Targeted single molecule sequencing methodology for ovarian hyperstimulation syndrome

Area of Science:

• Genomics and Bioinformatics
• Reproductive Medicine
• Molecular Biology

Background:

• Next-generation sequencing faces challenges with cost and short read lengths, hindering assembly and accuracy.
• Targeted capture enrichment using single molecule sequencing (SMS) offers potential improvements for sequence assembly and base-call accuracy.
• Few studies have applied these advances in translational research and clinical testing.

Purpose of the Study:

• To develop and validate a targeted single molecule sequencing (T-SMS) panel for genes associated with ovarian response to controlled ovarian hyperstimulation (COH).
• To assess the feasibility and effectiveness of combining emulsion PCR with SMS for long-read sequencing in human DNA samples.
• To establish a novel NGS panel for biomarker discovery in OHSS.

Main Methods:

• Developed a T-SMS panel using droplet-based multiplex PCR for 1kb amplicons targeting 44 loci.
• Performed SMS using Pacific Biosciences SMRT sequencing, achieving long average read lengths (1178 nt raw, 3200 nt CCS).
• Utilized Genome Analysis Toolkit and wANNOVAR for variant analysis, confirming 94.7% of functional variants with Sanger sequencing.

Main Results:

• Achieved high target enrichment (99.8% coverage) and long read lengths with high accuracy (97% CCS accuracy).
• Demonstrated effective detection of zygosity and coverage in GC-rich regions using long DNA fragments.
• Identified and verified a non-synonymous LHCGR variant in severe OHSS cases.

Conclusions:

• Combining emulsion PCR and SMRT DNA sequencing enhances T-SMS depth of coverage and simplifies sequence assembly.
• This study presents the first report of combining emulsion PCR and T-SMS for long-read sequencing in human DNA.
• The developed NGS panel shows promise for biomarker discovery in OHSS.