title: 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. Next-generation deep pyrosequencing, principally, allows for the highly sensitive detection of molecular mutations. However, standard 454 chemistry laboratory procedures lack efficient amplification of guanine-cytosine (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.
authors.affiliation: MLL Munich Leukemia Laboratory, Munich, Germany.
authors.forename: Vera
authors.initials: V
authors.lastname: Grossmann
authors.forename: Susanne
authors.initials: S
authors.lastname: Schnittger
authors.forename: Sonja
authors.initials: S
authors.lastname: Schindela
authors.forename: Hans-Ulrich
authors.initials: HU
authors.lastname: Klein
authors.forename: Christiane
authors.initials: C
authors.lastname: Eder
authors.forename: Martin
authors.initials: M
authors.lastname: Dugas
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Kern
authors.forename: Torsten
authors.initials: T
authors.lastname: Haferlach
authors.forename: Claudia
authors.initials: C
authors.lastname: Haferlach
authors.forename: Alexander
authors.initials: A
authors.lastname: Kohlmann
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001482:Base Composition; D001483:Base Sequence; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000175:diagnosis / Q000235:genetics; D008297:Male; D008875:Middle Aged; D016254:Mutagenesis, Insertional; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011379:Prognosis; D017384:Sequence Deletion
chemical_list: D022763:CCAAT-Enhancer-Binding Protein-alpha