This is the accepted manuscript. It is currently embargoed pending publication. ; High throughput somatic mutation screening using formalin-fixed paraffin-embedded (FFPE) tissues is a major challenge due to a lack of established methodology 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 optimised against a series of somatic mutations (147 substitutions, 12 indels ranging 1-33bp ) in 7 genes, previously detected by Sanger sequencing of DNA from 163 FFPE lymphoma biopsies. The optimised experimental protocol and variant calling algorithm were further ascertained in two separate experiments by including the 7 genes as a part of larger gene panels (22 or 15 genes) using FFPE and high molecular weight lymphoma DNAs respectively. We showed that most false positives were due to DNA degradation, deamination and Taq polymerase errors, but they were non-reproducible and could be efficiently eliminated by duplicate experiments. A small fraction of false positives 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. ; 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.