Bacterial flora-typing with targeted, chip-based Pyrosequencing

<p>Abstract</p> <p>Background</p> <p>The metagenomic analysis of microbial communities holds the potential to improve our understanding of the role of microbes in clinical conditions. Recent, dramatic improvements in DNA sequencing throughput and cost will enable such analyses on individuals. However, such advances in throughput generally come at the cost of shorter read-lengths, limiting the discriminatory power of each read. In particular, classifying the microbial content of samples by sequencing the < 1,600 bp 16S rRNA gene will be affected by such limitations.</p> <p>Results</p> <p>We describe a method for identifying the phylogenetic content of bacterial samples using high-throughput Pyrosequencing targeted at the 16S rRNA gene. Our analysis is adapted to the shorter read-lengths of such technology and uses a database of 16S rDNA to determine the most specific phylogenetic classification for reads, resulting in a weighted phylogenetic tree characterizing the content of the sample. We present results for six samples obtained from the human vagina during pregnancy that corroborates previous studies using conventional techniques.</p> <p>Next, we analyze the power of our method to classify reads at each level of the phylogeny using simulation experiments. We assess the impacts of read-length and database completeness on our method, and predict how we do as technology improves and more bacteria are sequenced. Finally, we study the utility of targeting specific 16S variable regions and show that such an approach considerably improves results for certain types of microbial samples. Using simulation, our method can be used to determine the most informative variable region.</p> <p>Conclusion</p> <p>This study provides positive validation of the effectiveness of targeting 16S metagenomes using short-read sequencing technology. Our methodology allows us to infer the most specific assignment of the sequence reads within the phylogeny, and to identify the most discriminative variable region to target. The analysis of high-throughput Pyrosequencing on human flora samples will accelerate the study of the relationship between the microbial world and ourselves.</p

Whole genome survey of coding SNPs reveals a reproducible pathway determinant of Parkinson disease

It is quickly becoming apparent that situating human variation in a pathway context is crucial to understanding its phenotypic significance. Toward this end, we have developed a general method for finding pathways associated with traits that control for pathway size. We have applied this method to a new whole genome survey of coding SNP variation in 187 patients afflicted with Parkinson disease (PD) and 187 controls. We show that our dataset provides an independent replication of the axon guidance association recently reported by Lesnick et al. [PLoS Genet 2007;3:e98], and also indicates that variation in the ubiquitin-mediated proteolysis and T-cell receptor signaling pathways may predict PD susceptibility. Given this result, it is reasonable to hypothesize that pathway associations are more replicable than individual SNP associations in whole genome association studies. However, this hypothesis is complicated by a detailed comparison of our dataset to the second recent PD association study by Fung et al. [Lancet Neurol 2006;5:911–916]. Surprisingly, we find that the axon guidance pathway does not rank at the very top of the Fung dataset after controlling for pathway size. More generally, in comparing the studies, we find that SNP frequencies replicate well despite technologically different assays, but that both SNP and pathway associations are globally uncorrelated across studies. We thus have a situation in which an association between axon guidance pathway variation and PD has been found in 2 out of 3 studies. We conclude by relating this seeming inconsistency to the molecular heterogeneity of PD, and suggest future analyses that may resolve such discrepancies

A Simple Method for Encapsulating Single Cells in Alginate Microspheres Allows for Direct PCR and Whole Genome Amplification

<div><p>Microdroplets are an effective platform for segregating individual cells and amplifying DNA. However, a key challenge is to recover the contents of individual droplets for downstream analysis. This paper offers a method for embedding cells in alginate microspheres and performing multiple serial operations on the isolated cells. <i>Rhodobacter sphaeroides</i> cells were diluted in alginate polymer and sprayed into microdroplets using a fingertip aerosol sprayer. The encapsulated cells were lysed and subjected either to conventional PCR, or whole genome amplification using either multiple displacement amplification (MDA) or a two-step PCR protocol. Microscopic examination after PCR showed that the lumen of the occupied microspheres contained fluorescently stained DNA product, but multiple displacement amplification with phi29 produced only a small number of polymerase colonies. The 2-step WGA protocol was successful in generating fluorescent material, and quantitative PCR from DNA extracted from aliquots of microspheres suggested that the copy number inside the microspheres was amplified up to 3 orders of magnitude. Microspheres containing fluorescent material were sorted by a dilution series and screened with a fluorescent plate reader to identify single microspheres. The DNA was extracted from individual isolates, re-amplified with full-length sequencing adapters, and then a single isolate was sequenced using the Illumina MiSeq platform. After filtering the reads, the only sequences that collectively matched a genome in the NCBI nucleotide database belonged to <i>R. sphaeroides</i>. This demonstrated that sequencing-ready DNA could be generated from the contents of a single microsphere without culturing. However, the 2-step WGA strategy showed limitations in terms of low genome coverage and an uneven frequency distribution of reads across the genome. This paper offers a simple method for embedding cells in alginate microspheres and performing PCR on isolated cells in common bulk reactions, although further work must be done to improve the amplification coverage of single genomes.</p></div

Illustration of the process workflow.

<p>1.) Cells are diluted in alginate polymer to a concentration of approximately 10⁵ cells per microliter, resulting in a 10% occupancy rate in 100 μm microspheres. 2.) Cells are lysed using heat, and the bulk microsphere solids are mixed with reagents for a 2-step whole genome amplification reaction. 3.) After amplification, microspheres are diluted to extinction in a 384 well plate, and scanned for presence of single microspheres that fluoresce with PicoGreen DNA stain. 4.) An isolated microsphere is transferred to a fresh tube and the DNA products are recovered by dissolving the alginate matrix. These amplified products are submitted to further rounds of amplification to add sequencing adapters. 5.) The products are prepared for high throughput sequencing using the Illumina MiSeq platform. Intermediate steps include fluorescence microscopy and quantitative PCR for quality control.</p

Results from Illumina MiSeq paired-end sequencing run, 2 × 150 bp.

<p>Results from Illumina MiSeq paired-end sequencing run, 2 × 150 bp.</p

Sequence coverage of the <i>Rhodobacter sphaeroides</i> genome after whole genome amplification.

<p>Sequence coverage of the <i>Rhodobacter sphaeroides</i> genome after whole genome amplification.</p

Strategy for WGA in two steps.

<p>This diagram outlines the whole genome amplification strategy in 2 steps, plus an additional step for adding the full length sequencing adapters. In Step 1, primers with 2 different tag sequences are added in a 50:50 mix. The primers anneal to the template, and a thermophilic strand-displacing enzyme (Vent exo- polymerase) is used to generate a population of fragments. For Step 2, the tag sequences are used as primers to further amplify the population of fragments. Half of the fragments are expected to have two different primer sequences on each end. After isolation of a microsphere containing fluorescent products, the sample is processed in a third reaction for addition of the full length sequencing adapters, or tails.</p

Primers for 2-step PCR whole genome amplification, tailing, and qPCR.

<p>Primers for 2-step PCR whole genome amplification, tailing, and qPCR.</p

Effects of cell loading rate, visualized before and after PCR.

<p>Microscope images of 100 μm alginate microspheres containing: (A) no cells, (B) one cell per microsphere, and (C) multiple cells per microsphere. After PCR or whole genome amplification (2-step method), the DNA is stained by GelGreen and visualized by fluorescence microscopy. Images show: (D) no amplification, (E) amplification that is characteristic for one cell per microsphere, and (F) amplification of many cells, showing multiple foci of amplification and “comet tails” from cells that are trapped in the border region of the microsphere.</p