Percent coverage of pooled sequencing result relative to barcoded sequencing result.

<p>Each of the 73 clones was categorized into Clone Types A, B, C, or D by the number of end-tags obtained (one or two), whether the end-tag retrieved a contig from the pool, and the completeness of the retrieved pooled sequencing result relative to the reference barcoded sequencing result (full or partial coverage). Clone Type descriptions are given above.</p

Evaluation of a Pooled Strategy for High-Throughput Sequencing of Cosmid Clones from Metagenomic Libraries

<div><p>High-throughput sequencing methods have been instrumental in the growing field of metagenomics, with technological improvements enabling greater throughput at decreased costs. Nonetheless, the economy of high-throughput sequencing cannot be fully leveraged in the subdiscipline of functional metagenomics. In this area of research, environmental DNA is typically cloned to generate large-insert libraries from which individual clones are isolated, based on specific activities of interest. Sequence data are required for complete characterization of such clones, but the sequencing of a large set of clones requires individual barcode-based sample preparation; this can become costly, as the cost of clone barcoding scales linearly with the number of clones processed, and thus sequencing a large number of metagenomic clones often remains cost-prohibitive. We investigated a hybrid Sanger/Illumina pooled sequencing strategy that omits barcoding altogether, and we evaluated this strategy by comparing the pooled sequencing results to reference sequence data obtained from traditional barcode-based sequencing of the same set of clones. Using identity and coverage metrics in our evaluation, we show that pooled sequencing can generate high-quality sequence data, without producing problematic chimeras. Though caveats of a pooled strategy exist and further optimization of the method is required to improve recovery of complete clone sequences and to avoid circumstances that generate unrecoverable clone sequences, our results demonstrate that pooled sequencing represents an effective and low-cost alternative for sequencing large sets of metagenomic clones.</p></div

Functional metagenomics reveals novel β-galactosidases not predictable from gene sequences

<div><p>The techniques of metagenomics have allowed researchers to access the genomic potential of uncultivated microbes, but there remain significant barriers to determination of gene function based on DNA sequence alone. Functional metagenomics, in which DNA is cloned and expressed in surrogate hosts, can overcome these barriers, and make important contributions to the discovery of novel enzymes. In this study, a soil metagenomic library carried in an IncP cosmid was used for functional complementation for β-galactosidase activity in both <i>Sinorhizobium meliloti</i> (<i>α-Proteobacteria</i>) and <i>Escherichia coli</i> (<i>γ-Proteobacteria</i>) backgrounds. One β-galactosidase, encoded by six overlapping clones that were selected in both hosts, was identified as a member of glycoside hydrolase family 2. We could not identify ORFs obviously encoding possible β-galactosidases in 19 other sequenced clones that were only able to complement <i>S</i>. <i>meliloti</i>. Based on low sequence identity to other known glycoside hydrolases, yet not β-galactosidases, three of these ORFs were examined further. Biochemical analysis confirmed that all three encoded β-galactosidase activity. Lac36W_ORF11 and Lac161_ORF7 had conserved domains, but lacked similarities to known glycoside hydrolases. Lac161_ORF10 had neither conserved domains nor similarity to known glycoside hydrolases. Bioinformatic and structural modeling implied that Lac161_ORF10 protein represented a novel enzyme family with a five-bladed propeller glycoside hydrolase domain. By discovering founding members of three novel β-galactosidase families, we have reinforced the value of functional metagenomics for isolating novel genes that could not have been predicted from DNA sequence analysis alone.</p></div

Metagenomic and genomic libraries used in this study.

<p>*from the Canadian MetaMicroBiome Library collection, <a href="http://www.cm2bl.org" target="_blank">http://www.cm2bl.org</a>.</p

12AC metagenomic clones complementing <i>E</i>. <i>coli</i> DH5α (<i>lacZYA</i>) and <i>S</i>. <i>meliloti</i> RmF728 (<i>lacEFGZ1K1</i>) grown in M9-lactose medium.

<p>12AC metagenomic clones complementing <i>E</i>. <i>coli</i> DH5α (<i>lacZYA</i>) and <i>S</i>. <i>meliloti</i> RmF728 (<i>lacEFGZ1K1</i>) grown in M9-lactose medium.</p

Bacterial strains, plasmids, and cosmids.

<p>Bacterial strains, plasmids, and cosmids.</p

Protein homology searches of novel β<i>-</i>galactosidase sequences of Lac161_ORF10, Lac161_ORF7 and Lac36W_ORF11 against aquatic, human gut, and soil metagenomic databases, normalized to the <i>rpoB</i> gene.

<p>Protein homology searches of novel β<i>-</i>galactosidase sequences of Lac161_ORF10, Lac161_ORF7 and Lac36W_ORF11 against aquatic, human gut, and soil metagenomic databases, normalized to the <i>rpoB</i> gene.</p

Overlapping clones assemble into one contig.

<p>Three overlapping clones as revealed by barcoded sequencing (above) and pooled sequencing (below). Locations of end-tags are indicated by vertical dashed lines. White dashed boxes indicate gaps in the pooled sequencing data; black boxes indicate a contig. Lengths of all contigs are given.</p

Heat map of clone sequence similarity and corresponding bar plots of clone coverage.

<p>Pair-wise sequence similarity is shown for all 73 clones (A), juxtaposed to their pooled sequencing coverage, showing both retrieved coverage (B) and actual coverage (C).</p

Conserved domains (<i>E</i> < 0.01) in β-galactosidases isolated from 12AC metagenomic library clones.

<p>Conserved domains (<i>E</i> < 0.01) in β-galactosidases isolated from 12AC metagenomic library clones.</p