Bioreactor virome metagenomics sequencing using DNA spike-ins

Area of Science:

• Environmental virology
• Metagenomics
• Bioinformatics

Background:

• Next Generation Sequencing (NGS) advances viral identification in natural environments.
• Limited viral DNA yields hinder bioinformatic analysis.
• Multiple Displacement Amplification (MDA) is a common but potentially biased method for amplifying low DNA amounts.

Purpose of the Study:

• To evaluate DNA-spiking as an alternative to MDA for viral metagenomic sequencing.
• To assess the bias introduced by MDA compared to DNA-spiking.
• To determine the efficiency of viral genome recovery using both methods.

Main Methods:

• Sequencing of bioreactor viromes using Ion Torrent technology.
• Comparison of DNA-spiking and MDA amplification protocols.
• Bioinformatic analysis including 16S rRNA gene read removal and genome assembly.
• Quantification of viral genome coverage using mapped reads.

Main Results:

• MDA amplification introduced a bias towards lower GC content in viral DNA.
• DNA-spiking resulted in significantly higher viral genome recovery (97.9%-100%) compared to MDA (6.3%-100%).
• Six viral genomes were successfully assembled from both methods, but DNA-spiking enabled near-complete retrieval.

Conclusions:

• DNA-spiking is a simple, inexpensive, and low-bias alternative for metagenomic sequencing of low-DNA samples.
• This method enhances viral genome recovery and accuracy in environmental virology studies.
• DNA-spiking overcomes the limitations associated with MDA bias in viral metagenomics.