ThruPLEX HV is a complete, fast, and accurate system that enables reproducible sequencing readouts from challenging sample types. With the addition of the ThruPLEX HV PLUS Enzymatic Fragmentation Module, ThruPLEX DNA-Seq HV PLUS performs size-tunable enzymatic fragmentation in tandem with the ThruPLEX DNA-Seq HV repair step to generate NGS-ready libraries with only 15 minutes of hands-on time (Figure 1). Similarly, ThruPLEX Tag-Seq HV PLUS shares the same great workflow as and benefits of ThruPLEX DNA-Seq HV PLUS, with the added advantage of unique molecular identifiers (UMIs).
Taking advantage of the ThruPLEX HV unique dual index (UDI) technology, ThruPLEX HV PLUS kits allow multiplexing of up to 96 libraries, ready for sequencing with Illumina® sequencing reagents and protocols. Both ThruPLEX HV and ThruPLEX HV PLUS kits 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 HV PLUS single-tube library preparation workflow. The ThruPLEX HV PLUS 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. In this latest version of the ThruPLEX technology, an enzymatic fragmentation step at the start of the protocol streamlines the generation of high complexity libraries from double-stranded DNA, up to 200 ng in 30 µl.
ThruPLEX HV PLUS
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 DNA-Seq HV 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.
The ThruPLEX HV PLUS library preparation system expands the coveted single-tube workflow of the ThruPLEX HV kits with a modified template preparation step that now includes an enzymatic fragmentation module in the same template preparation conditions (Figure 1, Table 1). Using this enzymatic fragmentation module, the ThruPLEX HV PLUS kits provide optimal protocols to generate DNA fragments of 300 and 450 bp (Figure 2). Fragment size can be modulated by simply varying the in‑reaction concentration of fragmentation enzyme, thereby eliminating the need for input-specific reaction times or extra time for mechanical fragmentation. Furthermore, the ThruPLEX HV PLUS system 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 HV PLUS. Libraries were prepared from 5 ng of Control Human gDNA using ThruPLEX DNA-Seq HV PLUS. 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, the ThruPLEX HV PLUS library preparation system accommodates a large input volume of 30 µl and utilizes enzymatic fragmentation.
Starting with samples of low concentration requires PCR amplification to enrich for a pool of sequenceable libraries and increase the overall yield. Regions of high GC content contain 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 and workflow optimization, ThruPLEX HV PLUS ensures accurate representation of the original material by removing bias and providing substantial improvements specifically for coverage of regions with extreme GC composition.
Uniform library coverage across input levels
Sequencing libraries must accurately and proportionally cover a given sample’s complete sequence to faithfully represent the input material. This becomes increasingly more challenging as input concentrations are reduced, as the chances of uniformly covering the sample decreases. Additionally, at lower input concentrations, reproducibility can be compromised. The ThruPLEX DNA-Seq HV PLUS kit demonstrates coverage uniformity across the input range (Figure 3). Importantly, at the lowest recommended input value, 5 ng, the ThruPLEX HV PLUS Kit still performs well with superb reproducibility (Figure 3).
Figure 3. Reproducibility and uniform coverage across input levels. Correlation plots are shown for replicate library preparations generated by ThruPLEX DNA-Seq HV PLUS with 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 HV PLUS show coverage much closer to ideal normalized coverage (Figure 4). In intermediate GC compositions, all three kits perform similarly. As the GC content increases, the ThruPLEX DNA-Seq HV PLUS Kit data remain true to the ideal normalized coverage, while those of KAPA and NEB kits diverge dramatically. This holds true for both 5-ng and 50-ng sample inputs, further highlighting the ThruPLEX HV PLUS advantage in uniform library coverage 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 HV PLUS, 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 alligned
% reads aligned
% chimera
% duplicate
ThruPLEX DNA-Seq HV PLUS
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
The ThruPLEX HV PLUS kits are the newest members of the ThruPLEX HV family. ThruPLEX HV 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 the addition of the ThruPLEX HV PLUS Enzymatic Fragmentation Module, ThruPLEX HV PLUS kits perform size-tunable enzymatic fragmentation in tandem with the ThruPLEX HV repair step to generate NGS-ready libraries in a single tube in about 2.5 hours with only 15 minutes hands-on time. In head-to-head comparison experiments, ThruPLEX DNA-Seq HV PLUS outperforms both KAPA HyperPLUS and NEBNext Ultra II FS in coverage of regions with increasing GC content. ThruPLEX DNA-Seq HV PLUS offers 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 HV PLUS, 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.
