The application of a one-day hybridisation-based enrichment protocol for NGS incorporating a rapid (30 minute) hybridisation step

Methods

Preparation of purified DNA to sequencer-ready libraries in 7 hours, 45 minutes

• An enhanced version of the SureSeq™ LPK (OGT) was utilised which incorporates an enzymatic DNA fragmentation in combination with a rapid hybridisation of just 30 minutes. This enhanced protocol reduces the overall processing time by 6 hours, resulting in a streamlined, 1-day workflow. 
• This kit 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.

Study design

• Four different haematological panels have been used, with a size range from 0.5 Kb to 138 Kb.
• Data quality comparison was performed in terms of Mean Target Coverage (MTC) and % on-target bases achieved with the 1-day and the standard workflow. More specifically, we compared the uniformity of coverage achieved with both protocols for difficult to sequence genes such as CALR, CEBPAand FLT3, as well as the coverage at the key myeloproliferative neoplasm mutation sites: JAK2 V617F, JAK2 exon 12, MPL W515K/L and CALR exon 9. 
• Sequencing was performed on a MiSeq^® using a V2 300 bp cartridge (Illumina).

Results

Comparison of the data generated by the 1-day and standard NGS protocols

• Data presented here are from 24* samples that were processed using the enhanced LPK in combination with four haematological panels on an Illumina MiSeq. 
• The quality of the data generated with the 1-day protocol is comparable to the standard 4-hour hybridisation protocol. 
• OGT 1-day protocol generated >85% of the % on-target bases generated with the standard protocol. The % change is consistent for all panel sizes.

The MTC generated is dependent on the size of each panel. Overall, both workflows generated very good coverage. The MTC generated with the 1-day protocol is >80% of the MTC generated with the standard protocol. The % change is consistent for all panels.

All panels meet the following uniformity specifications: >99% of bases covered at >20% of the mean (after de-duplication). This permits the reliable detection of more complex rearrangements (i.e.) indels and ITDs.

Accurate and reproducible variant detection even in heterogeneous samples

The SureSeq Core MPN panel has been validated with samples from the National Institute for Biological Standards and Control (NIBSC) and we have shown the accurate detection of JAK2 V617F is possible down to the 1% Variant Allele Frequency (VAF) level at a de-duplicated read depth of >1000x (Table 2).

Accurate detection of difficult to sequence genes

Mutations in the CEBPA and FLT3 genes are among the most common molecular alterations in AML. Sequencing of the CEBPA gene is often hampered by a repetitive nucleotide sequence and a very high GC-rich content. Genes such as FLT3 ITDs are challenging to target because they are by nature repetitive, can be long and are generally masked in most panel designs.

Accurate detection of deletions

Using the enhanced workflow we were 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).