The accurate detection by next-generation sequencing (NGS) of difficult to sequence genes (CALR, CEBPA, FLT3) associated with myeloid disorders using a hybridisation-based enrichment approach

Monday 4 September 2017

Lyudmila Georgieva, Ezam Uddin, Jacqueline Chan and Graham Speight

Presented at CGC 2017, this poster highlights the excellent uniformity of coverage obtained from the hybridisation-based enrichment using the SureSeq myPanel NGS Custom AML Panel.

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The application of short read NGS for research into myeloid disorders such as myeloproliferative neoplasms (MPNs) and acute myeloid leukaemia (AML) has been hampered by the difficulty in sequencing certain genes.

Amongst the difficult to sequence genes are calreticulin (CALR), CCAAT/enhancer binding protein alpha (CEBPA) and fms-related tyrosine kinase 3 (FLT3).

The development of robust assays for CEBPA mutation testing is challenging due to the high GC content of the gene (75% in the coding region), the presence of a trinucleotide repeat region, the complexity of the mutations, and the frequent occurrence of mutations in mononucleotide repeats. Genes such as FLT3 are challenging to target because they contain complex repetitive elements that can be long and are generally masked in most panel designs. CALR sequencing is challenging due to the presence of low complexity regions making the detection of insertions and deletions difficult.

Mutations in the CEBPA and FLT3 genes are among the most common molecular alterations in AML. Two mutations on separate alleles of CEBPA with a specific combination of an N-terminal frameshift mutation on one allele and a C-terminal in frame mutation on the other allele are important for prognosis1,2. The prevalence of an internal tandem duplication (ITD) of the juxtamembrane domain-coding sequence and a missense mutation of D835 within the kinase domain of the FLT3 gene occurs in 15-35% and 5-10% of adults with AML, respectively3 . A quarter of patients with essential thrombocythemia or primary myelofibrosis carry a driver mutation of CALR. A 52-bp deletion (type 1) and a 5-bp insertion (type 2 mutation) are the most frequent variants4 .


The SureSeq™ hybridisation-based approach was used throughout this study; the workflow of this is outlined below in Figure 1.

OGT SureSeq NGS hybridisation workflowFigure 1: OGT SureSeq workflow. The SureSeq workflow allows users to go from extracted DNA to sequencer in 1.5 days with minimal handling time.

Panel content

We have utilised a hybridisation-based enrichment approach in combination with a SureSeq myPanel™ NGS Custom AML Panel.

Study Design

The panel was used to confirm variants in research samples* containing variants in each of these difficult to sequence genes. Sequencing was conducted on a MiSeq® using a V2 300 bp cartridge (Illumina).

Hybridisation-based enrichment generates highly uniform coverage of key targets

  • Heterogeneous cancer samples pose significant challenges as alleles are likely to be present at a lower fraction than what would be expected for germline variants. Samples also typically contain a mixture of cancer and normal cells; moreover, the tumour may 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. 
  • Uniformity of coverage is a useful value with which to compare this distribution and can be expressed as the percentage of target bases that have greater than 20% of the mean coverage. 
  • The SureSeq myPanel NGS Custom AML Panel in our hands met the following uniformity specifications: >99% of bases covered at >20% of the mean (after removal of PCR duplicates). This permits the reliable detection of more complex rearrangements (i.e.) indels and ITDs.

Accurate detection of variants in difficult to sequence genes

Excellent uniformity of coverage of the CEBPA geneFigure 2: Excellent uniformity of coverage of the CEBPA gene (average depth ~2000x). Depth of coverage per base (grey). GC percentage (red). Repeat regions and GCrich regions (pink).

Detection of insertions and deletions in CEBPAFigure 3: Detection of insertions and deletions in CEBPA.

Detection of FLT3-ITDs with different sizes.Figure 4: Detection of FLT3-ITDs with different sizes. Wild-type sample (bottom panel).

Detection of insertions and deletions in exon 9 of CALRFigure 5: Detection of insertions and deletions in exon 9 of CALR. Wildtype sample (panel A) is compared to a 25bp (panel B), 34bp (panel C), 52bp somatic deletions (panel D) and 5bp insertion (panel E).


  • Excellent uniformity of coverage was obtained from the hybridisation-based enrichment using the SureSeq myPanel NGS Custom AML Panel. 
  • This approach is a robust method that can be used successfully in sequencing difficult genes (including CEBPA, FLT3 and CALR). 
  • High levels of uniformity are maintained across all genes permitting the reliable detection and accurate sizing (including low allele frequency) of key CALR variants (including 52 bp deletions and 5 bp insertions), SNVs, indels and other in frame variants throughout CEBPA with a de-duplicated depth of greater than 2000x as well as ITDs of between 24 and 201 bp in FLT3
  • This approach may therefore remove the requirements for supplementary approaches to analyse these difficult genes, such as Sanger sequencing (CEBPA) and fragment analysis (CALR and FLT3).


  1. Behdad A ,et al. A clinical grade sequencing-based assay for CEBPA mutation testing: report of a large series of myeloid neoplasms. J Mol Diagn. 2015 Jan;17(1):76-84. 
  2. Spencer DH, et al. Detection of FLT3 internal tandem duplication in targeted, short-read-length, next-generation sequencing data. J Mol Diagn. 2013 Jan;15(1):81-93. 
  3. Dufour A, et al. Acute myeloid leukemia with biallelic CEBPA gene mutations and normal karyotype represents a distinct genetic entity associated with a favorable clinical outcome. J Clin Oncol. 2010 Feb 1;28(4):570-7. 
  4. Pietra D, et al. Differential clinical effects of different mutation subtypes in CALR-mutant myeloproliferative neoplasms. Leukemia. 2016 Feb; 30(2): 431–438.


*Research sample provided by the National Genetics Reference Laboratories - Wessex, University of Virginia, University of Newcastle and UK NEQAS.

SureSeq: For research use only. Not for use in diagnostic procedures.

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