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.

Familiar workflow, improved with fragmentation

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.

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).

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).

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.

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.