Targeted gene panels for next generation sequencing technologies (NGS) are useful tools to analyse specific mutations in a given DNA sample. Focused panels contain only a select set of genes or gene regions allowing a cost-effective approach to focus your research.
NGS uses parallel short-read DNA sequencing, genome alignment and assembly methods to digitally and rapidly analyse genomic sequence information. This fast-sequencing approach allows researchers to study the genetic causes of rare diseases and cancers, or make genome-level comparative analysis.
The two most common methods for library preparation are amplicon sequencing and hybridisation capture
With the hybridisation method DNA is fragmented and tagged with appropriate barcodes and then subsequently amplified. Hybridisation is a more robust technique with the advantage of providing better uniformity of coverage, fewer false positives, and superior variant detection due to fewer PCR cycles.
The amplicon-based library preparation method creates a targeted sequencing library from your DNA sample using two basic steps: 1) capture and amplification of the targeted sequences of interest to yield a pool of appropriately sized fragments, 2) addition of sequencing adapters that will later interact with the NGS platform of choice.
Probes used in the capture step of the library preparation are designed based on the targeted sequences of interest.
The adapter ligation step uniquely tags the DNA fragments with specific oligonucleotide sequences that will interact with the surface of a sequencing flow cell. If multiple samples are to be sequenced in a single sequencing run, a unique identifier, or barcode, is additionally ligated to the targeted fragments. The resulting completed libraries can be pooled into a single sequencing run that is then “demultiplexed” during data analysis.
Classic library preparation protocols consist of lengthy multistep processes that require costly reagents and substantial hands-on-time.
Universal NGS Complete Workflow Solution includes a combined multi-enzymatic fragmentation, end repair and A-tailing step together with convenient bead concentration steps, delivering increased convenience and flexibility for our highest quality library preparation.
Universal NGS Complete Workflow Solution is our latest and most advanced system for capture of targeted genomic regions and generation of NGS libraries, tested and optimised with both SureSeq™ and CytoSure® NGS panels.
OGT’s Universal NGS Complete Workflow Solution has a combined multi-enzymatic fragmentation, end repair and A-tailing step followed by convenient bead concentration steps, leading to the need of fewer clean-up and QC steps, to deliver scalability and reproducibility while minimising human errors.
Additionally, the inclusion of Unique Dual Index (UDI)/Unique Molecular Index (UMI) adapters in the first phase of library preparation increases the kit multiplexing efficiency and confidence, enhancing capabilities to include sensitive applications. OGT’s Universal Hyb & Wash buffer simplifies this key step while offering excellent coverage uniformity and reproducibility.
The new Universal NGS Complete Workflow Solution uses a Unique Dual Indexing strategy to ensure accurate demultiplexing and avoid index hopping and Unique Molecular Identifier (UMI) for reliable identification of low-frequency variants.
Figure 2: Combinatorial dual indexing barcoding (A) uses a plate matrix approach of i5 and i7 adapters that leads to unique combinations of non-unique indexes. In contrast, Unique Dual Indexing (B) uses completely unique indexes across the whole plate, avoiding any repetition in sequence (eg, 96 unique i5s and 96 unique i7s per 96-well plate).
Figure 3: UMIs are short arbitrary oligonucleotides sequences that are attached to the library of DNA fragments by ligation prior to the amplification step. Reads that have the same UMI tag are from the same original DNA fragment and so the deriving PCR amplicons should be identical. Without the use of UMIs, low-frequency variants can be confused with DNA polymerase errors produced during the amplification step as well as sequencing errors produced during the sequencing step.
The Universal NGS Complete Workflow Solution works hand-in-hand with both CytoSure NGS Constitutional kit as well as with SureSeq targeted cancer enrichment panels, ensuring you get the most sensitive and reproducible variant detection and industry-leading coverage uniformity.
Figure 4: Interpret is able to call duplications with the same precision as microarrays, in this example a 1.59 Mb duplication on chromosome 7 is detected using the CytoSure Constitutional NGS Panel.
Interpret is OGT’s powerful, easy-to-use and customisable next-generation sequencing analysis solution delivers accurate calling of SNVs and indels, as well as structural aberrations, including ITDs, PTDs, CNVs, LOH and translocations.
Interpret is designed to work seamlessly with SureSeq and CytoSure NGS panels offering flexible accessibility for data analysis; whether through a stand-alone computer, laboratory server or another web-enabled device.
OGT partnership approach is key to providing the highest level of service, working closely with you to understand your unique challenges, customising our approach to meet your exact needs.
We were delighted with the performance of the SureSeq panel. It showed complete concordance with our other techniques, detecting all known mutations with excellent sensitivity down to 1% [MAF (minor allele frequency)], including, in one case, a JAK2 V617F mutation which was not detected by ddPCR due to a second mutation under the primer. The panel also demonstrated mutations in other genes in samples with low level JAK2 V617F and good correlation between allele frequencies and quantitative analysis by ddPCR. We are planning to adopt the panel in the near future.
Dr Anna Skowronska
R&D Scientist, Haemato-Oncology Team, West Midlands Regional Genetics Laboratory, Birmingham Women’s NHS Foundation Trust, UK