Dataset Information

PrimerProspector: de novo design and taxonomic analysis of barcoded polymerase chain reaction primers.

ABSTRACT:

Motivation

PCR amplification of DNA is a key preliminary step in many applications of high-throughput sequencing technologies, yet design of novel barcoded primers and taxonomic analysis of novel or existing primers remains a challenging task.

Results

PrimerProspector is an open-source software package that allows researchers to develop new primers from collections of sequences and to evaluate existing primers in the context of taxonomic data.

Availability

PrimerProspector is open-source software available at http://pprospector.sourceforge.net

Contact

rob.knight@colorado.edu

Supplementary information

Supplementary data are available at Bioinformatics online.

SUBMITTER: Walters WA

PROVIDER: S-EPMC3072552 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

PrimerProspector: de novo design and taxonomic analysis of barcoded polymerase chain reaction primers.

Walters William A WA Caporaso J Gregory JG Lauber Christian L CL Berg-Lyons Donna D Fierer Noah N Knight Rob R

Bioinformatics (Oxford, England) 20110223 8

<h4>Motivation</h4>PCR amplification of DNA is a key preliminary step in many applications of high-throughput sequencing technologies, yet design of novel barcoded primers and taxonomic analysis of novel or existing primers remains a challenging task.<h4>Results</h4>PrimerProspector is an open-source software package that allows researchers to develop new primers from collections of sequences and to evaluate existing primers in the context of taxonomic data.<h4>Availability</h4>PrimerProspector ...[more]

PMID: 21349862

Similar Datasets

Project description:Choosing appropriate primers is probably the single most important factor affecting the polymerase chain reaction (PCR). Specific amplification of the intended target requires that primers do not have matches to other targets in certain orientations and within certain distances that allow undesired amplification. The process of designing specific primers typically involves two stages. First, the primers flanking regions of interest are generated either manually or using software tools; then they are searched against an appropriate nucleotide sequence database using tools such as BLAST to examine the potential targets. However, the latter is not an easy process as one needs to examine many details between primers and targets, such as the number and the positions of matched bases, the primer orientations and distance between forward and reverse primers. The complexity of such analysis usually makes this a time-consuming and very difficult task for users, especially when the primers have a large number of hits. Furthermore, although the BLAST program has been widely used for primer target detection, it is in fact not an ideal tool for this purpose as BLAST is a local alignment algorithm and does not necessarily return complete match information over the entire primer range.We present a new software tool called Primer-BLAST to alleviate the difficulty in designing target-specific primers. This tool combines BLAST with a global alignment algorithm to ensure a full primer-target alignment and is sensitive enough to detect targets that have a significant number of mismatches to primers. Primer-BLAST allows users to design new target-specific primers in one step as well as to check the specificity of pre-existing primers. Primer-BLAST also supports placing primers based on exon/intron locations and excluding single nucleotide polymorphism (SNP) sites in primers.We describe a robust and fully implemented general purpose primer design tool that designs target-specific PCR primers. Primer-BLAST offers flexible options to adjust the specificity threshold and other primer properties. This tool is publicly available at http://www.ncbi.nlm.nih.gov/tools/primer-blast.

Project description:Nitrification, the oxidation of ammonia via nitrite to nitrate, has always been considered to be catalyzed by the concerted activity of ammonia- and nitrite-oxidizing microorganisms. Only recently, complete ammonia oxidizers ("comammox"), which oxidize ammonia to nitrate on their own, were identified in the bacterial genus Nitrospira, previously assumed to contain only canonical nitrite oxidizers. Nitrospira are widespread in nature, but for assessments of the distribution and functional importance of comammox Nitrospira in ecosystems, cultivation-independent tools to distinguish comammox from strictly nitrite-oxidizing Nitrospira are required. Here we developed new PCR primer sets that specifically target the amoA genes coding for subunit A of the distinct ammonia monooxygenase of comammox Nitrospira. While existing primers capture only a fraction of the known comammox amoA diversity, the new primer sets cover as much as 95% of the comammox amoA clade A and 92% of the clade B sequences in a reference database containing 326 comammox amoA genes with sequence information at the primer binding sites. Application of the primers to 13 samples from engineered systems (a groundwater well, drinking water treatment and wastewater treatment plants) and other habitats (rice paddy and forest soils, rice rhizosphere, brackish lake sediment and freshwater biofilm) detected comammox Nitrospira in all samples and revealed a considerable diversity of comammox in most habitats. Excellent primer specificity for comammox amoA was achieved by avoiding the use of highly degenerate primer preparations and by using equimolar mixtures of oligonucleotides that match existing comammox amoA genes. Quantitative PCR with these equimolar primer mixtures was highly sensitive and specific, and enabled the efficient quantification of clade A and clade B comammox amoA gene copy numbers in environmental samples. The measured relative abundances of comammox Nitrospira, compared to canonical ammonia oxidizers, were highly variable across environments. The new comammox amoA-targeted primers enable more encompassing future studies of nitrifying microorganisms in diverse habitats. For example, they may be used to monitor the population dynamics of uncultured comammox organisms under changing environmental conditions and in response to altered treatments in engineered and agricultural ecosystems.

Dataset Information

PrimerProspector: de novo design and taxonomic analysis of barcoded polymerase chain reaction primers.

Motivation

Results

Availability

Contact

Supplementary information

Publications

PrimerProspector: de novo design and taxonomic analysis of barcoded polymerase chain reaction primers.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets