SLAI: a novel method for filtering stem-loop artifact variants in enzyme-fragmented FFPE samples

Compared to traditional sonication-based DNA fragmentation, enzyme-based DNA fragmentation has many advantages: no upfront equipment investment, fast and efficient processing, easy for automation, etc. However, enzyme-fragmentation can introduce artifacts into DNA sequences and complicates somatic variant calling from formalin-fixed paraffin-embedded (FFPE) samples. Existing bioinformatics algorithms struggle to identify and remove these artifacts, leading to false somatic variants. This study introduces a novel filtering algorithm, Stem-Loop Artifact Identifier (SLAI), to address this problem. By understanding the molecular mechanism that generates the artifact reads in the enzymatic fragmentation process, SLAI identifies and eliminates artifact-associated reads, a common byproduct of enzymatic fragmentation, thereby reducing false positives. We validated SLAI in two batches of samples, one normal-quality batch and the other low-quality batch, using a 769-gene cancer panel, demonstrating its ability to improve variant accuracy in enzyme-fragmented samples, esp. for low variant allele frequency (VAF) variants, on par with that of sonication-fragmented samples. SLAI effectively reduces false positive somatic variants introduced by enzyme-based DNA fragmentation, improving the accuracy of variant calling, particularly in low-quality FFPE samples. This enhances the reliability of genomic studies utilizing enzyme-based fragmentation methods.