Validation and implementation of a custom 21-gene panel next-generation sequencing assay for myeloid neoplasms

Rapid and reliable mutational analysis of myeloid neoplasms is increasingly important for diagnostic, prognostic and therapeutic reasons. In this article we describe the development and validation of a custom next-generation sequencing (NGS) assay that reliably tests across a broad range of myeloid neoplasms, including AML, MDS, and myeloproliferative neoplasms. The lllumina TruSeq Custom Amplicon panel was designed to detect variants in 21 genes. The validation protocol included sequencing of cell lines (n=3) and patient samples (n=36) on an Illumina MiSeq platform. A read depth ≥100x was observed for &gt;97% of targeted bases. After filtering for artifacts, a specificity of 100% was obtained. A detection limit of ≤5% was observed for variants present in cell lines. On average two reportable variants were present in samples from patients with a myeloid neoplasm. In conclusion, the custom NGS assay provides an adequate routine assay for genetic analysis of variants present in myeloid neoplasms. Practical considerations on choice of targeted genes, type of assay and method of data analysis are provided in this report.</p

Validation and implementation of a custom 21-gene panel next-generation sequencing assay for myeloid neoplasms

Rapid and reliable mutational analysis of myeloid neoplasms is increasingly important for diagnostic, prognostic and therapeutic reasons. In this article we describe the development and validation of a custom next-generation sequencing (NGS) assay that reliably tests across a broad range of myeloid neoplasms, including AML, MDS, and myeloproliferative neoplasms. The lllumina TruSeq Custom Amplicon panel was designed to detect variants in 21 genes. The validation protocol included sequencing of cell lines (n=3) and patient samples (n=36) on an Illumina MiSeq platform. A read depth ≥100x was observed for &gt;97% of targeted bases. After filtering for artifacts, a specificity of 100% was obtained. A detection limit of ≤5% was observed for variants present in cell lines. On average two reportable variants were present in samples from patients with a myeloid neoplasm. In conclusion, the custom NGS assay provides an adequate routine assay for genetic analysis of variants present in myeloid neoplasms. Practical considerations on choice of targeted genes, type of assay and method of data analysis are provided in this report.</p

Validation and implementation of a custom 21-gene panel next-generation sequencing assay for myeloid neoplasms

Rapid and reliable mutational analysis of myeloid neoplasms is increasingly important for diagnostic, prognostic and therapeutic reasons. In this article we describe the development and validation of a custom next-generation sequencing (NGS) assay that reliably tests across a broad range of myeloid neoplasms, including AML, MDS, and myeloproliferative neoplasms. The lllumina TruSeq Custom Amplicon panel was designed to detect variants in 21 genes. The validation protocol included sequencing of cell lines (n=3) and patient samples (n=36) on an Illumina MiSeq platform. A read depth ≥100x was observed for &gt;97% of targeted bases. After filtering for artifacts, a specificity of 100% was obtained. A detection limit of ≤5% was observed for variants present in cell lines. On average two reportable variants were present in samples from patients with a myeloid neoplasm. In conclusion, the custom NGS assay provides an adequate routine assay for genetic analysis of variants present in myeloid neoplasms. Practical considerations on choice of targeted genes, type of assay and method of data analysis are provided in this report.</p

Antineutrophil cytoplasmic antibodies to proteinase 3 in Wegener's granulomatosis:Epitope analysis using synthetic peptides

Background. Antineutrophil cytoplasmic antibodies (ANCA) to proteinase 3 (PR3) are strongly associated with Wegener's granulomatosis (WG) and are thought to be involved in its pathogenesis. Levels of PR3-ANCA do not always correspond to clinical disease activity nor to functional effects of these antibodies in vitro, suggesting differences in epitope specificity. To define relevant epitopes for PR3-ANCA, sera of WG patients were analyzed on their reactivity to linear peptides of PR3. Methods. Fifty linear peptides of 15 amino acids in length with an overlap of 10 aa spanning the entire PR3 sequence were synthesized. Sera of 27 WG patients with active disease and 27 age- and sex-matched healthy controls, eight anti-PR3 monoclonal antibodies (mAbs), and a rabbit anti-PR3 serum were tested by enzyme-linked immunosorbent assay for reactivity to PR3 peptides. Results. Rabbit anti-PR3 serum recognized three distinct peptide areas, whereas none of the anti-PR3 mAbs bound PR3 peptides. Sera of both WG patients and healthy controls recognized a restricted number of PR3 peptides. Four of these peptide areas were recognized significantly more strongly by WG sera than by control sera. Sera drawn at the initial presentation of WG mainly recognized these peptides. Two of the recognized peptide areas were located near the active center of PR3. Conclusion. A restricted number of epitope areas of PR3 are recognized both by WG patient sera and control sera. Four peptide areas were bound stronger by sera of WG patients at initial presentation than by healthy controls