SureSeq™ NGS Library Preparation Kit

The SureSeq NGS Library Preparation Kit generates NGS libraries suitable for the capture of targeted genomic regions using hybridisation. With a streamlined workflow, significantly reduced hands-on steps and hybridisation times as low as 30 minutes, SureSeq offers all of the benefits of hybridisation in as little as a 1-day workflow. For increased convenience and flexibility, the SureSeq workflow can be performed with an enzymatic or Covaris^® DNA fragmentation and Speedvac^® or bead concentration steps, whilst still delivering libraries of the highest quality. The inclusion of the SureSeq Hyb & Wash buffer, optimised for use with SureSeq NGS panels, simplifies this key step while offering excellent coverage uniformity and reproducibility. Additionally, a new automation workflow enables standardisation of OGT’s library preparation, hybridisation and washing steps for up to 96 samples in a single batch, improving laboratory productivity.

The SureSeq Library Preparation Kit offers:

• Greater trust in your data - high performance with low duplication rates, high sequence quality and high percentage of on-target bases
• Simpler hybridisation - all components of the SureSeq Hyb & Wash buffer are ready-to-use with no requirement for multiple wash buffers
• Rapid process - streamlined protocol, minimal manual handling, automation and a rapid hybridisation step offers increased reliability as well as throughput
• Reliable results - NGS targeted panel, complete NGS library preparation solution and powerful NGS analysis software are fully optimised for excellent results

Greater trust in your data

NGS data is increasingly being relied upon as a front-line technology for the generation of data for scientific and medical research. A combination of quality metrics are used to give confidence that variants called are real and the number of false positive calls are low. The SureSeq NGS Library Preparation Kit delivers high performance in the quality metrics that really matter, giving more reliable, more trustworthy data.

Low levels of duplication

High duplication rates reduce the complexity of your NGS library and can lead to poor coverage of targeted genomic regions even though average coverage rates appear high.

The SureSeq NGS Library Preparation Kit gives exceptionally low levels of duplication (Figure 1). This ensures more accurate calling, more even coverage and higher levels of confidence in the data produced.

Figure 1. The SureSeq NGS Library Preparation Kit delivers low levels of sequence duplication. The duplication rates are shown for samples fragmented by mechanical or enzymatic methods. Samples were prepared using the SureSeq NGS Library Preparation kit and hybridised with a SureSeq myPanel Custom Myeloid 49 Gene Plus panel.

High quality sequence data

For complete confidence in your data, the sequence quality scores should be high and consistent across the length of each read. The SureSeq NGS Library Preparation Kit delivers exceptional sequence quality scores even 150 bases into the read (Figure 2). This reduces the requirement for post-acquisition bioinformatics, such as read size reduction, to remove poor quality sequence.

Figure 2. The SureSeq NGS Library Preparation Kit delivers high sequence quality. Data shown was generated using 4 samples of human genome DNA, the OGT SureSeq Myeloid Panel, and the SureSeq NGS Library Preparation Kit.

High levels of on-target bases

In order to maximize the amount of useful information that can be obtained for a given sequencing lane, high coverage depth and low coefficients of variation are desirable. The benefits of superior levels of specific sequence capture include increased sequence complexity and greater depth of coverage of the target regions. The SureSeq NGS Library Preparation Kit delivers excellent reproducibility (Figure 3).

Figure 3. High depth of coverage and a low coefficient of coverage variation from 16 replicates. A SureSeq myPanel Myeloid 49 gene plus and SureSeq Library Preparation Kit were utilised in the evaluation of 336 coding targets in the custom panel (excluded targets on X or Y chromosome) to illustrate the low CV (coefficient of variation) and excellent uniformity of SureSeq.

Simpler hybridisation with the SureSeq NGS Hyb & Wash Kit

The SureSeq NGS Hyb & Wash kit is an easy to use, robust and high performing hybridisation solution, containing all the components required to perform the hybridisation and wash steps in SureSeq sequence capture protocols. All components in the kit are ready-to-use, eliminating the requirement to dilute buffers and the possibility of cross-contamination during buffer preparation. There is no need to use multiple wash buffers, as in other manufacturer’s kits, the simplified protocol utilises just a single wash buffer. With hybridisation times as little as 30 minutes, it offers excellent quality sequencing data, when used in conjunction with the SureSeq NGS Library Preparation Kit and SureSeq NGS panels (Figure 4).

Figure 4. Superior performance. Comparison of the mean target coverage generated using the SureSeq Hyb & Wash Buffer Kit (light blue) compared to an alternative supplier kit (dark blue). [A] SureSeq myPanel custom CLL panel and [B] SureSeq myPanel custom CLL plus CNV panel.

Rapid process with streamlined protocol

By extensive testing of enzymes and buffer optimisation, it has been possible to streamline the standard library preparation protocol to minimise the number of hands-on steps and the overall processing time (Figure 5). For added convenience, OGT now offers Dynabeads™ M270 Streptavidin and AMPure^® XP beads for use with SureSeq NGS panels. While the addition of a workflow for automation on the Agilent Bravo A^® Automated Liquid Handling Platform offers greater flexibility and productivity for your laboratory.

• End repair and ligation are combined into a single step
• Purification steps, where material is lost, are reduced
• Fewer steps reduce handling errors for increased reliability
• Reduced technician time and cost and increased sample throughput
• Workflow amenable to automation

Figure 5. A streamlined protocol, including enrichment by hybridisation. With hybridisation times as low as 30 minutes, the procedure can be completed in as little as one day with minimal handling time.

Reliable results with SureSeq targeted NGS panels and analysis software

The SureSeq NGS Library Preparation Kit and Hyb & Wash Kit work hand-in-hand with SureSeq targeted enrichment panels, ensuring you get the most sensitive and reproducible variant detection, (Figure 6), and industry-leading coverage uniformity.

SureSeq Interpret is a powerful and easy-to-use next generation sequencing analysis solution. Sequencing data can be quickly processed to deliver accurate identification of single nucleotide variants (SNVs) and Indels as well as structural variants such as copy number variants (CNVs) and internal tandem duplications (ITDs). Coupled with a comprehensive and powerful filtering framework, the software delivers accurate mutation calling with 100% sensitivity and 99.9% specificity at >1% variant allele frequency (VAF)*. It is designed to work with Oxford Gene Technology’s (OGT) extensive range of SureSeq NGS panels and offers flexible accessibility to analyse your data whether through a stand-alone computer^†, laboratory server or another web enabled device.

Figure 6. Excellent reproducibility with low allele frequency detection and CV distribution. The SureSeq workflow and software were validated using OncoSpan Reference Standards (Horizon Discovery). A SureSeq myPanel NGS Custom panel was utilised to detect 46 variants in 15 genes present in the OncoSpan Reference Standard. Three different DNA inputs with three independent replicates were processed in parallel. Data generated was analysed using SureSeq Interpret Software.

Ordering Information

* Sensitivity and specificity determined using Horizon Discovery OncoSpan and TruQ7 and HapMap (NA12878) standards.

^† Computer specification: RAM: min 16Gb, suggested 24Gb; HDD: min 500Gb, suggested 2TB; CPU: 8+ logical cores at 2+ GHz; OS: (Windows 7 or newer), Virtualization (VT-x) needs to be enabled in the BIOS.

^‡ The SureSeq™ FFPE DNA Repair Mix can only be purchased in conjunction with SureSeq NGS panels, not as a standalone product.

** Only for use with OGT's NGS panels

Disclaimer

SureSeq: For Research Use Only; Not for Diagnostic Procedures. This webpage and its contents are © Oxford Gene Technology IP Limited – 2019. All rights reserved. OGT™ and SureSeq™ are trademarks of Oxford Gene Technology IP Limited. The SureSeq NGS Library Preparation Kit was jointly developed between Oxford Gene Technology and Bioline Reagents Limited. SpeedVac^® is a registered trademark of Thermo Fisher Scientific, Covaris™ is a registered trademark of Covaris, Inc., Bravo^® is a registered trademark of Agilent Technologies, Inc., Dynabeads is a trademark of Thermo Fisher Scientific and AMPure^® is a registered trademark of Beckman Coulter Inc.