Low-host double MDA workflow for uncultured ASFV positive blood and serum sample sequencing

Area of Science:

• Virology
• Genomics
• Molecular Biology

Background:

• African swine fever (ASF) is a severe, contagious disease caused by the African swine fever virus (ASFV).
• Whole-genome sequencing of ASFV is crucial for understanding viral evolution, transmission, and mutation.
• Analyzing uncultured samples is challenging due to high levels of contaminating host DNA, impacting sequencing efficiency.

Purpose of the Study:

• To develop an effective strategy for reducing background DNA in uncultured ASFV samples.
• To optimize a workflow compatible with next-generation sequencing (NGS) and targeted sequencing (TGS) technologies.
• To enable efficient whole-genome sequencing of ASFV from challenging clinical specimens.

Main Methods:

• An improved C18 spacer Multiple Displacement Amplification (MDA) combined with a host DNA exhaustion strategy was employed.
• The workflow was designed to remove non-ASFV DNA, thereby enriching for viral genetic material.
• Developed custom software for real-time base calling and analysis of ASFV TGS sequencing reads on a cloud server.

Main Results:

• Successfully sequenced two uncultured ASFV-positive samples using the developed workflow.
• Demonstrated a significant reduction in the percentage of background DNA compared to conventional methods.
• The new method proved effective for both NGS and TGS platforms.

Conclusions:

• The improved C18 spacer MDA-combined host DNA exhaustion strategy is highly effective for ASFV whole-genome sequencing from uncultured samples.
• This approach conserves sequencing resources and improves data quality.
• The integrated software facilitates rapid analysis of sequencing data, aiding in ASFV surveillance and control efforts.