BMC Genomics 2008, 9:122 http://www.biomedcentral.com/1471-2164/9/122 Table 1: Bi-codon frequencies. The third column lists those codons c for the first amino acid (AA1) in a consecutive pair which lead to a stop in frame -1. The overall relative frequency of c as a codon for AA1 is given in the fifth column. The relative frequency of c in the specific context AA1-AA2 is given in column six. The penultimate column shows the enrichment of the codon c in this context; an enrichment > 1 indicates that this codon is more frequently used for AA1 when it leads to a stop in frame -1 than in the average case. Probability (P) values were assigned using the χ2 test, and significance determined with a Bonferroni multiple testing correction (15 analyses). Single (*) and triple (***) asterisks indicate significance at the 0.05 and 0.001 levels, respectively. AA1 AA2 First codon leading to stop in frame -1 Overall frequency Frequency when leading to stop Enrichment P A C D F G H I L N P R S T V Y W Q M K E D D D D D D D D D D D D D D D D D D D D GCT TGT GAT TTT GGT CAT ATT CTT AAT CCT CGT TCT, AGT ACT GTT TAT - - - - - 27 45 46 46 16 42 36 13 47 29 8 34 25 18 44 - - - - - 38 80 78 80 23 69 55 12 78 36 9 51 31 19 75 - - - - - chemokine family. Chemokines are typically quite poorly conserved, and as such provide an indication of how much sequences may diverge while retaining similar func- tions. For instance, half of mouse-human chemokine orthologs in Homologene [10,11] have an amino acid sequence identity of 60–70% (from 32 entries in total). Since a 60% cut-off seems suitable for this family, and we were interested in detecting possible new peptide ligands, it was chosen as a threshold for the matreshkas. A number of matreshkas mapped to alternative, frameshifted splice variants (see Additional file 2). In some cases, there is evidence that these variants are func- tionally distinct. Among these are the paired box gene 8 (PAX8) isoform c [12], and X-box protein (XBP1) isoform U [13]. Given the nucleotide sequence similarity between tran- scripts in orthologous triplets, conserved translation prod- ucts in alternative reading frames would be expected to occur by chance. Additional parameters were required to differentiate likely matreshkas from false positives. Gener- ally, longer matreshkas are less likely to arise by chance, therefore matreshka length was chosen as a first parameter for selection. A second useful parameter gave an approxi- mate measure of the selection pressure maintaining a potential matreshka: this was the number of amino acid positions where a stop codon could have arisen, truncat- ing the matreshka but leaving the parent (RefSeq) protein Table 2: Matreshka statistics. Matreshka statistics for tORFs and met-tORFs (generated from a separate run of the analysis, see text), broken down by frame. Note that tORFM are redundant (see Methods). # Human-Mouse-Rat ortholog triplets 9163 Frame +1 Frame -1 Frame -1 as a percentage of total # tORFX # matreshkaX # tORFM # matreshkaM 24 746 5 764 18 030 1 689 20 240 1 915 1 711 104 Total 44 986 7679 19 741 1793 45% 25% 9% 6% 1.4 1.8 1.7 1.7 1.4 1.6 1.5 0.9 1.7 1.2 1.1 1.5 1.2 1.1 1.7 - - - - - 0.034 1.814e-07 *** 2.38e-06 *** 5.358e-07 *** 0.08 3.097e-05 *** 0.002 * 0.782 6.131e-06 *** 0.194 0.724 0.004 * 0.230 0.814 2.962e-06 *** Page 6 of 16 (page number not for citation purposes)