Optimizing protocols for extraction of bacteriophages prior to metagenomic analyses of phage communities in the human gut

The human gut is densely populated with archaea, eukaryotes, bacteria, and their viruses, such as bacteriophages. Advances in high-throughput sequencing (HTS) as well as bioinformatics have opened new opportunities for characterizing the viral communities harbored in our gut. However, limited attention has been given to the efficiency of protocols dealing with extraction of phages from fecal communities prior to HTS and their impact on the metagenomic dataset.

Two procedures (TFF and PEG) for extraction of bacteriophages from fecal samples were optimized using a set of spiked bacteriophages as process control. These protocols are highly efficient tools for extraction and purification of PPs prior to HTS in phage-metavirome studies. Our methods can be easily modified, being thus applicable and adjustable for in principle any solid environmental material in dissolution.

We describe two optimized methods for extraction of phages from fecal samples based on tangential-flow filtration (TFF) and polyethylene glycol precipitation (PEG) approaches using an adapted method from a published protocol as control (literature-adapted protocol (LIT)). To quantify phage recovery, samples were spiked with low numbers of c2, ϕ29, and T4 phages (representatives of the Siphoviridae, Podoviridae, and Myoviridae families, respectively) and their concentration (plaque-forming units) followed at every step during the extraction procedure. Compared with LIT, TFF and PEG had higher recovery of all spiked phages, yielding up to 16 times more phage particles (PPs) and up to 68 times more phage DNA per volume, increasing thus the chances of extracting low abundant phages. TFF- and PEG-derived metaviromes showed 10% increase in relative abundance of Caudovirales and unclassified phages infecting gut-associated bacteria (>92% for TFF and PEG, 82.4% for LIT). Our methods obtained lower relative abundance of the Myoviridae family (<16%) as compared to the reference protocol (22%). This decline, however, was not considered a true loss of Myoviridae phages but rather a greater level of extraction of Siphoviridae phages (TFF and PEG >32.5%, LIT 22.6%), which was achieved with the enhanced conditions of our procedures (e.g., reduced filter clogging). A high degree of phage diversity in samples extracted using TFF and PEG was documented by transmission electron microscopy. Conclusions: Two procedures (TFF and PEG) for extraction of bacteriophages from fecal samples were optimized using a set of spiked bacteriophages as process control. These protocols are highly efficient tools for extraction and purification of PPs prior to HTS in phage-metavirome studies. Our methods can be easily modified, being thus applicable and adjustable for in principle any solid environmental material in dissolution.