and 50-ng sample inputs, further highlighting the coverage uniformity advantage provided by ThruPLEX DNA-Seq FLEX EF across sample inputs (Table 2).
Figure 4. Superior coverage uniformity. Libraries were prepared in triplicate from 5-ng and 50-ng inputs of NA12878 gDNA. Libraries were generated following ThruPLEX DNA-Seq FLEX EF, KAPA HyperPlus, or NEBNext Ultra II FS protocols. Paired-end sequencing was performed on a NextSeq® 500/550 Mid Output Kit v2.5 (150 Cycles), and total reads were downsampled to 5 million total reads. The vertical blue bars represent the expected GC content distribution using 100-bp windows.
Input
Total reads aligned
% reads aligned
% chimera
% duplicate
ThruPLEX DNA-Seq FLEX EF
50 ng
4.83E+06
96.69%
0.49%
0.75%
5 ng
4.84E+06
96.73%
0.50%
0.80%
NEBNext Ultra II FS
50 ng
4.78E+06
95.86%
1.38%
0.89%
5 ng
4.79E+06
96.16%
1.68%
1.06%
KAPA HyperPlus
50 ng
4.76E+06
95.55%
1.71%
1.24%
5 ng
4.71E+06
94.80%
1.06%
1.47%
Table 2. Comparison of different library preparation kits with integrated fragmentation modules. Sample inputs used were 50 ng or 5 ng. % reads aligned refers to those successfully aligned to a reference genome. % chimera refers to the percentage of reads that align to two distinct portions of the genome. % duplicate refers to the percentage of reads originated from a single fragment of DNA, typically during library construction via PCR.
Conclusion
ThruPLEX FLEX chemistry is engineered and optimized to generate DNA libraries with high molecular complexity and balanced GC representation from input volumes of up to 200 ng in 30 µl. Through workflow optimization, reformulation, and incorporation of an enzymatic fragmentation module, ThruPLEX DNA-Seq FLEX EF kits perform size-tunable enzymatic fragmentation and template repair in parallel to generate NGS-ready libraries in a single tube in about 2.5 hours with only 15 minutes of hands-on time. In head-to-head comparison experiments, ThruPLEX DNA-Seq FLEX EF outperforms both KAPA HyperPLUS and NEBNext Ultra II FS in coverage of regions with increasing GC content. ThruPLEX DNA-Seq FLEX EF provides efficiency, simplicity, and reliability in a single-tube workflow, suitable for challenging whole genome sequencing experiments.
Methods
DNA preparation
The concentration of human genomic DNA (NA12878) was measured using a Qubit 2.0 Fluorometer with Quant-IT dsDNA Assay Kit, high sensitivity (Thermo Fisher Scientific).
Library preparation
Libraries were prepared according to the manufacturer's instructions using ThruPLEX DNA-Seq FLEX EF, KAPA HyperPlus or NEBnext Ultra II FS. All libraries were generated using dual indexes. Amplified libraries were purified using AMPure XP (Beckman Coulter) and eluted in low TE buffer for whole genome sequencing (WGS). Size of purified libraries was assessed by Agilent 2100 BioAnalyzer using High Sensitivity DNA Reagents. Libraries were quantified by Qubit 2.0 Fluorometer with Quant-IT dsDNA Assay Kit, high sensitivity (Thermo Fisher Scientific).
Illumina sequencing
Quantified post-PCR libraries were pooled and loaded onto a NextSeq® 500/550 v2.5 flow cell for sequencing. Libraries were loaded following Illumina’s recommended loading concentrations.
Data analysis
Raw sequencing reads were downsampled to equal numbers across all samples using seqtk (v1.3-r106) and quality processed to remove adapters and low-quality bases using trimmomatic (v0.36). Quality processed reads were aligned to the UCSC hg19 reference genome with bowtie2 (v2.3.4.3) with default parameters. Resulting SAM files were coordinate sorted using Picard SortSam (v2.18.3) and converted to BAM files with samtools view (v1.8). Duplicate reads were identified and marked from sorted BAM files with Picard MarkDuplicates (v2.18.3) and used as input to collect alignment, insert size, GC bias, and various WGS metrics with Picard AlignmentSummaryMetrics (v2.18.3), Picard CollectInsertSizeMetrics (v2.18.3), Picard CollectGcBiasMetrics (v2.18.3), and Picard CollectWgsMetrics (v2.18.3), respectively.
