DNA-based microarrays are increasingly central to biomedical research. Selecting oligonucleotide sequences that will behave consistently across experiments is essential to the design, production and performance of DNA microarrays. Here our aim was to improve on probe design parameters by empirically and systematically evaluating probe performance in a multivariate context. We used experimental data from 19 array CGH hybridizations to assess the probe performance of 385,474 probes tiled in the Duchenne muscular dystrophy (DMD) region of the X chromosome. Our results demonstrate that probe melting temperature, single nucleotide polymorphisms (SNPs), and homocytosine motifs all have a strong effect on probe behavior. These findings, when incorporated into future microarray probe selection algorithms, may improve microarray performance for a wide variety of applications.
The ability to distinguish microbial pathogens from closely related but nonpathogenic strains is key to understanding the population biology of these organisms. In this regard, Bacillus anthracis, the bacterium that causes inhalational anthrax, is of interest because it is closely related and often difficult to distinguish from other members of the B. cereus group that can cause diverse diseases. We employed custom-designed resequencing arrays (RAs) based on the genome sequence of Bacillus anthracis to generate 422 kb of genomic sequence from a panel of 41 Bacillus cereus sensu lato strains. Here we show that RAS represent a "one reaction" genotyping technology with the ability to discriminate between highly similar B. anthracis isolates and more divergent strains of the B. cereus s I Clade 1. Our data show that RAs can be an efficient genotyping technology for pre-screening the genetic diversity of large strain collections to selected the best candidates for whole genome sequencing.
Background
Fragile X syndrome (FXS) is caused by loss of function mutations in the FMR1 gene. Trinucleotide CGG-repeat expansions, resulting in FMR1 gene silencing, are the most common mutations observed at this locus. Even though the repeat expansion mutation is a functional null mutation, few conventional mutations have been identified at this locus, largely due to the clinical laboratory focus on the repeat tract.
Methodology/Principal Findings
To more thoroughly evaluate the frequency of conventional mutations in FXS-like patients, we used an array-based method to sequence FMR1 in 51 unrelated males exhibiting several features characteristic of FXS but with normal CGG-repeat tracts of FMR1. One patient was identified with a deletion in FMR1, but none of the patients were found to have other conventional mutations.
Conclusions/Significance
These data suggest that missense mutations in FMR1 are not a common cause of the FXS phenotype in patients who have normal-length CGG-repeat tracts. However, screening for small deletions of FMR1 may be of clinically utility.
by
Peter E. Chen;
Kristin M. Willner;
Amy Butani;
Shakia Dorsey;
Matroner George;
Andrew Stewart;
Shannon M. Lentz;
Christopher E. Cook;
Arya Akmal;
Lance B. Price;
Paul S. Keim;
Alfred Mateczun;
Trupti N. Brahmbhatt;
Kimberly A. Bishop-Lilly;
Michael Zwick;
Timothy Read;
Shanmuga Sozhamannan
Background: The anthrax letter attacks of 2001 highlighted the need for rapid identification of biothreat agents not only for epidemiological surveillance of the intentional outbreak but also for implementing appropriate countermeasures, such as antibiotic treatment, in a timely manner to prevent further casualties. It is clear from the 2001 cases that survival may be markedly improved by administration of antimicrobial therapy during the early symptomatic phase of the illness; i.e., within 3 days of appearance of symptoms. Microbiological detection methods are feasible only for organisms that can be cultured in vitro and cannot detect all genetic modifications with the exception of antibiotic resistance. Currently available immuno or nucleic acid-based rapid detection assays utilize known, organism-specific proteins or genomic DNA signatures respectively. Hence, these assays lack the ability to detect novel natural variations or intentional genetic modifications that circumvent the targets of the detection assays or in the case of a biological attack using an antibiotic resistant or virulence enhanced Bacillus anthracis, to advise on therapeutic treatments. Methodology/Principal Findings: We show here that the Roche 454-based pyrosequencing can generate whole genome draft sequences of deep and broad enough coverage of a bacterial genome in less than 24 hours. Furthermore, using the unfinished draft sequences, we demonstrate that unbiased identification of known as well as heretofore-unreported genetic modifications that include indels and single nucleotide polymorphisms conferring antibiotic and phage resistances is feasible within the next 12 hours. Conclusions/Significance: Second generation sequencing technologies have paved the way for sequence-based rapid identification of both known and previously undocumented genetic modifications in cultured, conventional and newly emerging biothreat agents. Our findings have significant implications in the context of whole genome sequencing-based routine clinical diagnostics as well as epidemiological surveillance of natural disease outbreaks caused by bacterial and viral agents.
by
David J Cutler;
Michael Zwick;
David T. Okou;
Sampath Prahalad;
Thomas Walters;
Stephen L. Guthery;
Marla Dubinsky;
Robert Baldassano;
Wallace V. Crandall;
Joel Rosh ;
James Markowitz;
Michael Stephens;
Richard Kellermayer;
Marian Pfefferkon;
Melvin B. Heyman;
Neal LeLeiko;
David Mack;
Dedrick Moulton;
Michael D. Kappelman;
Archana Kumar;
Jarod Prince;
Promita Bose;
Kajari Mondal;
Dhanya Ramchandran;
John F. Bohnsack;
Anne M. Griffiths ;
Yael Haberman;
Jonah Essers;
Susan D. Thompson;
Bruce Aronow;
David J. Keljo;
Jeffrey S. Hyams;
Lee A. Denson;
Subra Kugathasan
The inflammatory bowel diseases (IBD) are common, complex disorders in which genetic and environmental factors are believed to interact leading to chronic inflammatory responses against the gut microbiota. Earlier genetic studies performed in mostly adult population of European descent identified 163 loci affecting IBD risk, but most have relatively modest effect sizes, and altogether explain only ~20% of the genetic susceptibility. Pediatric onset represents about 25% of overall incident cases in IBD, characterized by distinct disease physiology, course and risks. The goal of this study is to compare the allelic architecture of early onset IBD with adult onset in population of European descent.