Sources of erroneous sequences and artifact chimeric reads in next generation sequencing of genomic DNA from...

Area of Science:

• Pathology
• Genomics
• Molecular Biology

Background:

• Formalin-fixed, paraffin-embedded (FFPE) tissues are standard in pathology labs but pose challenges for next-generation sequencing (NGS).
• Nucleic acid damage during FFPE processing can lead to sequencing artifacts, including chimeric reads.
• Strand-split artifact reads (SSARs), which align to both DNA strands, are a significant artifact class in FFPE-derived NGS data.

Purpose of the Study:

• To elucidate the mechanistic basis of SSARs and other chimeric artifacts in FFPE NGS data.
• To develop and validate methods for reducing SSARs and associated sequencing artifacts.
• To establish an analytical approach for quantifying SSARs in NGS datasets.

Main Methods:

• Investigated the alignment patterns of chimeric reads in NGS data from FFPE samples.
• Developed a conceptual framework for SSAR genesis.
• Applied S1 nuclease treatment to remove single-stranded DNA fragments and overhangs.
• Evaluated the impact of S1 nuclease treatment on sequencing bias, error rates, and variant detection.
• Created an analytical method for SSAR quantification.

Main Results:

• A substantial proportion of chimeric reads in FFPE NGS data align to both Watson and Crick strands, identified as SSARs.
• S1 nuclease treatment significantly reduced SSAR levels.
• S1 nuclease treatment also decreased sequence bias, base error rates, and false positive variant calls (copy number and single nucleotide variants).

Conclusions:

• SSARs are a prevalent artifact in FFPE NGS, arising from specific molecular mechanisms.
• S1 nuclease treatment is an effective strategy to mitigate SSARs and improve overall NGS data quality from FFPE samples.
• The developed analytical approach enables accurate quantification of SSARs, facilitating artifact assessment in FFPE-derived NGS studies.