Optimized, 1-day hybridization-based NGS protocol yields 1% variant detection in MPN samples, as quickly and cost-effectively as multiplex PCR

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Contributors

Graham Speight¹ , Ephrem Chin¹ , Lyudmila Georgieva¹, Nick Cross² and David Cook¹

¹OGT, Oxford, UK and ² National Genetics Reference Laboratory - Wessex, UK

Introduction

• Myeloproliferative neoplasms (MPNs) are a group of diseases that affect blood cell production in the bone marrow resulting in the overproduction of one or more cell types. The key MPN driver mutations involve JAK2 (V617F [occurrence of 50-98% depending on the MPN subtype]), JAK2 exon 12, MPL W515K/L and CALR exon 9 insertion and deletions. 
• Designed for research into the diagnosis, aetiology and prognosis of MPNs, the SureSeq™ Core MPN Panel has been developed by OGT in collaboration with recognized cancer experts to deliver accurate detection (down to a 1% variant allele fraction [VAF]) of somatic variants of these key MPN driver mutations. 
• The aim of this study is to evaluate the SureSeq Core MPN Panel in conjunction with a new streamlined 1-day hybridization-based Next Generation Sequencing (NGS) library preparation kit (LPK).

Methods

• An enhanced version of the SureSeq Library Preparation Kit (OGT) was utilized which incorporated an enzymatic DNA fragmentation in combination with a rapid hybridization of just 30 minutes. This enhanced protocol reduces the overall processing time by 6 hours, and now offers a streamlined, 1-day workflow from purified DNA sample to sequencer. 
• This protocol offers a similar turn-around time to amplicon-based enrichment protocols, without the associated disadvantages, such as PCR bias, allelic bias (indels) and drop-outs, as well as poor uniformity of coverage.

Figure 1: Workflow of SureSeq NGS library preparation, from DNA to sequencer.

Panel content 

• The SureSeq Core MPN Panel covers 3 genes important in MPN research: JAK2 (V617F and exon 12), MPL (W515K/L/R/A and S505N) and CALR (exon 9).

Study design

• The SureSeq Core MPN Panel was technically validated using the JAK2 V617F Genotyping Sensitivity Panel provided by the National Institute for Biological Standards and Control (NIBSC) in order to confirm the lower levels of analytical allele detection and confidence. 
• The SureSeq Core MPN Panel was also used to confirm a broader set of variants in 14 research samples (provided by the National Genetics Reference Laboratory - Wessex, UK) containing variants for each of the targeted regions. Sequencing was conducted on a MiSeq^® using a V2 300 bp cartridge (Illumina).

Results

Accurate and reproducible variant detection even in heterogeneous samples

• Heterogeneous cancer samples pose significant challenges as alleles are likely to be present at a lower fraction than would be expected for standard germline variants. Samples typically contain a mixture of cancer and normal cells; moreover, cancer can consist of several molecularly distinct clones. In order to detect alleles that contribute only a small percentage to the reads at any locus, a highly uniform and sensitive enrichment is required. 
• The SureSeq Core MPN Panel has been technically validated with samples from the NIBSC and has been shown to accurately detect alleles down to a 1% VAF in JAK2 (V617F) at a read depth of >1000x (Table 1).

Table 1: Data generated from a 48 sample run on an Illumina MiSeq. The SureSeq Core MPN Panel permitted the detection of alleles at 1% VAF with high confidence.

Results

The Core MPN Panel in combination with the enhanced workflow is able to reliably detect single nucleotide variants (SNVs) as well as insertions (5 bp insertion in JAK2 exon 12 and CALR exon 9) and deletions (5 bp deletion exon 12 JAK2 and 52 bp deletion CALR exon 9) (see below).

Figure 2: Detection of a 52 bp deletion (exon 9 CALR). Wild-type sample (top panel) is compared to a 52 bp somatic deletion (bottom panel).

Figure 3: Detection of a 5 bp deletion (exon 12 JAK2). Wild-type sample (top panel) is compared to a 5 bp somatic deletion (bottom panel).

Results

Technical validation of the Core MPN Panel and enhanced library preparation approach with 14 research samples

• Data presented here are from 14 research samples (including 2 wild-type controls) that were processed using the enhanced LPK in combination with the Core MPN Panel. 
• See below (Table 2) for the range of variants and VAFs detected from 14 research samples. These include SNVs as well as 5 bp insertions in JAK2 (exon 12) and deletions of up to 52 bp in CALR (exon 9) (Table 2). No variants were identified in the control samples.

Table 2: Data generated using the SureSeq Core MPN Panel in combination with the enhanced LPK was 100% concordant with independent findings (National Genetics Reference Laboratory – Wessex, UK).

Conclusions

• We have successfully utilized the OGT 1-day hybridization-based SureSeq LPK protocol in combination with the SureSeq Core MPN Panel to reliably and routinely detect somatic SNVs by NGS down to a 1% VAF. 
• The uniformity of coverage of this approach permitted the detection of CALR and JAK2 indels (including 52 bp deletions and 5 bp insertions). 
• This enhanced protocol incorporates an enzymatic fragmentation which permits the high-throughput preparation of 48 samples from genomic DNA to sequencer in a 1-day workflow. 
• To achieve >1000x de-duplicated depth (required for confident detection of 1% VAF), 48 samples can be reliably sequenced in a single MiSeq (V2 300 bp) run. This allows the generation of high quality data in a cost effective and timely manner.

SureSeq: For Research Use Only; Not for Diagnostic Procedures.