Mutation screening using formalin-fixed paraffin-embedded tissues: a stratified approach according to DNA quality

Laboratory Investigation; a Journal of Technical Methods and Pathology
Francesco CuccoMing-Qing Du

Abstract

DNA samples from formalin-fixed paraffin-embedded tissues are highly degraded with variable quality, and this imposes a big challenge for targeted sequencing due to false positives, largely caused by PCR errors and cytosine deamination. To eliminate false positives, a common practice is to validate the detected variants by Sanger sequencing or perform targeted sequencing in duplicate. Technically, PCR errors could be removed by molecular barcoding of template DNA prior to amplification as in the HaloPlexHS design. Nonetheless, it is uncertain to what extent variants detected using this approach should be further validated. Here, we addressed this question by correlating variant reproducibility with DNA quality using HaloPlexHS target enrichment and Illumina HiSeq4000, together with an in-house validated variant calling algorithm. The overall sequencing coverage, as shown by analyses of 70 genes in 266 cases of large B-cell lymphoma, was excellent (98%) in DNA samples amenable for PCR of ≥400 bp, but suboptimal (92%) and poor (80%) in those amenable for PCR of 300 bp and 200 bp respectively. By mutation analysis in duplicate in 93 cases, we demonstrated that 20 alternative allele depth (AAD) was an optimal cut-off value for sepa...Continue Reading

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Methods Mentioned

BETA
PCR
deamination
Illumina sequencing
biopsies

Software Mentioned

bwa mem
UnifiedGenotyper
MuTect2
samtools
SNV
Fluidigm Access
SurecallTrimmer
Fluidigm
bedtools
AGeNT LocatIt

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