Polymerase chain reaction (PCR) is a basic molecular biology technique with a multiplicity of uses, including deoxyribonucleic acid cloning and sequencing, functional analysis of genes, diagnosis of diseases, genotyping and discovery of genetic variants. Reliable primer design is crucial for successful PCR, and for over a decade, the open-source Primer3 software has been widely used for primer design, often in high-throughput genomics applications. It has also been incorporated into numerous publicly available software packages and web services. During this period, we have greatly expanded Primer3’s functionality. In this article, we describe Primer3’s current capabilities, emphasizing recent improvements. The most notable enhancements incorporate more accurate thermodynamic models in the primer design process, both to improve melting temperature prediction and to reduce the likelihood that primers will form hairpins or dimers. Additional enhancements include more precise control of primer placement—a change motivated partly by opportunities to use whole-genome sequences to improve primer specificity. We also added features to increase ease of use, including the ability to save and re-use parameter settings and the ability to require that individual primers not be used in more than one primer pair. We have made the core code more modular and provided cleaner programming interfaces to further ease integration with other software. These improvements position Primer3 for continued use with genome-scale data in the decade ahead.

Summary: The determination of annealing temperature is a critical step in PCR design. This parameter is typically derived from the melting temperature of the PCR primers, so for successful PCR work it is important to determine the melting temperature of primer accurately. We introduced several enhancements in the widely used primer design program Primer3. The improvements include a formula for calculating melting temperature and a salt correction formula. Also, the new version can take into account the effects of divalent cations, which are included in most PCR buffers. Another modification enables using lowercase masked template sequences for primer design. Availability: Features described in this article have been implemented into the development code of Primer3 and will be available in future versions (version 1.1 and newer) of Primer3. Also, a modified version is compiled under the name of mPrimer3 which is distributed independently. The web-based version of mPrimer3 is available at

Supplementary data are available at Bioinformatics online.

BackgroundFast, accurate and high-throughput identification of bacterial isolates is in great demand. The present work was conducted to investigate the possibility of identifying isolates from unassembled next-generation sequencing reads using custom-made guide trees.ResultsA tool named StrainSeeker was developed that constructs a list of specific k-mers for each node of any given Newick-format tree and enables the identification of bacterial isolates in 1–2 min. It uses a novel algorithm, which analyses the observed and expected fractions of node-specific k-mers to test the presence of each node in the sample. This allows StrainSeeker to determine where the isolate branches off the guide tree and assign it to a clade whereas other tools assign each read to a reference genome. Using a dataset of 100 Escherichia coli isolates, we demonstrate that StrainSeeker can predict the clades of E. coli with 92% accuracy and correct tree branch assignment with 98% accuracy. Twenty-five thousand Illumina HiSeq reads are sufficient for identification of the strain.ConclusionStrainSeeker is a software program that identifies bacterial isolates by assigning them to nodes or leaves of a custom-made guide tree. StrainSeeker’s web interface and pre-computed guide trees are available at . Source code is stored at GitHub: .

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Extended-spectrum beta-lactamases (ESBL) and AmpC producing-Escherichia coli have spread worldwide, but data about ESBL-producing-E. coli in the Northern and Eastern regions of Europe is scant. The aim of this study has been to describe the phenotypical and molecular epidemiology of different ESBL/AmpC/Carbapenemases genes in E. coli strains isolated from the Baltic States (Estonia, Latvia, and Lithuania), Norway and St. Petersburg (Russia), and to determine the predominant multilocus sequence type and single nucleotide polymorphisms diversity of E. coli isolates deduced by whole genome sequencing (WGS). A total of 10,780 clinical E. coli strains were screened for reduced sensitivity to third-generation cephalosporins. They were collected from 21 hospitals located in Estonia, Latvia, Lithuania, Norway and St. Petersburg during a 5 month period in 2012. The overall prevalence of ESBL/AmpC strains was 4.7% by phenotypical test and 3.9% by sequencing. We found more strains with the ESBL/AmpC phenotype and genotype in St. Petersburg and Latvia than other countries. Of phenotypic E. coli strains, 85% contained confirmed ESBL genes (including blaCTX–M, blaTEM–29, blaTEM–71), AmpC genes (blaCMY–59, blaACT–12/–15/–20, blaESC–6, blaFEC–1, blaDHA–1), or carbapenemase genes (blaNDM–1). blaCTX–M–1, blaCTX–M–14 and blaCTX–M–15 were found in all countries, but blaCTX–M–15 prevalence was higher in Latvia than in St. Petersburg (Russia), Estonia, Norway and Lithuania. The dominating AmpC genes were blaCMY–59 in the Baltic States and Norway, and blaDHA–1 in St. Petersburg. E. coli strains belonged to 83 different sequence types, of which the most prevalent was ST131 (40%). In conclusion, we generally found low ESBL/AmpC/Carbapenemase prevalence in E. coli strains isolated in Northern/Eastern Europe. However, several inter-country differences in distribution of particular genes and multilocus sequence types were found.

Motivation: The concentration of pathogen DNA in biological samples is often very low. Therefore, the sensitivity of diagnostic tests is always a critical factor.Results: We have developed a novel computational method that identifies species-specific repeats from microbial organisms and automatically designs species-specific PCR primers for these repeats. We tested the methodology on 30 randomly chosen microbial species and we demonstrate that species-specific repeats longer than 300 bp exist in all these genomes. We also used our methodology to design species-specific PCR primers for 86 repeats from five medically relevant microbial species. These PCR primers were tested experimentally. We demonstrate that using species-specific repeats as a PCR template region can increase the sensitivity of PCR in diagnostic tests.Availability and Implementation: A web version of the method called MultiMPrimer3 was implemented and is freely available at http://bioinfo.ut.ee/multimprimer3/.Contact: maido.remm@ut.eeSupplementary information: Supplementary data are available at Bioinformatics online.