Contributions
Abstract: PB1711
Type: Publication Only
Background
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 inability to sequence certain genes. These genes can harbour key mutations so it is desirable to ensure suitable sequencing coverage is obtained. These genes amongst others include: CALR exon 9 insertions and deletions (up to 52 bp), CEBPA single nucleotide variants (SNVs) and FLT3 Internal Tandem Duplications (ITDs) and SNVs. Each of these regions contain certain challenging DNA sequences that can impact the quality of the data generated, e.g. large indels and low complexity regions (CALR), high GC content (75% on average for the whole gene with specific regions at 100%) and repetitive regions (CEBPA), and complex repetitive elements (FLT3).
Aims
To determine whether a hybridisation-based enrichment approach overcomes the difficulties associated with these genes, and permits the generation of high quality (sufficient de-duplicated depth) data to allow these targets to be accurately interrogated.
Methods
We utilised a hybridisation-based enrichment approach for library preparation in combination with a SureSeq myPanel™ NGS Custom AML panel. The library was then sequenced using a 2x150 bp read length protocol on an Illumina MiSeq®.
Results
Here we present the coverage and variants generated from numerous research samples for each of these difficult to sequence genes. The results clearly show that this approach can reliably detect and accurately size (including low allele frequency) insertions and deletions of up 52 bp in CALR (exon 9), SNVs and deletions in CEBPA with a de-duplicated depth in excess of 2000x as well as ITDs of between 24 and 201 bp in FLT3.
Conclusion
This approach is suitable for the analysis by NGS of these difficult genes and therefore removes the requirements for supplementary approaches to analyse these difficult genes, such as Sanger sequencing (CEBPA) and fragment analysis (CALR and FLT3).
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): Acute Myeloid Leukemia, Cancer, Myeloid
Abstract: PB1711
Type: Publication Only
Background
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 inability to sequence certain genes. These genes can harbour key mutations so it is desirable to ensure suitable sequencing coverage is obtained. These genes amongst others include: CALR exon 9 insertions and deletions (up to 52 bp), CEBPA single nucleotide variants (SNVs) and FLT3 Internal Tandem Duplications (ITDs) and SNVs. Each of these regions contain certain challenging DNA sequences that can impact the quality of the data generated, e.g. large indels and low complexity regions (CALR), high GC content (75% on average for the whole gene with specific regions at 100%) and repetitive regions (CEBPA), and complex repetitive elements (FLT3).
Aims
To determine whether a hybridisation-based enrichment approach overcomes the difficulties associated with these genes, and permits the generation of high quality (sufficient de-duplicated depth) data to allow these targets to be accurately interrogated.
Methods
We utilised a hybridisation-based enrichment approach for library preparation in combination with a SureSeq myPanel™ NGS Custom AML panel. The library was then sequenced using a 2x150 bp read length protocol on an Illumina MiSeq®.
Results
Here we present the coverage and variants generated from numerous research samples for each of these difficult to sequence genes. The results clearly show that this approach can reliably detect and accurately size (including low allele frequency) insertions and deletions of up 52 bp in CALR (exon 9), SNVs and deletions in CEBPA with a de-duplicated depth in excess of 2000x as well as ITDs of between 24 and 201 bp in FLT3.
Conclusion
This approach is suitable for the analysis by NGS of these difficult genes and therefore removes the requirements for supplementary approaches to analyse these difficult genes, such as Sanger sequencing (CEBPA) and fragment analysis (CALR and FLT3).
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): Acute Myeloid Leukemia, Cancer, Myeloid