Patterns of positive selection on the mammalian tree Supplementary Material

To accurately identify orthologous sequences in the six mammalian species, we used syntenic whole-genome alignments produced by the MULTIZ program [1] on the following assemblies available through the UCSC genome browser [2]: human (hg18), chimpanzee (panTro2), macaque (rheMac2), mouse (mm8), rat (rn4), and dog (canFam2). The human genome was used as a reference genome. We used the pairwise syntenic nets [3] with respect to human genome as an input to MULTIZ pipeline to reduce the likelihood of paralogous alignments. We compensated for low sequence quality in some regions by masking the bases in chimpanzee, macaque, rat, and dog with sequence quality lower than 20. To identify genes within these alignments, we used the union of all human annotated genes in RefSeq [4], UCSC Known Genes [5], and VEGA [6], downloaded from the UCSC genome browser on February 19, 2007. We discarded transcripts without annotated coding regions (CDSs), CDSs shorter than 100 bp, as well as CDSs with length other than multiple of three. Clustering of the remaining 88,879 transcripts (some of them may be duplicates from different annotation databases) resulted in an initial set of 21,115 gene clusters in the human genome. We then used our syntenic alignments to map these genes to other five mammalian species. For the purpose of the positive selection analysis, we are interested in a high-confidence set of orthologous alignments. To ensure the quality of our data set, we have subjected the resulting alignments to a series of filters designed to minimize impact of annotation errors, sequence quality, and changes in gene structure on subsequent analysis. In particular, we require that: (1) a transcript maps to the non-human genome via a single chain of sequence alignments that includes at least 80% of CDS (as determined by building process of UCSC syntenic maps), (2) no more than 10% of CDS falls into known sequencing gaps or low quality sequence in the non-human genome, (3) there are no frame-shifts in the non-human CDS, unless they are compensated for within 15 bases, (4) and there are no in-frame stop codons in the non-human CDS, and all splice sites are conserved. In an earlier study [7], we have found that start sites and stop codons are often not conserved between species. Therefore, we also consider incomplete transcripts, where we remove ∼10% of coding sequence from both ends of the complete transcript, and subject them to the same series of filters. For each gene cluster we have selected the transcript (complete or incomplete) that has been successfully mapped to the largest number of species, using CDS length as a secondary criteria. This procedure resulted in 17,500 genes that were successfully mapped to at least two additional species besides human. For technical reasons, we have removed 11 additional transcripts, obtaining orthologous multiple alignments for 17,489 genes, with average of ∼4 non-human orthologs per gene. The number of genes in this data set for each species is shown in Table 1. For recently duplicated genes it is impossible to establish 1:1 orthologous relationships of human gene and its non-human lineage ortholog. Moreover, these genes often occur in tandem clusters, where it is hard to distinguish orthologous from paralogous relationships. We have developed a pairwise test for these recent duplications (i.e., duplication occurring after split from the non-human species lineage), and to avoid potential false positives, we have removed from our set orthologs that have such a recent non-pseudogenized