HaloPlex Targeted Resequencing for Mutation Detection in Clinical Formalin-Fixed, Paraffin-Embedded Tumor Samples.
In recent years, the advent of massively parallel next-generation sequencing technologies has enabled substantial advances in the study of human diseases. Combined with targeted DNA enrichment methods, high sequence coverage can be obtained for different genes simultaneously at a reduced cost per sample, creating unique opportunities for clinical cancer diagnostics. However, the formalin-fixed, paraffin-embedded (FFPE) process of tissue samples, routinely used in pathology departments, results in DNA fragmentation and nucleotide modifications that introduce a number of technical challenges for downstream biomolecular analyses. We evaluated the HaloPlex target enrichment system for somatic mutation detection in 80 tissue fractions derived from 20 clinical cancer cases with paired tumor and normal tissue available in both FFPE and fresh-frozen format. Several modifications to the standard method were introduced, including a reduced target fragment length and two strand capturing. We found that FFPE material can be used for HaloPlex-based target enrichment and next-generation sequencing, even when starting from small amounts of DNA. By specifically capturing both strands for each target fragment, we were able to reduce the number of false-positive errors caused by FFPE-induced artifacts and lower the detection limit for somatic mutations. We believe that the HaloPlex method presented here will be broadly applicable as a tool for somatic mutation detection in clinical cancer settings.
Department of Immunology Genetics and Pathology, Uppsala University, Science for Life Laboratory, Uppsala, Sweden.
Lotte N J
LN
Moens
Department of Immunology Genetics and Pathology, Uppsala University, Science for Life Laboratory, Uppsala, Sweden.
Elin
E
Falk-Sörqvist
Department of Immunology Genetics and Pathology, Uppsala University, Science for Life Laboratory, Uppsala, Sweden.
Viktor
V
Ljungström
Department of Immunology Genetics and Pathology, Uppsala University, Science for Life Laboratory, Uppsala, Sweden.
Johanna
J
Mattsson
Department of Immunology Genetics and Pathology, Uppsala University, Science for Life Laboratory, Uppsala, Sweden.
Magnus
M
Sundström
Department of Immunology Genetics and Pathology, Uppsala University, Science for Life Laboratory, Uppsala, Sweden.
Linnéa
L
La Fleur
Department of Immunology Genetics and Pathology, Uppsala University, Science for Life Laboratory, Uppsala, Sweden.
Lucy
L
Mathot
Department of Immunology Genetics and Pathology, Uppsala University, Science for Life Laboratory, Uppsala, Sweden.
Patrick
P
Micke
Department of Immunology Genetics and Pathology, Uppsala University, Science for Life Laboratory, Uppsala, Sweden; Department of Biochemistry and Biophysics, Stockholm University, Science for Life Laboratory, Stockholm, Sweden. Electronic address: mats.nilsson@scilifelab.se.
Mats
M
Nilsson
Department of Immunology Genetics and Pathology, Uppsala University, Science for Life Laboratory, Uppsala, Sweden. Electronic address: johan.botling@igp.uu.se.
Johan
J
Botling
D004247:DNA / Q000235:genetics; D005557:Formaldehyde / Q000737:chemistry*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009154:Mutation / Q000235:genetics*; D009369:Neoplasms / Q000235:genetics*; D010232:Paraffin / Q000737:chemistry*; D016612:Paraffin Embedding / Q000379:methods; D016707:Tissue Fixation / Q000379:methods
D005557:Formaldehyde; D010232:Paraffin; D004247:DNA