title: Somatic Mutation Screening Using Archival Formalin-Fixed, Paraffin-Embedded Tissues by Fluidigm Multiplex PCR and Illumina Sequencing.
abstract: High-throughput somatic mutation screening using FFPE tissues is a major challenge because of a lack of established methods and validated variant calling algorithms. We aimed to develop a targeted sequencing protocol by Fluidigm multiplex PCR and Illumina sequencing and to establish a companion variant calling algorithm. The experimental protocol and variant calling algorithm were first developed and optimized against a series of somatic mutations (147 substitutions, 12 indels ranging from 1 to 33 bp) in seven genes, previously detected by Sanger sequencing of DNA from 163 FFPE lymphoma biopsy specimens. The optimized experimental protocol and variant calling algorithm were further ascertained in two separate experiments by including the seven genes as a part of larger gene panels (22 or 13 genes) using FFPE and high-molecular-weight lymphoma DNAs, respectively. We found that most false-positive variants were due to DNA degradation, deamination, and Taq polymerase errors, but they were nonreproducible and could be efficiently eliminated by duplicate experiments. A small fraction of false-positive variants appeared in duplicate, but they were at low alternative allele frequencies and could be separated from mutations when appropriate threshold value was used. In conclusion, we established a robust practical approach for high-throughput mutation screening using archival FFPE tissues.
authors.affiliation: Division of Molecular Histopathology, Department of Pathology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Ming
authors.initials: M
authors.lastname: Wang
authors.affiliation: Division of Molecular Histopathology, Department of Pathology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Leire
authors.initials: L
authors.lastname: Escudero-Ibarz
authors.affiliation: Division of Molecular Histopathology, Department of Pathology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Sarah
authors.initials: S
authors.lastname: Moody
authors.affiliation: Division of Molecular Histopathology, Department of Pathology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Naiyan
authors.initials: N
authors.lastname: Zeng
authors.affiliation: Division of Molecular Histopathology, Department of Pathology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Clipson
authors.affiliation: Division of Molecular Histopathology, Department of Pathology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Yuanxue
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Division of Molecular Histopathology, Department of Pathology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Xuemin
authors.initials: X
authors.lastname: Xue
authors.affiliation: Division of Molecular Histopathology, Department of Pathology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Nicholas F
authors.initials: NF
authors.lastname: Grigoropoulos
authors.affiliation: Haematological Malignancy Diagnostic Service, St. James's Institute of Oncology, Leeds, United Kingdom.
authors.forename: Sharon
authors.initials: S
authors.lastname: Barrans
authors.affiliation: Haematological Malignancy Diagnostic Service, St. James's Institute of Oncology, Leeds, United Kingdom.
authors.forename: Lisa
authors.initials: L
authors.lastname: Worrillow
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Tim
authors.initials: T
authors.lastname: Forshew
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Jing
authors.initials: J
authors.lastname: Su
authors.affiliation: Division of Virology, Department of Pathology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Andrew
authors.initials: A
authors.lastname: Firth
authors.affiliation: Department of Molecular Genetics, Addenbrooke's Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom.
authors.forename: Howard
authors.initials: H
authors.lastname: Martin
authors.affiliation: Haematological Malignancy Diagnostic Service, St. James's Institute of Oncology, Leeds, United Kingdom.
authors.forename: Andrew
authors.initials: A
authors.lastname: Jack
authors.affiliation: Department of Molecular Genetics, Addenbrooke's Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom.
authors.forename: Kim
authors.initials: K
authors.lastname: Brugger
authors.affiliation: Division of Molecular Histopathology, Department of Pathology, University of Cambridge, Cambridge, United Kingdom. Electronic address: mqd20@cam.ac.uk.
authors.forename: Ming-Qing
authors.initials: MQ
authors.lastname: Du
mesh_terms: D000465:Algorithms; D019943:Amino Acid Substitution; D004252:DNA Mutational Analysis / Q000295:instrumentation / Q000379:methods; D005189:False Positive Reactions; D005557:Formaldehyde / Q000737:chemistry*; D005820:Genetic Testing / Q000295:instrumentation / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D054643:INDEL Mutation; D008223:Lymphoma / Q000235:genetics / Q000473:pathology; D060885:Multiplex Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods; D016612:Paraffin Embedding / Q000379:methods; D016707:Tissue Fixation / Q000379:methods
chemical_list: D005557:Formaldehyde