Abstract
Development and use of primer sets to amplify nucleic acid sequences of interest is fundamental to studies spanning many life science disciplines. As such, the validation of primer sets is essential. Several computer programs have been created to aid in the initial selection of primer sequences that may or may not require multiple nucleotide combinations (i.e., degeneracies). Conversely, validation of primer specificity has remained largely unchanged for several decades, and there are currently few available programs that allows for an evaluation of primers containing degenerate nucleotide bases. To alleviate this gap, we developed the program De-MetaST that performs an in silico amplification using user defined nucleotide sequence dataset(s) and primer sequences that may contain degenerate bases. The program returns an output file that contains the in silico amplicons. When De-MetaST is paired with NCBI’s BLAST (De-MetaST-BLAST), the program also returns the top 10 nr NCBI database hits for each recovered in silico amplicon. While the original motivation for development of this search tool was degenerate primer validation using the wealth of nucleotide sequences available in environmental metagenome and metatranscriptome databases, this search tool has potential utility in many data mining applications.
Highlights
PCR is one of the most fundamental and powerful molecular biology tools available
We have developed a computational method to generate in silico amplifications from degenerate primer sets searched against user defined nucleotide databases
To illustrate the utility of DeMetaST-BLAST, we demonstrate its performance using a novel degenerate primer set designed for use on environmental samples
Summary
PCR is one of the most fundamental and powerful molecular biology tools available. PCR primer sets that contain degenerate bases allow for the amplification of homologous sequences and have been used in various applications, including genetic diversity analyses (e.g., [1,2,3,4,5,6,7,8,9,10,11,12]). The most common practice for initial validation of degenerate primers is by direct sequence analysis of PCR amplicons (e.g., [33,34,35,36,37]). This can be both laborious and costly, and does not take advantage of the everincreasing publicly available nucleotide data, including that derived from natural samples. Environmental metagenomes and metatranscriptomes are especially attractive reference databases (e.g., CAMERA [38] [http://camera.calit2.net/] and MG-RAST [http://metagenomics.anl.gov/]) to perform in silico tests en masse to identify sequences a degenerate primer set might amplify
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