Myeloid malignancies are a heterogeneous group of diseases, associated with a wide range of variants ranging from mutations to structural variations. The SureSeq™ Myeloid Plus NGS Complete Workflow combines the rapid Universal NGS Complete Workflow hybridisation based target enrichment method together with OGT’s expert bait design to detect 49 key genes implicated in myeloid disorders.
Designed in collaboration with recognised cancer experts, SureSeq Myeloid Plus is a targeted gene panel for next generation sequencing technologies (NGS) to analyse specific mutations in key genes implicated in acute myeloid leukaemia (AML), myeloproliferative neoplasms (MPN), and myelodysplastic syndrome (MDS), providing researchers with a single NGS workflow delivering a comprehensive picture of the genetic make-up of each myeloid sample.
The SureSeq Myeloid Plus Panel accurately detects SNVs and indels in genes such as CEBPA, JAK2, CALR and MPL, as well as structural variants including FLT3-ITDs and KMT2A-PTDs, and is able to detect low-frequency SNVs and indels with confidence.
* Exon examples not yet available
Mutations in the CEBPA gene are among the most common molecular alterations in AML, which itself is the most common type of acute leukaemia in adults1,2. Sequencing of CEBPA is challenging due to the presence of repeat regions and the high GC-content of the gene, leading to poor coverage across these regions and potentially missed variants. OGT’s expert bait design overcomes these issues and provides exceptional coverage uniformity, enabling reliable detection of variants and eliminating the requirement for supplementary fill-in with Sanger sequencing (Figure 1).
FLT3 internal tandem duplications (ITDs) are challenging to target, and subsequently detect, because they are by nature repetitive and can be very long. As a result, FLT3-ITDs are generally masked in most panel designs, necessitating additional techniques to obtain a comprehensive genetic picture. OGT employs sophisticated bait designs to generate uniform coverage across, as well as upstream and downstream of the repetitive region. In combination with our complimentary NGS analysis software Interpret, this allows easy detection of FLT3-ITDs ranging from a handful of base pairs to >200 bp (Figure 2).
Other tandem duplications frequently observed in AML are partial tandem duplications (PTDs) in KMT2A (MLL). Similar to ITDs, KMT2A-PTDs are notoriously difficult to detect due to their size, with duplications spanning multiple exons. With OGT’s expertise in hybridisation-based panel design, SureSeq offers robust detection of all sizes of KMT2A-PTDs, alleviating the burden of running multiple assays (Figure 3).
Figure 1: Illustration of the excellent coverage uniformity of the CEBPA gene. Depth of coverage per base (grey). Targeted region (green). Gene coding region as defined by RefSeq (blue). GC percentage (red).
Figure 2: FLT3-ITDs of various sizes and even regions containing multiple ITDs can be confidently detected. ITD sizes are (A) 174 bp, (B) 225 bp, (C) 195 bp with additional 6 bp, (D) 120 bp and (E) 168 bp with additional 69 bp.
Hybridisation-based enrichment is now well recognised as providing superior results over ampliconbased enrichment technology. The recently launched OGT Universal Library Preparation Kit delivers a straightforward and robust workflow with fewer hands-on steps, and improved turnaround times. For increased convenience and flexibility, the Universal NGS Complete Workflow is performed with a combined enzymatic fragmentation, end repair and A tailing step, and convenient bead concentration steps, whilst still delivering libraries of the highest quality. The inclusion of unique Dual Index Adapters increases multiplexing efficiency and confidence, whilst enhancing capabilities to include sensitive applications. Universal Hyb & Wash buffer simplifies this key step while offering excellent coverage uniformity and reproducibility.
Interpret is OGT’s powerful and easy-to-use NGS analysis solution, facilitating analysis and visualisation of a wide range of somatic variants and structural aberrations. Designed to work seamlessly with all SureSeq panels, Interpret perfectly complements the SureSeq Myeloid Plus Panel, delivering fast and accurate detection of all SNVs, indels, ITDs and PTDs covered by the panel. Following detection, all variants can be easily visualised in the user-friendly variant browser, for an effortless translation of all your myeloid data into meaningful results.
You never have to sequence genes you’re not interested in and can always modify each panel to what’s relevant to your research. If the SureSeq Myeloid Plus Panel doesn’t meet your exact requirements, you can choose from our regularly updated, expert-curated library of pre-optimised cancer content to create your ideal custom SureSeq myPanel™ Myeloid Panel. Alternatively, have a look at the other myeloid panels we have available, including our focused 3-gene SureSeq Core MPN Panel and the SureSeq Pan-Myeloid Panel, incortporating key variants in 70 genes implicated in a wide range of myeloid disorders, or our disease-specific content, such as our SureSeq myPanel NGS Custom AML panels.
The 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