A high-performance NGS library preparation system must have a simple, streamlined workflow that can accommodate a range of sample inputs without compromising accuracy. ThruPLEX DNA-Seq FLEX satisfies these requirements, with a complete, fast, and accurate system that enables reproducible sequencing readouts from challenging sample types. ThruPLEX DNA-Seq FLEX was designed to accommodate a large input volume and a higher amount of starting material than our original ThruPLEX DNA-Seq Kit, which improves coverage and mutation detection by increasing the complexity of the sample input and eliminating the need to concentrate precious DNA samples prior to library preparation. A final important consideration for low-frequency mutations is the ability to achieve even coverage throughout the genome in order to ensure optimal read depth at all relevant loci. To facilitate the necessary even coverage, our system has been optimized for improved coverage uniformity across a broad range of GC content.

Single-tube ThruPLEX DNA-Seq FLEX workflow

The ThruPLEX DNA-Seq FLEX workflow consists of three simple steps of reagent addition that take place in a single well or PCR tube with just 15 minutes of hands-on time, and yields indexed libraries from fragmented DNA within two hours (Figure 1). The libraries generated can be used directly for whole-genome sequencing applications or enriched using a custom panel for the leading target enrichment platforms. The implementation of a single-tube workflow increases throughput and prevents the loss of precious samples by eliminating the need for time-consuming bead purification or transfer of sample material. Additionally, eliminating input amount-based adapter dilution further minimizes hands-on time and allows ThruPLEX DNA-Seq FLEX to be one of the quickest and most consistent library preparation kits when compared to others that are commercially available (Table 1).

Improved library preparation

Preparing NGS libraries from input material with a low starting concentration can lead to a low-complexity library pool. This complexity can diminish even further when hampered by a low input volume. The ThruPLEX DNA-Seq FLEX library preparation kit takes the coveted single-tube workflow of the ThruPLEX DNA-Seq Kit and elevates the input volume from 10 to 30 µl, while also increasing the input range to 200 ng.

Preparing NGS libraries from low-concentration samples makes PCR amplification essential in order to enrich a pool of sequenceable libraries and increase yield. Regions of high GC content contain strong secondary structures and can introduce bias by resisting denaturation and amplification. Introducing bias in the process of NGS library preparation can lead to low yields, uneven representation of coverage, and low coverage depth in regions of interest. Through reformulation and workflow optimization, ThruPLEX DNA-Seq FLEX ensures the accurate representation of the original material by removing bias and providing substantial improvements in coverage of regions with extreme base composition.

Uniform library coverage across input levels

Generating libraries which proportionally cover the complete sequence of a given sample is pivotal to creating an accurate representation of that input material. This becomes challenging when lower inputs are used, as the chances of uniformly covering the sample decreases. Additionally, at lower inputs, reproducibility is challenged. By consistently generating libraries of the recommended insert size for sequencing, the need for size selection is eliminated and the likelihood of achieving a higher base quality score is improved. The ThruPLEX DNA-Seq FLEX kit can generate sequenceable libraries with a consistent insert size across the input range (data not shown). ThruPLEX DNA-Seq FLEX also demonstrates coverage uniformity across the input range and coverage uniformity of replicates at the kit's lowest recommended input (Figure 2).

Uniform GC coverage

While consistent, uniform coverage can be challenging, the robustness of a library preparation kit depends on its ability to accurately and uniformly cover a variety of challenging genomes with varying GC content. ThruPLEX DNA-Seq FLEX is able to produce consistent GC coverage spanning the input range for gDNA (Figure 3), as well as inputs with a variety of GC contents (Figure 4).

Improved library coverage with the FLEX protocol

Libraries were generated from gDNA, with input amounts that span the overlapping input ranges of ThruPLEX DNA-Seq FLEX and ThruPLEX DNA-Seq kit, using their respective protocols. The ThruPLEX DNA-Seq FLEX libraries show substantially better coverage at both inputs, as indicated by the improvements in uniform coverage (Table 2). These improvements in uniform coverage were made without sacrificing mapping, chimera, or duplication rates.

The ThruPLEX DNA-Seq FLEX library preparation kit for Illumina sequencing platforms elevates the ThruPLEX family by increasing starting input volume and expanding the amount of starting material when compared to the previous ThruPLEX DNA-Seq Kit. Along with these improvements, this advanced system retains the coveted single-tube workflow with no intermediate cleanup, which is synonymous with ThruPLEX technology. Through workflow optimization and reformulation, this kit provides substantial improvements in coverage of regions with increasing GC content. ThruPLEX DNA-Seq FLEX is a simple, fast, and accurate system with three addition-only steps that can be completed in a single tube in just two hours.

DNA preparation

Human genomic DNA from EMD Millipore (69237) or Horizon Discovery (HD701) and bacterial genomic DNA from Haemophilus influenzae 51907D-5, Escherichia coli 11303, or Rhodopseudomonas palustris BAA-98D-5 (ATCC) were sheared on a Covaris M220 following the 250-bp shearing protocol. Sheared input material was evaluated for correct size on an Agilent 2100 BioAnalyzer using High Sensitivity DNA Reagents. The concentration of these samples was measured using a Qubit 2.0 Fluorometer with the dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific).

Library preparation

Libraries were prepared as per the manufacturer's instructions using the ThruPLEX DNA-Seq FLEX kit or the ThruPLEX DNA-Seq Kit. All libraries were generated using dual indexes. Amplified libraries were pooled and then purified using AMPure XP beads (Beckman Coulter) and eluted in low TE buffer for whole-genome sequencing (WGS). Purified libraries' size was assessed on the Agilent 2100 BioAnalyzer using High Sensitivity DNA Reagents. Libraries were quantified by qPCR using Takara Bio's Library Quantification Kit, or Qubit 2.0 Fluorometer with the dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific).

Illumina sequencing

Quantified post-PCR libraries were pooled and loaded onto an Illumina MiSeq® V3 or NextSeq 500/550 v2.5 flow cell for sequencing, following Illumina-recommended loading concentrations.

Data analysis

For WGS data, reads were merged using FastQC and downsampled to equal numbers across all samples using seqtk. Adapters were trimmed using trimmomatic and each library was aligned to hg19 using Bowtie 2 and converted to sam format. Sam files were converted to bam files using samtools. Library metrics were collected from bam files using the following: Picard Tools, MarkDuplicates, AlignmentSummaryMetrics, CollectInsertSizeMetrics, CollectGcBiasMetrics, and CollectWgsMetrics.

Chen Y. C., Liu T., Yu C. H., Chiang T. Y., Hwang C. C. Effects of GC bias in next-generation-sequencing data on de novo genome assembly. PLoS One 8, e62856 (2013).

Tan, G., Opitz, L., Schlapbach, R. et al. Long fragments achieve lower base quality in Illumina paired-end sequencing. Sci. Rep. 9, 2856 (2019).