Molecular probe technology detects bacteria without culture

<p>Abstract</p> <p>Background</p> <p>Our ultimate goal is to detect the entire human microbiome, in health and in disease, in a single reaction tube, and employing only commercially available reagents. To that end, we adapted molecular inversion probes to detect bacteria using solely a massively multiplex molecular technology. This molecular probe technology does not require growth of the bacteria in culture. Rather, the molecular probe technology requires only a sequence of forty sequential bases unique to the genome of the bacterium of interest. In this communication, we report the first results of employing our molecular probes to detect bacteria in clinical samples.</p> <p>Results</p> <p>While the assay on Affymetrix GenFlex Tag16K arrays allows the multiplexing of the detection of the bacteria in each clinical sample, one Affymetrix GenFlex Tag16K array must be used for each clinical sample. To multiplex the clinical samples, we introduce a second, independent assay for the molecular probes employing Sequencing by Oligonucleotide Ligation and Detection. By adding one unique oligonucleotide barcode for each clinical sample, we combine the samples after processing, but before sequencing, and sequence them together.</p> <p>Conclusions</p> <p>Overall, we have employed 192 molecular probes representing 40 bacteria to detect the bacteria in twenty-one vaginal swabs as assessed by the Affymetrix GenFlex Tag16K assay and fourteen of those by the Sequencing by Oligonucleotide Ligation and Detection assay. The correlations among the assays were excellent.</p

A direct comparison of the KB™ Basecaller and phred for identifying the bases from DNA sequencing using chain termination chemistry

<p>Abstract</p> <p>Background</p> <p>Relatively recently, the software KB™ Basecaller has replaced <it>phred </it>for identifying the bases from raw sequence data in DNA sequencing employing dideoxy chemistry. We have measured quantitatively the consequences of that change.</p> <p>Results</p> <p>The high quality sequence segment of reads derived from the KB™ Basecaller were, on average, 30-to-50 bases longer than reads derived from <it>phred</it>. However, microbe identification appeared to have been unaffected by the change in software.</p> <p>Conclusions</p> <p>We have demonstrated a modest, but statistically significant, superiority in high quality read length of the KB™ Basecaller compared to <it>phred</it>. We found no statistically significant difference between the numbers of microbial species identified from the sequence data.</p

DMSO and Betaine Greatly Improve Amplification of GC-Rich Constructs in De Novo Synthesis

In Synthetic Biology, de novo synthesis of GC-rich constructs poses a major challenge because of secondary structure formation and mispriming. While there are many web-based tools for codon optimizing difficult regions, no method currently exists that allows for potentially phenotypically important sequence conservation. Therefore, to overcome these limitations in researching GC-rich genes and their non-coding elements, we explored the use of DMSO and betaine in two conventional methods of assembly and amplification. For this study, we compared the polymerase (PCA) and ligase-based (LCR) methods for construction of two GC-rich gene fragments implicated in tumorigenesis, IGF2R and BRAF. Though we found no benefit in employing either DMSO or betaine during the assembly steps, both additives greatly improved target product specificity and yield during PCR amplification. Of the methods tested, LCR assembly proved far superior to PCA, generating a much more stable template to amplify from. We further report that DMSO and betaine are highly compatible with all other reaction components of gene synthesis and do not require any additional protocol modifications. Furthermore, we believe either additive will allow for the production of a wide variety of GC-rich gene constructs without the need for expensive and time-consuming sample extraction and purification prior to downstream application

Agarose gel images showing the effects of DMSO and betaine during LCR assembly (A and C) and amplification (B and D) of IGF2R and BRAF gene fragments.

<p>Based on a 20 µl reaction volume, both additives were introduced with increasing percentage (%) for DMSO, and molarity (M) for betaine; ‘No Additive’ lanes correspond to the control samples. A 1 kb DNA ladder in the outermost lanes marked at 400, 500 and 700 bp indicates the area of highest product band population. IGF2R and BRAF full-length gene fragments are shown at 517 bp and 512 bp, respectively.</p

Agarose gel images showing the effects of DMSO and betaine during the PCA assembly (A and C) and amplification (B and D) of IGF2R and BRAF gene fragments.

<p>Based on a 20 µl reaction volume, both additives were introduced with increasing percentage (%) for DMSO, and molarity (M) for betaine; ‘No Additive’ lanes correspond to the control samples. A 1 kb DNA ladder in the outermost lanes marked at 400, 500 and 700 bp indicates the area of highest product band population. IGF2R and BRAF full-length gene fragments are shown at 517 bp and 512 bp, respectively.</p

Diversity of the vaginal microbiome correlates with preterm birth.

Reproductive tract infection is a major initiator of preterm birth (PTB). The objective of this prospective cohort study of 88 participants was to determine whether PTB correlates with the vaginal microbiome during pregnancy. Total DNA was purified from posterior vaginal fornix swabs during gestation. The 16S ribosomal RNA gene was amplified using polymerase chain reaction primers, followed by chain-termination sequencing. Bacteria were identified by comparing contig consensus sequences with the Ribosomal Database Project. Dichotomous responses were summarized via proportions and continuous variables via means ± standard deviation. Mean Shannon Diversity index differed by Welch t test (P = .00016) between caucasians with PTB and term gestation. Species diversity was greatest among African Americans (P = .0045). Change in microbiome/Lactobacillus content and presence of putative novel/noxious bacteria did not correlate with PTB. We conclude that uncultured vaginal bacteria play an important role in PTB and race/ethnicity and sampling location are important determinants of the vaginal microbiome

Diversity of the vaginal microbiome correlates with preterm birth.

Reproductive tract infection is a major initiator of preterm birth (PTB). The objective of this prospective cohort study of 88 participants was to determine whether PTB correlates with the vaginal microbiome during pregnancy. Total DNA was purified from posterior vaginal fornix swabs during gestation. The 16S ribosomal RNA gene was amplified using polymerase chain reaction primers, followed by chain-termination sequencing. Bacteria were identified by comparing contig consensus sequences with the Ribosomal Database Project. Dichotomous responses were summarized via proportions and continuous variables via means ± standard deviation. Mean Shannon Diversity index differed by Welch t test (P = .00016) between caucasians with PTB and term gestation. Species diversity was greatest among African Americans (P = .0045). Change in microbiome/Lactobacillus content and presence of putative novel/noxious bacteria did not correlate with PTB. We conclude that uncultured vaginal bacteria play an important role in PTB and race/ethnicity and sampling location are important determinants of the vaginal microbiome