In addition, the variety of Cytophaga-like bacteria was greater in the metagenomic In contrast to the differences outlined above for the library than in the PCR library. More Cytophaga-like clus- Cytophaga-like and beta-proteobacteria groups, Actino- ters contained fosmid sequences than PCR sequences in bacteria in the metagenomic and PCR libraries were quite the phylogenetic tree of Cytophaga-like bacteria Fig.
Of Myroides groups Fig. One Cytophaga-like clone in the the three clusters of Actinobacteria clones A—C , each PCR library Sta2—97 was not similar to any of the included clones from both libraries Fig. Cluster B metagenomic clones. The Actinobacteria clones in associated with the Flavobacteriales group Fig. Sequence these clones could be grouped into three clusters A—C. Clusters A and B were related to different kinds Alpha-proteobacteria comprised a minor component of of Flavobacterium spp.
Cluster A included an isolate from the Elbe River clones in the metagenomic and PCR clones respectively and cluster B included Flavobacterium xinjiangensi. The Table 1. The alpha-proteobacteria clones in the two average sequence identity in these two clusters with cul- libraries were closely related to each other and to some tured members was Three respectively. Clusters C and D were associated with the clones from the PCR library and two from the metage- Myroides and Flectobacillus groups, respectively, and nomic library belonged to a cluster that included cultured included bacteria with a higher degree of sequence simi- Sphingomonas spp.
One alpha-proteobacteria clone from larity The libraries also included clones belonging to five Some of the Cytophaga-like bacteria were represented groups of bacteria that can be present in aquatic sys- in both libraries.
Half of the Cytophaga-like clones in the tems but are typically minor members of the community. PCR library were highly similar to clones in the metage- These groups included Firmicutes and Spirochaetaceae nomic library, and every Cytophaga-like group with clones that occurred in only the metagenomic library and epsi- from the PCR library also had representatives from the lon-proteobacteria and Bdellovibrio that were found metagenomic library.
Clones belong- prised of three PCR clones and four metagenomic clones ing to the Verrucomicrobiales group occurred in both Fig. Cluster A included three metagenomic clones libraries. However, similarity between Cytophaga-like bacteria in the two Library coverage and diversity libraries was restricted to these two clusters. Beta-proteobacteria in clone libraries. The metage- Library coverage increased with the genetic distance, as nomic and PCR libraries also differed in the number and expected.
At an evolutionary distance of 0. Kirchman Fig. Phylogenetic relationships of selected Cytophaga-like bacteria as depicted in the ARB database tree constructed of sequences longer than bp June Values on the trapezoids indicate the number of sequences in the group. Labels A, B, C and D refer to clusters of closely related bacteria that are discussed in the text.
The scale bar represents 0. Phylogenetic relationships of selected beta-proteobacteria as depicted in the ARB database tree constructed of sequences longer than bp June Delaware River beta-proteobacteria were added using the ARB parsimony quick add tool. Labels A, B, C, D and E refer to clusters of closely related bacteria that are discussed in the text.
Phylogenetic relationships of selected Actinobacteria as depicted in the ARB database tree constructed of sequences longer than bp June Labels A, B and C refer to clusters of closely related bacteria that are discussed in the text.
The error bars are one standard deviation. We examined the possibility that diversity based on PCR clone library composition is overestimated because of artefacts of the PCR. Re-conditioned PCR was used to test for potential artefacts introduced when heterologous respectively Fig.
Re-conditioning of the Coverage of the metagenomic library was higher than PCR amplicons reduced the number of RFLP types coverage of the PCR library at all levels of genetic dis- expected in a library of 80 clones from 50 in the standard tance with the greatest differences occurring at small PCR library to 40 in the library produced with re-condi- genetic distance. Coverage decreased rapidly with tioned PCR Fig. There was no difference between decreasing genetic distance, and at a genetic distance of rarefaction curves of replicate PCR libraries produced with 0.
The Simpson index was The difference was not as correspondence between clone library composition and substantial for the Shannon—Weaver index Table 2. Microscopic enumeration Similarly, measures of evenness were higher for the of bacteria hybridized with fluorescently labelled 16S metagenomic library than the PCR library.
Evenness cal- rRNA directed probes revealed a community dominated culated using the Shannon—Weaver index was 0. As expected for a freshwater sys- respectively, while the Simpson index of evenness was tem, Actinobacteria and beta-proteobacteria were the 0.
Cytophaga-like bacteria were also prominent, Table 2. It was not surprising that the metage- inferred from the PCR library was surprisingly similar. In contrast, the abundance of Kirchman, ; Kirchman et al. It was also not beta-proteobacteria clones in the PCR library was twice surprising that the metagenomic library had more clones the abundance of bacteria detected with the beta-proteo- from Cytophaga-like bacteria than would have been bacteria FISH probe Table 1.
Three types of bacteria, expected based on the FISH counts because the general including epsilon-proteobacteria, Bdellovibrio and bacte- FISH probe CFa for Cytophaga-like bacteria does not ria belonging to the candidate division TM6, were present recognize all Cytophaga-like bacteria Weller et al. If metagenomic libraries represent a The Cytophaga-like bacterial group includes a highly biased sample of microbial diversity, they will not yield an diverse collection of bacteria but not all of this diversity accurate assessment of metabolic diversity.
Assuming was present in our Delaware River sample. In fact, most they are unbiased, large insert metagenomic libraries may of the Cytophaga-like bacteria were most closely related be particularly powerful in examining the relationship to just two genera of Flavobacteriales Flavobacterium between community structure and metabolic capacities. Therefore, it is necessary to know how the Flectobacillus group belong to the proposed the diversity of 16S rRNA genes sampled by large insert PRD01aB cluster of Cytophaga-like bacteria identified metagenomic libraries corresponds to actual community by Zwart and colleagues Nine of the 12 Delaware structure.
The proposed examination revealed noteworthy differences between the PRD01aB cluster, which is currently comprised solely libraries and the community structure determined by of uncultivated bacteria, has also been found in Adiron- FISH. The metagenomic library sampled a broader diver- dack lakes, Lake Baikal and other freshwater environ- sity of Cytophaga-like bacteria than the PCR library, but it ments Zwart et al.
However, this group of missed some groups of potentially important beta-proteo- Cytophaga-like bacteria has not been detected in all bacteria. Zwart and D and E were highly similar to bacteria in the freshwater colleagues also did not find evidence for an impor- beta-Proteobacteria clusters proposed by Zwart and col- tant contribution of soil bacteria to bacterial communities leagues Sequences in Delaware River beta-pro- in rivers.
BAL47 cluster. Differences in group E probably belong to the proposed LD28 cluster. LIBSHUFF analysis conducted The clusters of freshwater beta-proteobacteria pro- with the V3 region alone also revealed higher coverage by posed by Zwart and colleagues were more preva- the metagenomic library data not shown.
Differences in lent in the PCR library than in the metagenomic library, coverage may reflect more comprehensive sampling of suggesting that they may represent bacteria that are pref- bacterial diversity by the metagenomic library. Delaware River beta- Lower coverage by the PCR library was consistent with proteobacteria group B included clones from both the our hypothesis that PCR artefacts lead to overestimates metagenomic library and the PCR library, but Delaware of diversity.
Different scenarios could lead to the not as pronounced as would be expected if heterodu- overrepresentation of bacterial groups in PCR libraries plexes were a substantial source of diversity in the PCR compared with libraries constructed without a PCR step, libraries.
The higher diversity in the metagenomic library including selective PCR amplification with general bacte- compared with the PCR library is also consistent with the rial primers, differences in rRNA gene copy number or idea that the PCR libraries do not overestimate bacterial variation in DNA extraction efficiency von Wintzingerode diversity. In fact, they probably underestimate diversity, et al.
Fluorescence in situ hybridization probes are because they miss certain groups, such as Cytophaga- needed to determine the abundance of these beta-proteo- like bacteria Cottrell and Kirchman, Additional fac- bacteria, which appear to be in a variety of freshwater tors such as cloning biases, variation in genome size environments. Fogel et al. All , could have resulted in larger differences than we three groups of Actinobacteria identified in this study observed between the two library approaches and FISH.
With the aid of compared with their abundance determined by FISH. MI and STA clusters identified by Zwart and colleagues Archaea make a substantial contribution to prokaryotic , were identified in the metagenomic and PCR diversity in the ocean depths well below the euphotic zone libraries. Delaware River Actinobacte- at least in marine waters, it appears that only a few clades ria group A, which included clones from both libraries, did dominate, including alpha-proteobacteria belonging to the not overlap with previously described clusters of freshwa- SAR11 group Morris et al.
Gonzalez and Moran, , Cytoph- detected represented a terrestrial bacterial signal aga-like bacteria in Delaware cluster 2 Kirchman et al. NOTE: Setting may vary depending on objective of analyses and quality of fastq reads. These freely-available databases enable the acquisition of information on bacterial pathogens that included genotype and phylogeny, antibiotic-resistance mutations, and the presence of known virulence genes.
A consensus sequence was generated for each of the isolates analysed in Geneious. The Geneious report provided information on the percentage coverage of test sequence to the reference genome and the mean read depth Table 1. Each contiguous sequence is viewable in Geneious and can be analysed for coverage with respect to the reference genome.
Quality control checks of raw sequence data were also performed using FastQC [ 22 ]. This freely-available software provided information regarding per base sequence content and quality, per base and sequence GC content, and highlighted the parameters of the sequence quality. Percentage sequence coverage and mean read depth for each of the sequenced genomes with respect to reference strains.
Coverage refers to the percentage of reference genome bases covered by mapped sequence reads. Mean read depth indicates the mean number of times each base is mapped by a sequence read. Reference genomes used were E. We used open source databases to analyze the sequence data. Mycobacterium tuberculosis complex raw fastq. This database also enabled detection of IS insertion sites, and 43 loci for variable number tandem repeat VNTR typing.
Possible problems and their troubleshooting solutions are listed in Table 2. There are a number of limitations associated with the protocol that should be noted. These include: effective results with the protocol are reliant on the efficacy of the extraction procedure in producing a sufficient quantity of genomic DNA; analysis of sequences generated on an Illumina platform can be affected by the presence of highly repetitive regions; and depending on the output information sought, genome assembly can be influenced by the reference genome selected for the mapping of reads.
Nevertheless, the protocol was effective in generating high quality sequencing data for the range of bacterial species tested. National Center for Biotechnology Information , U.
Journal List J Biol Methods v. J Biol Methods. Published online Mar Sanjay S. Ronan F. Author information Article notes Copyright and License information Disclaimer. Competing interests: The authors have declared that no competing interests exist. This article has been cited by other articles in PMC. Abstract Bacterial whole genome sequencing WGS is becoming a widely-used technique in research, clinical diagnostic, and public health laboratories. Keywords: whole genome sequencing, Enterococcus faecium , Haemophilus influenzae , Mycobacterium tuberculosis.
Open in a separate window. Figure 1. Perform a final spin at g for 1 min. Quantification of bacterial genomic DNA Nextera XT tagment amplicon construction Neutralization of Nextera XT tagment amplicon PCR amplification Gently pipette the content and perform a quick spin.
Incubate at room temperature for 5 min. Place the tube on a magnetic stand for 2 min. Aspirate out the supernatant carefully. Library normalization Pool the genomic DNA from all of the tubes. Preparing pooled library for loading onto MiSeq Mix using a pipette 5 times. Geneious was used to map the Fastq sequence reads to a publicly available reference genome for each species as follows: Mapping the isolate sequence to the reference genome Click Align Assemble Map to Reference.
Check the settings Results: Select all options Results Save consensus sequences Options When mapping to reference is complete, a new folder will be created containing four files: Like two other DNA extraction protocol the boiling method presents advantages.
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