Comprehensive de novo mutation discovery with HiFi long-read sequencing

Area of Science:

• Genomics and Genetic Variation Analysis
• Next-Generation Sequencing Technologies
• Human Genetics and Disease Research

Background:

• Long-read sequencing (LRS) excels at identifying structural variants (SVs).
• High error rates in traditional LRS hindered small variant detection (substitutions, indels < 20 bp).
• PacBio HiFi sequencing improves LRS accuracy for small variant detection.

Purpose of the Study:

• To evaluate PacBio HiFi LRS for detecting de novo mutations (DNMs) of all types.
• To assess the accuracy of HiFi LRS compared to short-read sequencing (SRS).
• To determine the utility of HiFi LRS in identifying disease-causing genetic variations.

Main Methods:

• Sequenced eight parent-child trios using PacBio HiFi LRS (~30x coverage) and Illumina SRS (~50x coverage).
• Called de novo substitutions, small indels, short tandem repeats (STRs), and SVs from both datasets.
• Compared variant calls between LRS and SRS; phased small DNMs to determine parent-of-origin.

Main Results:

• HiFi LRS identified 672 de novo substitutions/indels and 24 SVs; SRS identified 859 substitutions/indels and 1 SV.
• Concordance for small variants was 92% (HiFi LRS) vs. 85% (SRS); SV concordance was 4% (HiFi LRS) vs. 100% (SRS).
• HiFi LRS achieved 96% DNM phasing accuracy, significantly higher than SRS (20%); validated 52.6% of LRS-unique SVs as true de novo events.

Conclusions:

• PacBio HiFi LRS provides a comprehensive variant dataset, enabling accurate calling of substitutions, indels, STRs, and SVs.
• HiFi LRS facilitates sensitive de novo mutation calling across all variant types.
• The phasing capability of HiFi LRS aids in distinguishing true positive from false positive DNMs, crucial for genetic disease research.