16S rRNA gene contigs and chimeric contigs for simulated datasets.

<p>Open circle: non-chimeric contigs; solid circle: chimeric contigs containing one contaminating read; solid triangles: chimeric contigs containing more than one contaminating read. Arrow: chimera detected by UChime. (A) HC. (B) MC. (C) LC.</p

Reads, 16S rRNAcontigs, OTUs and chimera examination of the simulated communities.

<p>Reads, 16S rRNAcontigs, OTUs and chimera examination of the simulated communities.</p

The sponge tag-sequencing data sets.

<p>The sponge tag-sequencing data sets.</p

Shared and unique OTUs of the PCR-based and shotgun-based sponge datasets.

<p>Circle sizes are proportional to OTU number. (A) 0.01 phylogenetic distance OTU. (B) 0.03 phylogenetic distance OTU. (C) 0.05 phylogenetic distance OTU.</p

Abundance and primer-mismatches in the top OTUs at the 0.01 phylogenetic distance level for the sponge datasets.

<p>Asterisk, primer-mis-match event.</p

Taxonomic classification of assembled and unassembled shotgun 16S rRNA gene reads for simulated datasets.

<p>(A) HC. (B) MC. (C) LC.</p

The sponge metagenomic datasets.

<p>The sponge metagenomic datasets.</p

Phylum-level classification of the sponge pyro-tag-sequencing and shotgun sequencing datasets.

<p>(A) 16S rRNA gene PCR approach. (B) Unassembled shotgun 16S rRNA gene reads. (C) Assembled shotgun 16S rRNA gene reads. (D) Single-copy gene analysis.</p

Electron micrographs of GISH for <i>E</i>.<i>coli</i> cells after different times of gold enhancement by GOLDENHANCE EM Plus (Nanoprobes).

<p>Panels A-D correspond to 0 min, 1.5 min, 3 min, 6 min of gold enhancement, respectively. The top and bottom panels are shown for SE and BSE images, respectively. Scale bar = 1 μm.</p

Primer fabrication using polymerase mediated oligonucleotide synthesis

Background: Custom solid phase oligonucleotide synthesis is an important foundation supporting nearly every aspect of current genomics. In spite of the demand for oligonucleotide primers, their synthesis remains relatively expensive, time consuming and in many circumstances a wasteful process. In this methodology, described as polymerase mediated oligonucleotide synthesis (PMOS), a DNA polymerase is used to increase the hybridization affinity of one oligonucleotide by using another as a template for DNA synthesis. This self-assembly process provides an opportunity to instantly generate a very large number of useful gene-specific primers from a small library of simple precursors. PMOS can be used to generate primers directly in the end-users laboratory within the context of any DNA polymerase chemistry such as in PCR or sequencing reactions. Results: To demonstrate the utility of PMOS, a universal 768-member oligonucleotide library (UniSeq) was designed, fabricated and its performance optimized and evaluated in a range of PCR and DNA sequencing reactions. This methodology used to derive specific 11-mers, performed well in each of these activities and produced the desired amplification or sequencing analysis with results comparable to primers made by time consuming and expensive custom synthesis. Conclusion: On the basis of these experiments, we believe this novel system would be broadly applicable and could in many circumstances replace the need for conventional oligonucleotide synthesis