Targeted and highly multiplexed detection of microorganisms by employing an ensemble of molecular probes.

The vast majority of microscopic life on earth consists of microbes that do not grow in laboratory culture. To profile the microbial diversity in environmental and clinical samples, we have devised and employed molecular probe technology, which detects and identifies bacteria that do and do not grow in culture. The only requirement is a short sequence of contiguous bases (currently 60 bases) unique to the genome of the organism of interest. The procedure is relatively fast, inexpensive, customizable, robust, and culture independent and uses commercially available reagents and instruments. In this communication, we report improving the specificity of the molecular probes substantially and increasing the complexity of the molecular probe set by over an order of magnitude (>1,200 probes) and introduce a new final readout method based upon Illumina sequencing. In addition, we employed molecular probes to identify the bacteria from vaginal swabs and demonstrate how a deliberate selection of molecular probes can identify less abundant bacteria even in the presence of much more abundant species.

Molecular probe technology detects bacteria without culture.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

The dynamics of the vaginal microbiome during infertility therapy with in vitro fertilization-embryo transfer.

PURPOSE: To determine the vaginal microbiome in women undergoing IVF-ET and investigate correlations with clinical outcomes. METHODS: Thirty patients had blood drawn for estradiol (E(2)) and progesterone (P(4)) at four time points during the IVF-ET cycle and at 4-6 weeks of gestation, if pregnant. Vaginal swabs were obtained in different hormonal milieu, and the vaginal microbiome determined by deep sequencing of the 16S ribosomal RNA gene. RESULTS: The vaginal microbiome underwent a transition during therapy in some but not all patients. Novel bacteria were found in 33% of women tested during the treatment cycle, but not at 6-8 weeks of gestation. Diversity of species varied across different hormonal milieu, and on the day of embryo transfer correlated with outcome (live birth/no live birth). The species diversity index distinguished women who had a live birth from those who did not. CONCLUSIONS: This metagenomics approach has enabled discovery of novel, previously unidentified bacterial species in the human vagina in different hormonal milieu and supports a shift in the vaginal microbiome during IVF-ET therapy using standard protocols. Furthermore, the data suggest that the vaginal microbiome on the day of embryo transfer affects pregnancy outcome.

Multiplex identification of microbes.

We have adapted molecular inversion probe technology to identify microbes in a highly multiplexed procedure. This procedure does not require growth of the microbes. Rather, the technology employs DNA homology twice: once for the molecular probe to hybridize to its homologous DNA and again for the 20-mer oligonucleotide barcode on the molecular probe to hybridize to a commercially available molecular barcode array. As proof of concept, we have designed, tested, and employed 192 molecular probes for 40 microbes. While these particular molecular probes are aimed at our interest in the microbes in the human vagina, this molecular probe method could be employed to identify the microbes in any ecological niche.

Allelic resolution NGS HLA typing of Class I and Class II loci and haplotypes in Cape Town, South Africa.

The development of next-generation sequencing (NGS) methods for HLA genotyping has already had an impact on the scope and precision of HLA research. In this study, allelic resolution HLA typing was obtained for 402 individuals from Cape Town, South Africa. The data were produced by high-throughput NGS sequencing as part of a study of T-cell responses to Mycobacterium tuberculosis in collaboration with the University of Cape Town and Stanford University. All samples were genotyped for 11 HLA loci, namely HLA-A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3, -DRB4, and -DRB5. NGS HLA typing of samples from Cape Town inhabitants revealed a unique cohort, including unusual haplotypes, and 22 novel alleles not previously reported in the IPD-IMGT/HLA Database. Eight novel alleles were in Class I loci and 14 were in Class II. There were 62 different alleles of HLA-A, 72 of HLA-B, and 47 of HLA-C. Alleles A∗23:17, A∗43:01, A∗29:11, A∗68:27:01, A∗01:23, B∗14:01:01, B∗15:10:01, B∗39:10:01, B∗45:07, B∗82:02:01 and C∗08:04:01 were notably more frequent in Cape Town compared to other populations reported in the literature. Class II loci had 21 different alleles of DPA1, 46 of DPB1, 27 of DQA1, 26 of DQB1, 41 of DRB1, 5 of DRB3, 4 of DRB4 and 6 of DRB5. The Cape Town cohort exhibited high degrees of HLA diversity and relatively high heterozygosity at most loci. Genetic distances between Cape Town and five other sub-Saharan African populations were also calculated and compared to European Americans.

Next generation 1536-well oligonucleotide synthesizer with on-the-fly dispense.

Here we report the development of our Next Generation Automated Multiplexed Oligonucleotide Synthesizer (NG-1536-AMOS), capable of producing 1536 samples in a single run using a multi-well filtered titer plate. With the potential to synthesize up to 3456 samples per plate, we converted the BioRAPTR Flying Reagent Dispenser into an open-well system where spent reagents are drained to waste under vacuum. During synthesis, reagents are delivered on-the-fly to each micro-titer well at volumes ≤5 µl with plate speeds up to 150 mm/s. Using gas-phase cleavage and deprotection, a full plate of 1536 60 mers may be processed with same-day turnaround with an average yield per well at 3.5 nmol. Final product at only $0.00277/base is eluted into a low-volume collection plate for immediate use in downstream application (e.g. Biomek FX for versatile sample handling). Also, crude oligonucleotide quality is comparable to that of commercial synthesis instrumentation, with an error rate on the NG-1536-AMOS platform of 1.53/717 bases. Furthermore, mass spectral analysis on strands synthesized up to 80 bases showed high purity with an average coupling efficiency of 99.5%.

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.

Direct oligonucleotide synthesis onto super-paramagnetic beads.

Super-paramagnetic beads (SPMB)s used for a variety of molecular diagnostic assays are prepared by attaching pre-synthesized oligonucleotides to the surface via a cumbersome and low efficient method of carbodiimide-mediated amide bond formation. To mainstream the process, we describe a novel procedure of direct oligonucleotide synthesis onto the surface of SPMBs (e.g. MyOne Dynabeads). With the many challenges surrounding containment of paramagnetic beads (≤1 µm) during automated oligonucleotide synthesis, we show that by applying a magnetic force directly to the SPMBs we prevent their loss caused by high-pressure drain steps during synthesis. To date we have synthesized 40 mers using a Spacer 9 phosphoramidite (triethylene glycol) coupled to the surface of hydroxylated SPMBs. HPLC analysis shows successful product generation with an average yield of 200 pmol per sample. Furthermore, because of the versatility of this powerful research tool, we envision its use in any laboratory working with conventional synthesis automation, as employed for single columns and for multi-well titer plates. In addition to direct synthesis of oligodeoxynucleotides (DNA) onto SPMBs, this platform also has the potential for RNA and peptide nucleic acid synthesis.

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.

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

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

Microbes on the human vaginal epithelium.

Using solely a gene-based procedure, PCR amplification of the 16S ribosomal RNA gene coupled with very deep sequencing of the amplified products, the microbes on 20 human vaginal epithelia of healthy women have been identified and quantitated. The Lactobacillus content on these 20 healthy vaginal epithelia was highly variable, ranging from 0% to 100%. For four subjects, Lactobacillus was (virtually) the only bacterium detected. However, that Lactobacillus was far from clonal and was a mixture of species and strains. Eight subjects presented complex mixtures of Lactobacillus and other microbes. The remaining eight subjects had no Lactobacillus. Instead, Bifidobacterium, Gardnerella, Prevotella, Pseudomonas, or Streptococcus predominated.

The genome of the basidiomycetous yeast and human pathogen Cryptococcus neoformans.

Cryptococcus neoformans is a basidiomycetous yeast ubiquitous in the environment, a model for fungal pathogenesis, and an opportunistic human pathogen of global importance. We have sequenced its approximately 20-megabase genome, which contains approximately 6500 intron-rich gene structures and encodes a transcriptome abundant in alternatively spliced and antisense messages. The genome is rich in transposons, many of which cluster at candidate centromeric regions. The presence of these transposons may drive karyotype instability and phenotypic variation. C. neoformans encodes unique genes that may contribute to its unusual virulence properties, and comparison of two phenotypically distinct strains reveals variation in gene content in addition to sequence polymorphisms between the genomes.