Nucleic Acids Research, 2006, Vol. 34, Web Server issue W651–W655 doi:10.1093/nar/gkl125 SNPmasker: automatic masking of SNPs and repeats across eukaryotic genomes Reidar Andreson1,2, Tarmo Puurand1 and Maido Remm1,2,* 1Department of Bioinformatics, University of Tartu, Estonia and 2Estonian Biocentre, Tartu, Estonia Received February 14, 2006; Revised February 23, 2006; Accepted March 13, 2006 ABSTRACT SNPmasker is a comprehensive web interface for masking large eukaryotic genomes. The program is designed to mask SNPs from recent dbSNP database and to mask the repeats with two alternative pro- grams. In addition to the SNP masking, we also offer population-specific substitution of SNP alleles in genomic sequence according to SNP frequencies in HapMap Phase II data. The input to SNPmasker can be defined in chromosomal coordinates or inserted as a sequence. The sequences masked by our web server are most useful as a preliminary step for different primer and probe design tasks. The service is avail- able at http://bioinfo.ebc.ee/snpmasker/ and is free for all users. INTRODUCTION Human genome contains millions of single nucleotide poly- morphisms (SNPs). There are many different technologies for determining the alleles of SNP markers in human DNA samples (1). Most of these technologies use PCR and/or primer extension for analysis of SNPs. Unfortunately, primer-based technologies are sensitive to repeats and to variations in the genome. Repeats around SNPs may cause failure of assay or give mixed signals from different genomic regions. Variations may cause biased signal due to allele-speciﬁc binding of primers. The SNPs in human genome are not distributed uniformly. In the current dbSNP database (release 125) 2 million SNPs out of total number of ten million are located within 25 bp or less from another SNP. A previous study has demonstrated that closely located SNPs may have affected the performance of assays in the Human HapMap Project (2), because of the interference with primers/probes used in assays. Thus, it is important to avoid both repeats and SNPs within primers. The most efﬁcient way to avoid unwanted regions within primers is masking the template sequence before designing primers. DNA masking is typically done by replacing nucleotide regions with certain properties with ‘N’ characters, or by converting the nucleotides within the region to lower-case letters. Repeats are most frequently masked by the program called RepeatMasker (Smit,A.F.A., Hubley,R. and Green,P. http://www.repeatmasker.org/). Low- complexity regions are often masked by DUST program, a built-in part of the BLAST software package (3). Several programs exist that offer masking of SNP locations in user-deﬁned genomic regions. For example, the Genome Browser at UCSC website (http://genome.ucsc.edu/) allows retrieving masked DNA for user-speciﬁed regions. Both SNPs and the repeats can be masked with different options. Similar service with fewer options is offered by the SNP Research Facility at Washington University (http://snp. wustl.edu/bio-informatics/human-snp-annotation.html) and by the Institute of Human Genetics, GSF, Germany (http:// ihg.gsf.de/cgi-bin/snps/seq1.pl). All the previously mentioned websites offer retrieval of masked DNA by entering chromosomal coordinates. This is necessary because SNP locations are typically deﬁned by chromosomal coordinates. However, often users do not know the exact coordinates of their DNA region of interest in a given assembly. In this case the location of the query sequence within the genome should be determined before masking of SNPs. One way to do that is by sequence homology search. For example, SNP BLAST web interface at NCBI (http://www. ncbi.nlm.nih.gov/SNP/snpblastByChr.html) allows searching with user’s query sequence against the sequence database of SNP ﬂanking regions. After ﬁnding homologies, SNP BLAST highlights the differences between the query and the target sequence, including SNP positions. The server can also mask human repeats. Searching SNPs in other genomes represented in dbSNP (4) is possible, but the retrieval of longer genomic regions by coordinates is not possible with this program. Thus, the existing web pages allow the retrieval of masked sequences by chromosomal coordinates or by homology search, but none of them allows both (Table 1). We have put these functionalities for SNP and repeat- masking together into one web service. In addition, we have added a possibility to mask the repeats by a different and more specialized manner using a custom-made *To whom correspondence should be addressed. Tel: +372 7375001; Fax: +372 7420286; Email: maido.remm@ut.ee The Author 2006. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org