Uncovering a hidden diversity: optimized protocols for the extraction of bacteriophages from soil

Abstract

11Background 12Bacteriophages are the most numerous biological entities on earth and play a crucial role in 13 shaping microbial communities. Investigating the bacteriophage community from soil samples 14 will shed light not only on the yet largely unknown phage diversity, but also may result in novel 15 insights into phage biology and functioning. Unfortunately, the study of soil viromes lags far 16 behind any other ecological model system, due to the heterogeneous soil matrix that rises 17 major technical difficulties in the extraction process. Resolving these technical challenges and 18 establishing a standardized extraction protocol is therefore a fundamental prerequisite for 19 replicable results and comparative virome studies. 20 Results 21We here report the optimization of protocols for extraction of bacteriophage DNA from soil 22 preceding metagenomic analysis such that the protocol can equally be harnessed for phage 23 isolation. As an optimization strategy, soil samples were spiked with a viral community 24 consisting of phages from different families (10 6 PFU/g soil): Listeria phage ΦA511 (Myovirus), 25Staphylococcus phage Φ2638AΔLCR (Siphovirus), and Escherichia phage ΦT7 (Podovirus). 26The efficacy of bacteriophage (i) elution, (ii) filtration, (iii) concentration, and (iv) DNA extraction 27 methods was tested. Successful extraction routes were selected based on spiked phage 28 recovery and low bacterial 16S rRNA gene contaminants. Natural agricultural soil viromes 29 were then extracted with the optimized methods and shotgun sequenced. Our approach 30 yielded sufficient amounts of inhibitor-free viral DNA for non-amplification dependent 31 sequencing and low 16S rRNA gene contamination levels (≤ 0.2 ‰). Compared to previously 32 published protocols, the number of bacterial read contamination was decreased by 65 %. In 33 addition, 468 novel circularized soil phage genomes in size up to 235 kb were obtained from 34 over 29,000 manually identified viral contigs, promising the discovery of a large, previously 35 inaccessible viral diversity. 36Conclusion 37 3 We have shown a dramatically enhanced extraction of the soil phage community by protocol 38 optimization that has proven robustness in both a culture-depended as well as through 39 metaviromic analysis. Our huge data set of manually curated soil viral contigs roughly doubles 40 the amount of currently available soil virome data, and provide insights into the yet largely 41 undescribed soil viral sequence space. 42 Keywords 43Bacteriophage extraction, phage metaviromes, soil samples, bacterial contamination 44 4 6 contigs. This data set roughly doubles the amount of today's available viral contigs from soil, 101 and opens the door for discovery of a very large soil viral diversity. 102 Results 103 Protocol optimization strategy 104The selective criteria for optimized extraction routes relied on three major parameters: (i) phage 105 yield at each step of the extraction route, (ii) reduction of bacterial DNA contamination levels 106 and (iii) bias...

Choice of Ultrafilter affects Recovery Rate of Bacteriophages

“…The first step of most viral analysis pipelines is to separate the viral particles from bacteria and other contaminants. There are currently no accepted standard protocol, resulting in each phage research group having its own preferred methods [14,15,16,17]. Many pipelines include agitation to release the viral particles from the sample material and into a buffer, followed by centrifugation and filtration to remove larger particles and bacteria.…”