Metagenomic sequencing of DNA viruses in cystic fibrosis sputum. human metagenome

In this study, metagenomic sequencing was used to explore the viral diversity of sputum from a cystic fibrosis (CF) patient. Bacteriophages were isolated from a sputum sample using a sequential filtration technique with DNase treatment. Phage DNA was extracted and amplified by a rolling circle technique and then sheared before sequencing as a paired-end sequencing run using Illumina technology. The data was assembled into contigs using the meta-IDBA assembler and annotated using BLASTX. Forty four contigs were over 10 kb and the largest contig was 61 kb. Four specimen contig assemblies were verified by PCR and sequencing from the original DNA extract. Assigned reads and contigs were mainly of phage or prophage origin, but the majority of the sequence data obtained was classified as “unassigned”. Phages from bacteria of the genera Streptococcus, Veillonella, Pseudomonas, Actinobacillus and Prevotella comprised the most frequently assembled contigs. A complete human Torque Teno-like virus was detected in the metagenome and by PCR. A substantial number of assigned sequences could be affiliated with species typically found in the oral cavity, as well as CF samples, reflecting the nature of sputum. Numerous antimicrobial resistance genes were detected flanked by phage sequences, suggesting sputum of cystic fibrosis patients is a source for transmissible antimicrobial resistance.