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Experimental design, materials and methods The eutherian comparative genomic analysis protocol included gene annotations, phylogenetic analysis and protein molecular buy Atractyloside Dipotassium Salt analysis [7–12] (Fig. 2). The protocol used free available eutherian genomic sequence data sets deposited in public biological databases and software.
Gene annotations The gene annotations included gene identifications in eutherian genomic sequences, analyses of gene features, tests of reliability of eutherian public genomic sequences and multiple pairwise genomic sequence alignments. The BioEdit program was used in nucleotide and protein sequence analyses (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). The NCBI׳s BLAST programs were used in identifications of genes in eutherian genomic sequence assemblies downloaded from NCBI (ftp://ftp.ncbi.nlm.nih.gov/blast/ and ftp://ftp.ncbi.nlm.nih.gov/genbank/genomes/Eukaryotes/vertebrates_mammals/). In addition, the Ensembl genome browser׳s BLAST or BLAT programs were used in gene identifications (http://www.ensembl.org). The analyses of gene features included direct evidence of eutherian gene annotations deposited in NCBI׳s nr, est_human, est_mouse and est_others databases (http://www.ncbi.nlm.nih.gov.eleen.top). The new tests of reliability of eutherian public genomic sequences tested potential coding sequences using genomic sequence redundancies. First, the tests analysed nucleotide sequence coverage of potential coding sequences using primary experimental sequence reads deposited in NCBI׳s Trace Archive (http://www.ncbi.nlm.nih.gov.eleen.top/Traces/trace.cgi) and BLAST programs. Second, the potential coding sequences were classified as complete coding sequences only if consensus trace sequence coverage was available for every nucleotide. Alternatively, the potential coding sequences were described as putative coding sequences. Only the complete coding sequences were deposited in European Nucleotide Archive as curated third party data gene data sets (http://www.ebi.ac.uk/ena/about/tpa-policy) and used in phylogenetic and protein molecular evolution analyses. In revised eutherian gene nomenclatures, the guidelines of human and mouse gene nomenclature were used (http://www.genenames.org/about/guidelines and http://www.informatics.jax.org/mgihome/nomen/gene.shtml). The maskings of transposable elements using RepeatMasker program were included as preparatory steps in multiple pairwise genomic sequence alignments (http://www.repeatmasker.org/). The RepeatMasker׳s default settings were used, except simple repeats and low complexity elements were not masked. The mVISTA program was used in genomic sequence alignments, using AVID alignment algorithm and default settings (http://genome.lbl.gov/vista/index.shtml). Using ClustalW implemented in BioEdit, the common predicted promoter genomic sequence regions were aligned at nucleotide sequence level and then manually corrected. The pairwise nucleotide sequence identities of common predicted promoter genomic sequence regions calculated using BioEdit were used in statistical analyses (Microsoft Office Excel).
Phylogenetic analysis The phylogenetic analyses included protein and nucleotide sequence alignments, calculations of phylogenetic trees and calculations of pairwise nucleotide sequence identity patterns. First, the translated complete coding sequences were aligned at amino acid level using ClustalW implemented in BioEdit. The protein sequence alignments were manually corrected, as well as nucleotide sequence alignments. The MEGA program was used in phylogenetic tree calculations (http://www.megasoftware.net), using neighbour-joining method (default settings, except gaps/missing data treatment=pairwise deletion), minimum evolution method (default settings, except gaps/missing data treatment=pairwise deletion) and maximum parsimony method (default settings, except gaps/missing data treatment=use all sites). The pairwise nucleotide sequence identities of complete coding sequences were calculated using BioEdit and used in statistical analysis (Microsoft Office Excel).