Interpreting and de-noising genetically engineered barcodes in a DNA virus.

Area of Science:

• Virology
• Genomics
• Molecular Biology

Background:

• Nucleic acid barcoding offers potential for pathogen genome tracking.
• Challenges include barcode-induced fitness changes, mutations, and construction errors.

Purpose of the Study:

• To generate and analyze randomized nucleic acid barcodes in a viral genome.
• To develop a robust post-sequencing method for accurate barcode identification.

Main Methods:

• Generation of ~5,000 randomized barcodes in murine polyomavirus.
• Illumina NextSeq sequencing of barcoded viral libraries.
• Development of a read-clustering method using control viral genomes.

Main Results:

• Observed significant variation in sequencing reads beyond expected barcode numbers.
• Identified library processing and sequencing errors as sources of variation.
• Successfully clustered erroneous reads to identify true viral genome barcodes.

Conclusions:

• Accurate interpretation of randomized nucleic acid barcodes in pathogens is complex.
• A validated post-sequencing method can mitigate sequencing and processing errors.
• Findings offer insights for barcoding applications in microbial systems.