Strategy for Robust Detection of Insertions, Deletions, and Point Mutations in CEBPA, a GC-Rich Content Gene, Using 454 Next-Generation Deep-Sequencing Technology

Abstract

CEBPA mutations are of prognostic relevance in acute myeloid leukemia (AML) and are currently detected using a combination of denaturing high-performance liquid chromatography (DHPLC), gene scan/fragment length analysis, and direct Sanger sequencing. Nextgeneration deep pyrosequencing, principally, allows for the highly sensitive detection of molecular mutations. However, standard 454 chemistry laboratory procedures lack efficient amplification of guaninecytosine (GC)-rich amplicons during the emulsion PCR (emPCR) steps allowing direct massively parallel clonal amplification of PCR products. To solve this problem, we investigated six distinct emPCR conditions. The coding sequence of CEBPA was subdivided into four overlapping amplicons: GC content for amplicon 1, 74%; amplicon 2, 76%; amplicon 3, 77%; and amplicon 4, 69%. A new emPCR condition, improving the standard titanium assay, presents a robust solution to sequence amplicons with a GC content of up to 77%. Moreover, this assay was subsequently tested on a larger independent cohort of 23 AML patients. For each patient, a median of 737 reads was generated (coverage range, 397-fold to 1194-fold) and therefore allowed a robust detection of insertions, deletions, and point mutations. In conclusion, next-generation amplicon sequencing enables the highly sensitive detection of molecular mutations and is a feasible assay for routine assessment of GC-rich content amplicons.

The Interlaboratory RObustness of Next-generation sequencing (IRON) study: a deep sequencing investigation of TET2, CBL and KRAS mutations by an international consortium involving 10 laboratories

“…9,24 In addition, we recently reported that such a technique is applicable to all PCR products. Grossmann et al 25 published a study where this assay was applied to sequence CEBPA, a gene with high GC content. Using a modified PCR setup, amplicons with up to 77% GC content were successfully processed by deep sequencing.…”

A novel hierarchical prognostic model of AML solely based on molecular mutations

“…9,[30][31][32][33] The complete regions of CEBPA and RUNX1 were investigated by 454 deep sequencing (Roche Applied Science) as previously described. 34,35 Furthermore, ASXL1 exon 12 aberrations were investigated by Sanger sequencing. 33 The controversial ASXL1 variant p.Gly646TrpfsX12 was scored as a mutation as we and others have convincing data that this is a somatic mutation (for further details, see supplemental Methods).…”

Genotyping of 25 Leukemia-Associated Genes in a Single Work Flow by Next-Generation Sequencing Technology with Low Amounts of Input Template DNA

“…An alternative emPCR protocol previously established for CEBPA (454 GS FLX Titanium chemistry) uses Additive reagent instead of water in the emPCR master mix. This protocol increases the amount of PCR product in the amplicon library by up to 4-fold (18 ). Beads enriched according to this protocol have also been analyzed on the GS Junior platform.…”