Abstract
The clinical sequencing of tumors is usually performed on formalin-fixed, paraffin-embedded samples and results in many sequencing errors. We identified that most of these errors are detected in chimeric reads caused by single-strand DNA molecules with microhomology. During the end-repair step of library preparation, mutations are introduced by the mis-annealing of two single-strand DNA molecules comprising homologous sequences. The mutated bases are distributed unevenly near the ends in the individual reads. Our filtering pipeline, MicroSEC, focuses on the uneven distribution of mutations in each read and removes the sequencing errors in formalin-fixed, paraffin-embedded samples without over-eliminating the mutations detected also in fresh frozen samples. Amplicon-based sequencing using 97 mutations confirmed that the sensitivity and specificity of MicroSEC were 97% (95% confidence interval: 82–100%) and 96% (95% confidence interval: 88–99%), respectively. Our pipeline will increase the reliability of the clinical sequencing and advance the cancer research using formalin-fixed, paraffin-embedded samples.
Highlights
The clinical sequencing of tumors is usually performed on formalin-fixed, paraffin-embedded samples and results in many sequencing errors
microhomology-induced chimeric read (MICR) are formed during the endrepair step of library preparation for clinical sequencing, wherein a considerable amount of extracted DNA is denatured to single-stranded DNA (ssDNA) and behaves as site-directed mutagenesis polymerase chain reaction (PCR) primers[15]
We examined the performance of MicroSEC in distinguishing true mutations from formalin-fixed and paraffin-embedded (FFPE) artifacts with our custom‐made multigene panel test, “Todai OncoPanel”[2]
Summary
The clinical sequencing of tumors is usually performed on formalin-fixed, paraffin-embedded samples and results in many sequencing errors. MicroSEC, focuses on the uneven distribution of mutations in each read and removes the sequencing errors in formalin-fixed, paraffin-embedded samples without overeliminating the mutations detected in fresh frozen samples. Based on our theory that artifacts are derived from ssDNA-annealing, we have developed a MICRoriginating Sequence Error Cleaning pipeline (MicroSEC), a post hoc filtering pipeline to predict whether a given mutation is an MICR-derived error. This pipeline allows the processing of thousands of mutations of target sequencing data within hours on a standard PC with 16 gigabytes of memory. MicroSEC requires a list of mutations and corresponding BAM files, rather than FASTQ files as it uses the positional bias of reads mapped against mutations
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