Whole Genome Shotgun Sequencing of Environmental Samples

New approaches to environmental sampling are emerging (6–9). One of these used a microarray to discover and assist in the isolation of new viruses (6); another used a shotgun clone and sequencing method to explore marine viral communities (9). Two others have used whole genome shotgun (WGS) sequencing on a population of bacteria, obviating the need to isolate each organism before sequencing can begin (7,8). These methods, used in combination with existing methods, may provide shortcuts to the discovery of new genes and give a holistic persective to microbial populations.

One recent study used a WGS approach to explore a sample from an acid mine drainage biofilm (7; AADL00000000). These investigators report that near-complete genomes for Leptospirillum Group II and Ferroplasma Type II were assembled, along with more fragmentory assemblies for Leptospirillum Group III, Thermoplasmatales archaeon gpl, and Ferroplasma acidarmanus Type I. Analysis of the results provided some insight into how such organisms survive in an extreme environment.

In another test case of the WGS method, Venter et al. (8) sampled water from the Sargasso Sea—one of the most well-characterized regions of ocean in the world. The major set of samples produced 1.66 million short sequences, some of which could be grouped together into larger genomic pieces. There remained about 400,000 paired-end reads and singleton reads.

Finding the Data

Using a WGS method to sequence an undefined population as opposed to a single organism adds significant complexity to the assembly process and to the identification of genes. About 25% of the assembled data from the Sargasso Sea had 3X coverage or greater; these well-sampled portions were used to cluster the sequence by “organism”.

The assembled sequences have been deposited in the WGS division of GenBank, with the project Accession number AACY01000000; thus, there are 811,372 WGS contigs in GenBank with the Accession numbers AACY01000001-AACY01811372. 498,641 of the WGS contigs are assembled into 232,442 scaffolds, the rest remain “singleton” WGS contigs; all but 10,685 of the scaffolds are made up of two contigs only. For the organism genomes listed in Table 1, 301 of the total scaffolds plus 36 singleton WGS contigs were used; the remainder have not been associated with any particular organism.

Table 1

The organism bins assembled from the Sargasso Sea WGS environmental sample dataset (8).

All of the short sequence reads, including those that were not included in the assembly, can be found in the Trace Archive.

The assemblies were then further clustered into 30 tentative organism “bins” based on depth of coverage, oligonucleotide frequencies and similarities to previously sequenced genomes. Of these, 12 are of sufficient size to be considered a genome assembly, while the remaining 16 are relatively small single scaffolds (Table 1). All organism bins have been assigned a taxonomy ID, and have been placed in the taxonomic tree. Figure 1 shows the graphical representation of the cf. Shewanella SAR-1 “genome” sequence.

Figure 1

(a) Genome view of cf. Shewanella SAR-1, constructed from the whole genome shotgun sequence derived from Sargasso Sea environmental samples (8). Genes have been classified according to the COG functional categories of the protein products, and color-coded (more...)

A variety of approaches suggested that there are at least 1000 species represented in the Sargasso Sea samples (8). Burkholderia species were represented in a high proportion (a genus that includes human and plant pathogens and some environmentally important bacteria), as were two distinct species closely related to Shewanella oneidensis. Both of these genera require a more nutrient-rich environment than the open ocean can offer, suggesting that they originated from microhabitats such as marine snow. The cyanobacterium Prochlorococcus was also relatively abundant in some samples.

Although the primary focus of this study was on bacterial populations, WGS environmental sampling may be an equally valid approach for exploring plasmids (Table 2), phage, viruses, and eukaryotic microbes.

Table 2

The plasmid bins assembled from the Sargasso Sea WGS environmental sample dataset (8).

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