Repository logo
 

Somatic Mutation Screening Using Archival Formalin-Fixed, Paraffin-Embedded Tissues by Fluidigm Multiplex PCR and Illumina Sequencing.

Accepted version
Peer-reviewed

Repository DOI


Type

Article

Change log

Authors

Wang, Ming 
Escudero-Ibarz, Leire 
Moody, Sarah 
Zeng, Naiyan 
Clipson, Alexandra 

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.

Description

Keywords

Algorithms, Amino Acid Substitution, DNA Mutational Analysis, False Positive Reactions, Formaldehyde, Genetic Testing, High-Throughput Nucleotide Sequencing, Humans, INDEL Mutation, Lymphoma, Multiplex Polymerase Chain Reaction, Paraffin Embedding, Tissue Fixation

Journal Title

J Mol Diagn

Conference Name

Journal ISSN

1525-1578
1943-7811

Volume Title

17

Publisher

Elsevier BV
Sponsorship
The research was supported by grants [LLR10006, LLR13006] from Leukaemia & Lymphoma Research, U.K. and Kay Kendal Leukaemia Fund. SM is a PhD student supported by Medical Research Council, Department of Pathology, University of Cambridge, and Addenbrooke’s Charitable Trust. LEI is a PhD student supported by the Pathological Society of UK & Ireland. XX was supported by a visiting fellowship from the China Scholarship Council, Ministry of Education, P.R. China. NG was supported by a Kay Kendal Leukaemia Fund [KKL649] and an Addenbrooke’s Charitable Trust fellowship.