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

Area of Science:

• Genomics and Molecular Biology
• Cancer Genomics
• Next-Generation Sequencing Technologies

Background:

• DNA from formalin-fixed paraffin-embedded (FFPE) tissues is highly degraded, leading to challenges in targeted sequencing.
• False positives in variant detection are common, often due to PCR errors and cytosine deamination in FFPE samples.
• Current validation methods like Sanger sequencing or duplicate sequencing are time-consuming and may not fully address FFPE DNA quality issues.

Purpose of the Study:

• To evaluate the effectiveness of molecular barcoding (HaloPlexHS) in mitigating PCR errors for FFPE DNA.
• To determine the optimal variant calling strategy based on DNA quality and reproducibility.
• To establish a stratified approach for accurate mutation screening using HaloPlexHS and Illumina HiSeq4000.

Main Methods:

• Targeted sequencing using HaloPlexHS enrichment and Illumina HiSeq4000 on 266 large B-cell lymphoma FFPE samples.
• Analysis of sequencing coverage based on DNA fragment length amenable for PCR (≥400 bp, 300 bp, 200 bp).
• Mutation analysis in duplicate for 93 cases to assess variant reproducibility and establish alternative allele depth (AAD) cut-offs.

Main Results:

• Sequencing coverage was excellent (98%) for DNA ≥400 bp, suboptimal (92%) for 300 bp, and poor (80%) for 200 bp.
• An alternative allele depth (AAD) cut-off of 20 demonstrated 97% sensitivity and 100% specificity for reproducible variants in DNA ≥300 bp.
• The HaloPlexHS protocol showed high sensitivity and specificity when cross-validated with a Fluidigm-PCR/Illumina MiSeq protocol.

Conclusions:

• A stratified mutation screening approach based on DNA quality is proposed for HaloPlexHS and Illumina HiSeq4000.
• Good quality DNA (≥400 bp) allows single replicate analysis, with variants at 15-20 AAD needing further validation.
• Suboptimal quality DNA (300 bp) benefits from duplicate analysis, with variants >15 AAD considered true genetic changes.