Metagenomic Characterisation of the Viral Community of Lough Neagh, the Largest Freshwater Lake in Ireland

Abstract

Lough Neagh is the largest and the most economically important lake in Ireland. It is also one of the most nutrient rich amongst the world's major lakes. In this study, 16S rRNA analysis of total metagenomic DNA from the water column of Lough Neagh has revealed a high proportion of Cyanobacteria and low levels of Actinobacteria, Acidobacteria, Chloroflexi, and Firmicutes. The planktonic virome of Lough Neagh has been sequenced and 2,298,791 2×300 bp Illumina reads analysed. Comparison with previously characterised lakes demonstrates that the Lough Neagh viral community has the highest level of sequence diversity. Only about 15% of reads had homologs in the RefSeq database and tailed bacteriophages (Caudovirales) were identified as a major grouping. Within the Caudovirales, the Podoviridae and Siphoviridae were the two most dominant families (34.3% and 32.8% of the reads with sequence homology to the RefSeq database), while ssDNA bacteriophages constituted less than 1% of the virome. Putative cyanophages were found to be abundant. 66,450 viral contigs were assembled with the largest one being 58,805 bp; its existence, and that of another 34,467 bp contig, in the water column was confirmed. Analysis of the contigs confirmed the high abundance of cyanophages in the water column.

Fig 1. Major bacterial groups found in Lough Neagh (phylum level).

Partial sequences of 16S rRNA genes were amplified and sequenced using 454 pyrosequencing. The sequences were clustered into OTUs at the 97% sequence similarity level and taxonomic annotation of OTUs was carried out using QIIME; the results obtained were used to generate the distribution of bacteria at the phylum level.

Fig 2. Seasonal changes in abundance of six major bacterial phyla in Lough Neagh over 12-month period.

Partial sequences of 16S rRNA genes from the additional water samples collected on 1 April 2014, 23 June 2014, 2 September 2014, 10 November 2014, and 18 February 2015 were amplified and sequenced using 454 pyrosequencing. The sequences were clustered into OTUs at the 97% sequence similarity level and taxonomic annotation of OTUs was carried out using QIIME; the results obtained were used to generate the distribution of bacteria at the phylum level.

Fig 3. Taxonomic composition of Lough Neagh virome.

Composition was computed at the MetaVir server from a BLAST comparison with the RefSeq complete viral genomes protein sequences database. Abundance of the major viral groups shown with the numbers of mapped sequences at the right ends of the corresponding bars.

Fig 4. Functional analysis of Lough Neagh virome.

The analysis was carried using SEED subsystems hierarchical functional annotation on the MG-RAST webserver. (A) Relative abundance of level one functional categories. (B) Distribution of minor functional categories. (C) Distribution of functional categories in the “Phages, Prophages, Transposable elements, Plasmids” group at levels 2 and 3.

Fig 5. Maps of putative phage genomes identified in Lough Neagh.

Genome regions amplified using PCR and genome specific primers are indicated with horizontal bars. Identified ORF shown by arrows. (A) Genome map of putative phage LNW4-c0. (B) Genome map of putative phage LNW4-c12.