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.

/pdf/limma-linear-models-for-microarray-data-29cymqv608.pdf

limma: Linear Models for Microarray Data

High-Resolution Restriction Mapfora240-Kilobase Region Spanning 91to96Minutes ontheSalmonella typhimurium LT2Chromosome

: Serendipitously, the bacterium Salmonella accumulates 1000-fold more in tumors than in normal tissue and we have shown that Salmonella sometimes cures cancer in animal models. We wished to improve Salmonella as a therapeutic system for cancer. In this first funding period there were three objectives and all three were achieved. First, in Aim 1, task 1, we were able to identify over 100 gene mutants of Salmonella that are better than any current vaccine strains (safe bacteria) at surviving in tumors but were less virulent than wild type bacteria. These mutants are potentially useful for an improved delivery agent. Second, we had previously identified promoters that were preferentially activated in tumors when compared to spleen. In Aim 2, task 1, we further characterized these regions, which are potentially useful to activate therapeutic genes engineered into the bacterium only when the bacterium is in the tumor. Third, in Aim 2, task 2, we screened promoters in vitro for their response to pH (tumors are more acidic than the rest of the body) and anoxia (tumors have lower O2 levels than the rest of the body). A comprehensive survey was performed of all candidate promoters. We also developed a set of bioinformatics programs to help perform, store, analyze, and interpret these experiments, as a foundation for year two of the project.

Engineering Improvements in a Bacterial Therapeutic Delivery System for Breast Cancer

La presente invention concerne une methode pour mesurer le niveau de deux molecules d'acide nucleique ou plus dans une cible par mise en contact d'une sonde et d'une cible renfermant au moins deux molecules d'acide nucleique. Ces molecules d'acide nucleique sont prelevees de facon arbitraire et renferment un sous-ensemble de molecules d'acide nucleique dans une population de molecules d'acide nucleique. Cette methode permet egalement de detecter l'importance de la liaison specifique entre la cible et la sonde. De plus, l'invention concerne une methode pour mesurer le niveau de deux molecules d'acide nucleique ou plus dans une cible par mise en contact d'une sonde et d'une cible renfermant au moins deux molecules d'acide nucleique. Ces molecules d'acide nucleique sont prelevees de facon statistique et renferment un sous-ensemble de molecules d'acide nucleique dans une population de molecules d'acide nucleique. Cette methode permet egalement de detecter l'importance de la liaison specifique entre la cible et la sonde.

Cibles d'acide nucleique de moindre complexite et leurs methodes d'utilisation

La presente invention concerne une methode permettant de determiner un ensemble de paires d'amorces destinees a l'amplification d'un groupe de sequences nucleotidiques associees. La methode de l'invention consiste a identifier un groupe de sequences nucleotidiques associees; a produire l'ensemble d'amorces correspondant a chacune des sequences nucleotidiques associees; a determiner pour chaque appariement systematique de chacune des amorces quelles sont les sequences nucleotidiques associees amplifiees; et a selectionner a partir des appariements systematiques, un sous-ensemble amplifiant toutes les sequences nucleotidiques associees. L'invention concerne egalement une methode d'utilisation d'un ensemble de paires d'amorces, methode qui consiste a amplifier un groupe de sequences nucleotidiques associees pour identifier des sequences nucleotidiques associees au groupe de sequences nucleotidiques d'origine. L'invention concerne aussi un dispositif informatique permettant de realiser les differentes etapes, executees de maniere informatisee, de l'invention. L'invention concerne enfin un logiciel comprenant un milieu vecteur de signaux permettant de mettre en oeuvre la methode precitee.

Michael McClelland

Papers

High-Resolution Restriction Mapfora240-Kilobase Region Spanning 91to96Minutes ontheSalmonella typhimurium LT2Chromosome

Engineering Improvements in a Bacterial Therapeutic Delivery System for Breast Cancer

Cibles d'acide nucleique de moindre complexite et leurs methodes d'utilisation

Selection de paires d'amorces pcr destinees a l'amplification d'un groupe de sequences nucleotidiques

Identifizierung von Organismen durch Detektion intronischer Nukleinsäuren oder kodierter Proteine