Immunoglobulin Genomics in the Guinea Pig Figure 5. Dot plot comparison of the guinea pig CH (m, e, c and a) region. A dot matrix representing repetitive sequences of guinea pig CH (m, e, c and a) genes. Switch regions are indicated by black-squared boxes and marked with arrowheads, and gaps are indicated by grey-squared boxes. Positions of CH genes are indicated as grey vertical lines. The dots represent homologies with a search length of 30 bp and maximum of 9 bp mismatches. doi:10.1371/journal.pone.0039298.g005 Sequence and Phylogenetic Analysis Multiple sequence alignments were edited and handled with the Megalign software program [16], and the Clustal W and Clustal X algorithms [17,18], before being analysed using BioEdit [19]. Comparative phylogenetic trees were constructed using the PHYLIP 3.67 [20] software and TreeView [21] based on the final nucleotide alignment. The neighbor-joining algorithm was used for phylogenetic analysis and bootstrap support was provided by 1000 replicates. Sequences from other species used in our phylogenetic analyses and sequence alignments are presented in Figures S1, S2, and S3 and Table S2. Dot Matrix Analysis Pairwise dot matrix comparisons were made using DNAMAN software (window size = 30-bp, mismatch limit = 9-bp) to identify potential alignment of nucleotide bases between the sequences. Definition of the VH/VL Gene Families In mammals, germline VH and VL genes are categorized into different families according to their amino acid or nucleotide sequences similarity [22]. Sequences with greater than 75% similarity are general considered to belong to the same family, while those with less than 70% similarity are placed in different gene families, and those possessing between 70% and 75% similarity are inspected on a case-by-case basis [23]. We placed potentially functional VH and VL gene segments sharing more than 70% similarity into the same family. Results Guinea Pig IgH Locus Analysis of the genomic sequence revealed that the guinea pig IgH locus is located within genomic scaffolds 54 and 75 (Figure 1, Figure 2). The entire IgH locus spans approximately 6,480 kb of the two scaffolds (4,302 kb in scaffold 54 and 2,178 kb in scaffold 75). The total length is an estimate due to the existence of sequence gaps (Figure 1, Figure 2). Six JH segments, 507 VH, 41 DH, four constant region genes (m, c, e and a) and a reverse d trace (marked with an arrow towards the left) were identified within the two scaffolds. Locations of the annotated IgH genes on the guinea pig genome are displayed in Table S3. PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e39298