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

High throughput screens reveal Salmonella behaviors required for persistence in tomatoes

Vertical arrays are microarrays that have complex mixtures of nucleic acids as array elements, and that are hybridized with single sequence probes. Like dot blots, many different experiments can be spotted on a single vertical array, allowing single genes to be compared across many conditions. Vertical arrays have two additional advantages over dot blots. First, they are printed on glass slides, allowing the use of low-volume, high-concentration hybridization reactions. Second, they can be made using low-complexity representations of the original nucleic acid population. This increases signal-to-noise relative to the usual use of dot blots, wherein the entire complexity of the population is usually spotted. Whereas standard microarrays achieve horizontal coverage of many genes and are repeated to cover many experiments, vertical arrays achieve vertical coverage of many experiments and are repeated to cover many genes. In cases where the number of genes is limited, but the number of experiments is very large, vertical arrays may be advantageous.

Vertical arrays: microarrays of complex mixtures of nucleic acids.

Methylation at overlapping dam (Gm6ATC) sites does not block cleavage by the NruI (TCGCGA) isoschizomer restriction endonucleases AmaI, SalDI and Sbo13I.

PROBLEM TO BE SOLVED: To provide methods for determining gene expression levels in one or more cell types in heterogeneous cell samples.SOLUTION: The method can include: (a) a step of determining the relative content of each cell type in two or more related heterogeneous cell samples, wherein at least two of the samples do not contain the same relative content of each cell type; (b) a step of measuring overall levels of one or more gene expression analytes in each sample; (c) a step of determining the regression correlation between the relative content of each cell type and the measured overall levels; and (d) a step of calculating the level of each of the one or more analytes in each cell type according to the regression correlation determined in the step (c), wherein gene expression levels correspond to the calculated levels of analytes.

Cell-type-specific pattern of gene expression

The regulation of gene expression in higher eukaryotes continues to be a primary area of investigation in molecular biology. Delineation of the mechanisms by which deoxyribonucleic acid (DNA) coding sequences are first transcribed into ribonucleic acid (RNA) and ultimately translated into proteins is essential to an understanding of cancer and other diseases as well as to rational drug design.

Michael McClelland

Papers

High throughput screens reveal Salmonella behaviors required for persistence in tomatoes

Vertical arrays: microarrays of complex mixtures of nucleic acids.

Methylation at overlapping dam (Gm6ATC) sites does not block cleavage by the NruI (TCGCGA) isoschizomer restriction endonucleases AmaI, SalDI and Sbo13I.

Cell-type-specific pattern of gene expression

RNA Arbitrarily Primed PCR