Minimum entropy decomposition : unsupervised oligotyping for sensitive partitioning of high-throughput marker gene sequences

© The Author(s), 2014. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in ISME Journal 9 (2015): 968–979, doi:10.1038/ismej.2014.195.Molecular microbial ecology investigations often employ large marker gene datasets, for example, ribosomal RNAs, to represent the occurrence of single-cell genomes in microbial communities. Massively parallel DNA sequencing technologies enable extensive surveys of marker gene libraries that sometimes include nearly identical sequences. Computational approaches that rely on pairwise sequence alignments for similarity assessment and de novo clustering with de facto similarity thresholds to partition high-throughput sequencing datasets constrain fine-scale resolution descriptions of microbial communities. Minimum Entropy Decomposition (MED) provides a computationally efficient means to partition marker gene datasets into ‘MED nodes’, which represent homogeneous operational taxonomic units. By employing Shannon entropy, MED uses only the information-rich nucleotide positions across reads and iteratively partitions large datasets while omitting stochastic variation. When applied to analyses of microbiomes from two deep-sea cryptic sponges Hexadella dedritifera and Hexadella cf. dedritifera, MED resolved a key Gammaproteobacteria cluster into multiple MED nodes that are specific to different sponges, and revealed that these closely related sympatric sponge species maintain distinct microbial communities. MED analysis of a previously published human oral microbiome dataset also revealed that taxa separated by less than 1% sequence variation distributed to distinct niches in the oral cavity. The information theory-guided decomposition process behind the MED algorithm enables sensitive discrimination of closely related organisms in marker gene amplicon datasets without relying on extensive computational heuristics and user supervision.AME was supported by a G. Unger Vetlesen Foundation grant to the Marine Biological Laboratory and the Alfred P Sloan Foundation

Genomic Data Reveal Toxoplasma gondii Differentiation Mutants Are Also Impaired with Respect to Switching into a Novel Extracellular Tachyzoite State

Toxoplasma gondii pathogenesis includes the invasion of host cells by extracellular parasites, replication of intracellular tachyzoites, and differentiation to a latent bradyzoite stage. We present the analysis of seven novel T. gondii insertional mutants that do not undergo normal differentiation to bradyzoites. Microarray quantification of the variation in genome-wide RNA levels for each parasite line and times after induction allowed us to describe states in the normal differentiation process, to analyze mutant lines in the context of these states, and to identify genes that may have roles in initiating the transition from tachyzoite to bradyzoite. Gene expression patterns in wild-type parasites undergoing differentiation suggest a novel extracellular state within the tachyzoite stage. All mutant lines exhibit aberrant regulation of bradyzoite gene expression and notably some of the mutant lines appear to exhibit high proportions of the intracellular tachyzoite state regardless of whether they are intracellular or extracellular. In addition to the genes identified by the insertional mutagenesis screen, mixture model analysis allowed us to identify a small number of genes, in mutants, for which expression patterns could not be accounted for using the three parasite states – genes that may play a mechanistic role in switching from the tachyzoite to bradyzoite stage

Disruption of the Expression of a Non-Coding RNA Significantly Impairs Cellular Differentiation in Toxoplasma gondii

The protozoan parasite Toxoplasma gondii is an important human and veterinary pathogen. Asexual replication of T. gondii in humans and intermediate hosts is characterized by two forms: rapidly growing “tachyzoites” and latent “bradyzoite” tissue cysts. Tachyzoites are responsible for acute illness and congenital neurological birth defects, while the more slowly dividing bradyzoite form can remain latent within the tissues for many years, representing a threat to immunocompromised patients. We have developed a genetic screen to identify regulatory genes that control parasite differentiation and have isolated mutants that fail to convert to bradyzoites. One of these mutants has an insertion disrupting a locus that encodes a developmentally regulated non-coding RNA transcript, named Tg-ncRNA-1. Microarray hybridizations suggest that Tg-ncRNA-1 is involved in the early steps of bradyzoite differentiation. Since Tg-ncRNA-1 does not contain an open reading frame, we used the algorithm Coding Potential Calculator (CPC) that evaluates the protein-coding potential of a transcript, to classify Tg-ncRNA-1. The CPC results strongly indicate that Tg-ncRNA-1 is a non-coding RNA (ncRNA). Interestingly, a previously generated mutant also contains an insertion in Tg-ncRNA-1. We show that both mutants have a decreased ability to form bradyzoites, and complementation of both mutants with wild-type Tg-ncRNA-1 restores the ability of the parasites to differentiate. It has been shown that an important part of bradyzoite differentiation is transcriptionally controlled, but this is the first time that a non-coding RNA is implicated in this process

A functional polymorphism in the promoter of the progesterone receptor gene associated with endometrial cancer risk

Excessive estrogen stimulation unopposed by progesterone strongly predisposes to endometrial cancer. Because the antiproliferative effect of progesterone requires the progesterone receptor (PR), which exists in two isoforms, PR-A and -B, we reasoned that variants in the PR gene may predispose to endometrial cancer. We found six variable sites, including four polymorphisms in the hPR gene and five common haplotypes. One promoter region polymorphism, +331G/A, creates a unique transcription start site. Biochemical assays showed that the +331G/A polymorphism increases transcription of the PR gene, favoring production of hPR-B in an endometrial cancer cell line. Using a case-control study nested within the Nurses' Health Study cohort, we observed a statistically significant association between the +331G/A polymorphism and the risk of endometrial cancer, which was even greater in overweight women carriers. After including a second population of controls, these associations remained intact. Our findings suggest that the +331G/A hPR gene polymorphism may contribute to endometrial cancer risk by increasing expression of the hPR-B isoform