GENOMEMASKER package for designing unique genomic PCR primers

Abstract

Background: The design of oligonucleotides and PCR primers for studying large genomes is complicated by the redundancy of sequences. The eukaryotic genomes are particularly difficult to study due to abundant repeats. The speed of most existing primer evaluation programs is not sufficient for large-scale experiments.

Results: In order to improve the efficiency and success rate of automatic primer/oligo design, we created a novel method which allows rapid masking of repeats in large sequence files, for example in eukaryotic genomes. It also allows the detection of all alternative binding sites of PCR primers and the prediction of PCR products. The new method was implemented in a collection of efficient programs, the GENOMEMASKER package. The performance of the programs was compared to other similar programs. We also modified the PRIMER3 program, to be able to design primers from lowercase-masked sequences.

Conclusion: The GENOMEMASKER package is able to mask the entire human genome for non-unique primers within 6 hours and find locations of all binding sites for 10,000 designed primer pairs within 10 minutes. Additionally, it predicts all alternative PCR products from large genomes for given primer pairs.

Figure 1

The workflow of the two separate units in GENOMEMASKER package: GenomeMasker (A) and GenomeTester (B). Boxes with rectangular corners describe data structures, rounded boxes describe procedures performed by different programs and boxes with bold outlines denote the main input files. Procedures indicated by dashed lines are optional.

Figure 2

Examples of masking style of different masking programs. Masked nucleotides are shown by red lowercase letters. There are some DNA regions that are extensively masked by RepeatMasker (A) and some regions where RepeatMasker does not find any repetitive motifs (B). Both programs are executed at similar sensitivity level.

Figure 3

The speed test between two masking methods: RepeatMasker and GenomeMasker. Both programs were tested with several parameters and different template sizes. Masking of the entire human genome takes 6–7 hours with the GenomeMasker. Programs were executed on a 2.66 GHz Intel Xeon™ processor with 6 GB of RAM.

Figure 4

The performance test between various alignment and e-PCR methods. We compared the speed of seven different methods with five datasets, consisting of 1, 10, 100, 1000 and 10000 randomly selected primer pairs. All datasets were subjected to the e-PCR against the human genome with different programs and the computing time was recorded. All programs were executed on a 2.66 GHz Intel Xeon™ processor with 6 GB of RAM.