Enhanced whole exome sequencing by higher DNA insert lengths

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Whole exome sequencing (WES) is vital for variant calling and copy number variation (CNV) analysis.
• Uneven read coverage in WES, caused by GC-rich regions and off-target enrichment, hinders accurate genetic analysis.
• Optimizing WES protocols is essential for reliable downstream applications.

Purpose of the Study:

• To investigate the impact of DNA insert size on WES coverage uniformity.
• To identify factors contributing to uneven read coverage in exome sequencing.
• To improve the accuracy of variant calling and CNV analyses through optimized WES.

Main Methods:

• Applied Agilent SureSelectXT exome capture and Illumina sequencing (2 × 101 bp paired-end mode).
• Sheared 12 genomic DNA samples to two insert sizes: 130 bp and 170 bp.
• Analyzed coverage evenness and performed mutation analysis on isogenic subclones.

Main Results:

• Samples with 170 bp inserts exhibited more uniform read coverage compared to 130 bp inserts, despite lower mean coverage.
• Merging overlapping paired-end reads significantly improved coverage evenness.
• The false negative rate for mutation detection was nearly double in 130 bp samples compared to 170 bp samples.

Conclusions:

• Generating longer DNA inserts is a key strategy for achieving uniform WES coverage.
• Enhanced coverage evenness improves the reliability of variant calling and CNV analyses.
• Optimizing insert size and utilizing overlapping reads can increase effective sequencing yield and data quality.