limma is an R/Bioconductor software package that provides an integrated solution for analysing data from gene expression experiments. It contains rich features for handling complex experimental designs and for information borrowing to overcome the problem of small sample sizes. Over the past decade, limma has been a popular choice for gene discovery through differential expression analyses of microarray and high-throughput PCR data. The package contains particularly strong facilities for reading, normalizing and exploring such data. Recently, the capabilities of limma have been significantly expanded in two important directions. First, the package can now perform both differential expression and differential splicing analyses of RNA sequencing (RNA-seq) data. All the downstream analysis tools previously restricted to microarray data are now available for RNA-seq as well. These capabilities allow users to analyse both RNA-seq and microarray data with very similar pipelines. Second, the package is now able to go past the traditional gene-wise expression analyses in a variety of ways, analysing expression profiles in terms of co-regulated sets of genes or in terms of higher-order expression signatures. This provides enhanced possibilities for biological interpretation of gene expression differences. This article reviews the philosophy and design of the limma package, summarizing both new and historical features, with an emphasis on recent enhancements and features that have not been previously described.

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limma powers differential expression analyses for RNA-sequencing and microarray studies

A novel DNA fingerprinting technique called AFLP is described. The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The technique involves three steps: (i) restriction of the DNA and ligation of oligonucleotide adapters, (ii) selective amplification of sets of restriction fragments, and (iii) gel analysis of the amplified fragments. PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. The selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites. Using this method, sets of restriction fragments may be visualized by PCR without knowledge of nucleotide sequence. The method allows the specific co-amplification of high numbers of restriction fragments. The number of fragments that can be analyzed simultaneously, however, is dependent on the resolution of the detection system. Typically 50-100 restriction fragments are amplified and detected on denaturing polyacrylamide gels. The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity.

AFLP: a new technique for DNA fingerprinting.

The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online ( http://bioinf.spbau.ru/spades ). It is distributed as open source software.

SPAdes, a new genome assembly algorithm and its applications to single-cell sequencing ( 7th Annual SFAF Meeting, 2012)

The 4,639,221-base pair sequence of Escherichia coli K-12 is presented. Of 4288 protein-coding genes annotated, 38 percent have no attributed function. Comparison with five other sequenced microbes reveals ubiquitous as well as narrowly distributed gene families; many families of similar genes within E. coli are also evident. The largest family of paralogous proteins contains 80 ABC transporters. The genome as a whole is strikingly organized with respect to the local direction of replication; guanines, oligonucleotides possibly related to replication and recombination, and most genes are so oriented. The genome also contains insertion sequence (IS) elements, phage remnants, and many other patches of unusual composition indicating genome plasticity through horizontal transfer.

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The Complete Genome Sequence of Escherichia coli K-12

A survey is given of differential expression analyses using the linear modeling features of the limma package. The chapter starts with the simplest replicated designs and progresses through experiments with two or more groups, direct designs, factorial designs and time course experiments. Experiments with technical as well as biological replication are considered. Empirical Bayes test statistics are explained. The use of quality weights, adaptive background correction and control spots in conjunction with linear modelling is illustrated on the β7 data.

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limma: Linear Models for Microarray Data

The effect of site-specific methylation on restriction-modification enzymes

Background
Many cutting-edge microarray analysis tools and algorithms, including commonly used limma and affy packages in Bioconductor, need sophisticated knowledge of mathematics, statistics and computer skills for implementation. Commercially available software can provide a user-friendly interface at considerable cost. To facilitate the use of these tools for microarray data analysis on an open platform we developed an online microarray data analysis platform, WebArray, for bench biologists to utilize these tools to explore data from single/dual color microarray experiments.

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WebArray: an online platform for microarray data analysis.

Background Although chronic infections by typhoidal Salmonella are well-known, prolonged human infections by nontyphoidal Salmonella (NTS) are poorly characterized. Methods We retrospectively analyzed 48 345 culture-confirmed NTS infections that occurred in Israel 1995-2012. A case-control study was performed to identify risk factors associated with persistent infections. Whole-genome-sequencing, pulsed-field gel electrophoresis (PFGE), and a mouse infection model were used to study genetic and phenotypic differences between same-patient persistent, recurring isolates. Results In total, 1047 cases of persistent NTS infections, comprising 2.2% of all reported cases of salmonellosis, were identified. The persistence periods ranged between 30 days to 8.3 years. The majority (93%) of the persistently infected patients were immunocompetent, and 65% were symptomatic with relapsing diarrhea, indicating a distinct clinical manifestation from the asymptomatic carriage of typhoidal Salmonella. Four NTS serovars (Mbandaka, Bredeney, Infantis and Virchow) were found to be significantly more frequently associated with persistence than others. Comparative genomics between early and later isolates obtained from the same patients confirmed clonal infection and showed 0 to 10 SNPs between persistent isolates. A different composition of mobile genetic elements (plasmids and phages) or amino acid substitutions in global regulators was identified in multiple cases. These changes resulted in differences in phenotype and virulence between early and later same-patient isolates. Conclusions These results illuminate the overlooked clinical manifestation of persistent salmonellosis that can serve as a human reservoir for NTS infections. Additionally, we demonstrate mechanisms of in-host microevolution and exhibit their potential to shape Salmonella pathogenicity, antimicrobial resistance and host-pathogen interactions.

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Persistent Infections by Nontyphoidal Salmonella in Humans: Epidemiology and Genetics

We present in Table I an updated list of the sensitivities of 298 restriction endonucleases and 20 DNA methyltransferases to sitespecific modification at 4-methylcytosine (\"HT), 5-methylcytosine C^C), 5-hydroxymethylcytosine OTM^), and 6-methyladenine (^A) (McC14), four modifications that are common in the DNA of prokaryotes, eukaryotes, and their viruses (Mc2,Mc5,Mc8,Mcll,Ne3,Ne4). In addition, new information is included on restriction endonuclease cleavage at sites modified with 5-hydroxymethyluracil ( ^ U ) . Knowledge of the sensitivity of restriction endonucleases to site-specific modification can be used to study cellular DNA methylation. Several restriction-modification enzymes share the same recognition sequence specificity, but have different sensitivities to site-specific methylation. Table II lists 32 known isoschizomer pairs and one isomethylator pair, along with the modified recognition sites at which they differ. The data presented here and an additional three other tables are available in printed form or as a text file on a 3.5\" Macintosh diskette. The extra tables include Table HI which is a list of over 205 characterized DNA methyltransferases. A detailed list of cloned restriction-modification genes has been made by Wilson (Wi4). Table IV lists the sensitivities of over 20 Type II DNA methyltransferases to \"^C, C, C, and \"^A modification. Most DNA methyltransferases are sensitive to non-canonical modifications within their recognition sequences (Bu9,MclO, Ne3,Po4), and this sensitivity often differs from that of their restriction endonuclease partners. Table V gives a list of restriction systems in this review alphabetized by recognition sequence. The data can be supplied as a Microsoft Word, Macwrite or MS-DOS file. Please contact Michael McClelland at CTBR, phone 619 535 5486, FAX 619 535 5472.

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Effect of site-specific methylation on restriction endonucleases and DNA modification methyltransferases.

Cyclooxygenase-II (Cox-II) overexpression is involved in the progression of various subtypes of cancer. We investigated the significance of Cox-II in the progression of malignant melanomas (MMs). Using immunohistology we determined that Cox-II is not expressed in 70 benign and malignant melanocytic tumours. Basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) were also analysed as controls: the BCCs were consistently Cox-II negative (n = 11), whereas the SCCs showed moderate to strong Cox-II expression in 53% (n = 17). Reverse transcription-polymerase chain reaction and Western blotting of MM cell lines and MM tissues confirmed the lack of Cox-II expression in MM. However, in vitro the Cox-inhibiting non-steroidal anti-inflammatory drug (NSAID) sulindac sulphide (SIS) was significantly more effective in inducing apoptosis than sulindac sulphone (SOS), a derivative with a negligible effect on Cox (P < 0.01). The SIS doses needed for the induction of apoptosis were not significantly different in MM cell lines versus a Cox-II-positive colon carcinoma cell line (HT29). Furthermore, add-back experiments with high doses of the prostaglandins PGE2 and PGF2beta, major Cox-II products, did not abrogate this effect. We conclude that Cox-II expression is not involved in the progression of MM, and NSAID-induced apoptosis in MM cell lines seems to follow pathways independent of Cox-II. Nevertheless, Cox-II inhibitors are still candidates for therapy, though they act via an unknown mechanism.

Michael McClelland

Papers

The effect of site-specific methylation on restriction-modification enzymes

WebArray: an online platform for microarray data analysis.

Persistent Infections by Nontyphoidal Salmonella in Humans: Epidemiology and Genetics

Effect of site-specific methylation on restriction endonucleases and DNA modification methyltransferases.

Progression and NSAID-induced apoptosis in malignant melanomas are independent of cyclooxygenase II.