W652 Nucleic Acids Research, 2006, Vol. 34, Web Server issue Table 1. Comparison of different web pages for masking SNPs and repeats Genome browser UCSC SNP Research Facility Washington University in St. Louis GSF Munich, Germany SNP BLAST NCBI SNPmasker University of Tartu SNP masking types lower-case, by color, bold/italic IUPAC ‘N’ IUPAC any character, IUPAC, lower-case, by HapMap frequency Repeat-masking programs RepeatMasker RepeatMasker RepeatMasker RepeatMasker RepeatMasker, GenomeMasker Region defined by coordinates Region found by sequence homology search Yes Yes Yes No Yes No No No Yes Yes program called GenomeMasker (5) and a possibility to change SNP alleles in a sequence. IMPLEMENTATION Input SNPmasker is currently able to mask sequences from two genomes: human and mouse. However, the program can easily be conﬁgured to accept additional genomes from the ENSEMBL database (6). The sequence of interest can be deﬁned in two principally different ways: by chromosomal coordinates and by sequence (Figure 1). The sequence can be inserted by pasting it into the text box or uploading ﬁle in FASTA format. Databases SNPmasker uses sequence databases from ENSEMBL (ftp:// ftp.ensembl.org/pub/release-35/). For each genome, all tables from database homo_sapiens_core_35_35h and from mus_musculus_35_34c are installed. SNP locations are retrieved from dbSNP database (ftp://ftp.ncbi.nih.gov/snp/). Additional database is required for storing HapMap (7) allele frequency data, which we create locally. The HapMap data- base is created by counting and storing allele frequencies of each SNP in each population. The counting is done by using tables downloaded from the Phase 2 database (http://www. hapmap.org/genotypes/2006-01/non-redundant/). Localization of input sequences SNP masking can only be done if the location of a sequence in the genome is known. If the location is not deﬁned by the user, then it must be found by a homology search. The homology search is performed by MEGABLAST (8) program against chromosome sequences. The location is considered unique if MEGABLAST ﬁnds a single match with length of 90% of query sequence that must have >90% identity with the target genome. The query sequence must contain at least one 100 bp long exact match (or 16 bp, if the user-given sequence is shorter than 100 bp). If a unique location cannot be determined by MEGABLAST, the masking is cancelled. In this case MEGABLAST alignments are presented to the user for further analysis and corrections of the input sequences/coordinates. Masking of SNPs The program has two major functionalities that can be used either together or separately—the masking of SNP positions and the masking of repeats. SNP masking is implemented as follows. Once the location is determined by the user or by a homology search, the program compares the coordinates of sequence with the coordinates of known SNPs and veriﬁes whether the given sequence region contains any SNPs. If a SNP is found within the sequence, it is masked by replacing the existing character with a lower-case character, any user- deﬁned symbol or an IUPAC symbol. A unique option, not offered by other similar services, is changing the sequence according to SNP major allele nucleotide—the nucleotide that is most frequent in certain human populations. This option is available for the human genome only and is based on the HapMap Phase2 data (public release #20), offering separate masking for CEPH, Japanese, Chinese and African (Yoruban) populations. When comparing the human genomic sequence with the HapMap genotype data, we discovered that in the current Golden Path sequence about 25% of SNP positions ( 900 000 nt over the whole genome) are representing a minor allele—the less frequent variant of a nucleotide. These ﬁgures are similar for all four populations represented in the HapMap. Changing sequence at these positions to represent most frequent allele in a given population might be useful for research projects, which are concentrating on individuals (or cell lines) from a given population only. Deletions or insertions are currently not masked. Masking of repeats Masking of repeats is optional. If the repeats are masked, the masking can be performed with either RepeatMasker or GenomeMasker. GenomeMasker is a novel masking program that was developed speciﬁcally for the PCR primer design and therefore has several differences compared to the traditional masking programs like DUST or RepeatMasker. Genome- Masker exhaustively counts all 16 nt motifs in a given genome and masks the abundant (>10 occurrences by default) motifs. Because PCR primers are single-stranded, this masking method is also strand-speciﬁc—the ‘upper’ and ‘lower’ strands can be masked separately, if necessary. This is useful for the PCR primer design around the markers or for the other target regions in the genome. For the PCR primer design only the upper strand should be masked on the left side of the target region and only the lower strand should be masked on the right side of the target region. Primer design from the lower- case-masked sequences is facilitated by the program called GM_Primer3 (executable available at http://bioinfo.ebc.ee/ download/, an on-line version can be found at http:// bioinfo.ebc.ee/cgi-bin/primer3_www.cgi). The GM_Primer3 is essentially a modiﬁed version of Primer3 with additional