Description

This track shows a measure of evolutionary conservation in 9 vertebrates, including mammalian, amphibian, bird, and fish species, based on a phylogenetic hidden Markov model, phastCons (Siepel et al., 2005). Multiz alignments of the following assemblies were used to generate this track:

• rat (Nov. 2004 (Baylor 3.4/rn4), rn4)
• mouse (Feb 2006, mm8)
• human (Mar 2006, hg18)
• dog (May 2005, canFam2)
• cow (Mar 2005, bosTau2)
• opossum (Jan 2006, monDom4)
• chicken (Feb 2004, galGal2)
• frog (Oct 2004, xenTro1)
• zebrafish (May 2005, danRer3)

Display Conventions and Configuration

In full and pack display modes, conservation scores are displayed as a "wiggle" (histogram), where the height reflects the size of the score. Pairwise alignments of each species to the rat genome are displayed below as a grayscale density plot (in pack mode) or as a "wiggle" (in full mode) that indicates alignment quality. In dense display mode, conservation is shown in grayscale using darker values to indicate higher levels of overall conservation as scored by phastCons.

The conservation wiggle can be configured in a variety of ways to highlight different aspects of the displayed information. Click the Graph configuration help link for an explanation of the configuration options.

Checkboxes in the track configuration section allow excluding species from the pairwise display; however, this does not remove them from the conservation score display. To view detailed information about the alignments at a specific position, zoom in the display to 30,000 or fewer bases, then click on the alignment.

Gap Annotation

The "Display chains between alignments" configuration option enables display of gaps between alignment blocks in the pairwise alignments in a manner similar to the Chain track display. The following conventions are used:

• Single line: No bases in the aligned species. Possibly due to a lineage-specific insertion between the aligned blocks in the rat genome or a lineage-specific deletion between the aligned blocks in the aligning species.
• Double line: Aligning species has one or more unalignable bases in the gap region. Possibly due to excessive evolutionary distance between species or independent indels in the region between the aligned blocks in both species.
• Pale yellow coloring: Aligning species has Ns in the gap region. Reflects uncertainty in the relationship between the DNA of both species, due to lack of sequence in relevant portions of the aligning species.

Genomic Breaks

Discontinuities in the genomic context (chromosome, scaffold or region) of the aligned DNA in the aligning species are shown as follows:

• Vertical blue bar: Represents a discontinuity that persists indefinitely on either side, e.g. a large region of DNA on either side of the bar comes from a different chromosome in the aligned species due to a large scale rearrangement.
• Green square brackets: Enclose shorter alignments consisting of DNA from one genomic context in the aligned species nested inside a larger chain of alignments from a different genomic context. The alignment within the brackets may represent a short misalignment, a lineage-specific insertion of a transposon in the rat genome that aligns to a paralogous copy somewhere else in the aligned species, or other similar occurrence.

Base Level

When zoomed-in to the base-level display, the track shows the base composition of each alignment. The numbers and symbols on the Gaps line indicate the lengths of gaps in the rat sequence at those alignment positions relative to the longest non-rat sequence. If there is sufficient space in the display, the size of the gap is shown; if not, and if the gap size is a multiple of 3, a "*" is displayed, otherwise "+" is shown.

Codon translation is available in base-level display mode if the displayed region is identified as a coding segment. To display this annotation, select the species for translation from the pull-down menu in the Codon Translation configuration section at the top of the page. Then, select one of the following modes:

• No codon translation: The gene annotation is not used; the bases are displayed without translation.
• Use default species reading frames for translation: The annotations from the genome displayed in the Default species to establish reading frame pull-down menu are used to translate all the aligned species present in the alignment.
• Use reading frames for species if available, otherwise no translation: Codon translation is performed only for those species where the region is annotated as protein coding.
• Use reading frames for species if available, otherwise use default species: Codon translation is done on those species that are annotated as being protein coding over the aligned region using species-specific annotation; the remaining species are translated using the default species annotation.

Codon translation uses the following gene tracks as the basis for translation, depending on the species chosen:

Gene Track	Species
Known Genes	rat, mouse, human
RefSeq Genes	chicken
MGC Genes	X. tropicalis
mRNAs	dog, cow, zebrafish
not translated	opossum

Methods

Best-in-genome pairwise alignments were generated for each species using blastz, followed by chaining and netting. The pairwise alignments were then multiply aligned using multiz, following the ordering of the species tree diagrammed above. The resulting multiple alignments were then assigned conservation scores by phastCons, using a tree model with branch lengths derived from the ENCODE project Multi-Species Sequence Analysis group, September 2005 tree model. This tree was generated from TBA alignments over 23 vertebrate species and is based on 4D sites.

The phastCons program computes conservation scores based on a phylo-HMM, a type of probabilistic model that describes both the process of DNA substitution at each site in a genome and the way this process changes from one site to the next (Felsenstein and Churchill 1996, Yang 1995, Siepel and Haussler 2005). PhastCons uses a two-state phylo-HMM, with a state for conserved regions and a state for non-conserved regions. The value plotted at each site is the posterior probability that the corresponding alignment column was "generated" by the conserved state of the phylo-HMM. These scores reflect the phylogeny (including branch lengths) of the species in question, a continuous-time Markov model of the nucleotide substitution process, and a tendency for conservation levels to be autocorrelated along the genome (i.e., to be similar at adjacent sites). The general reversible (REV) substitution model was used. Note that, unlike many conservation-scoring programs, phastCons does not rely on a sliding window of fixed size, so short highly-conserved regions and long moderately conserved regions can both obtain high scores. More information about phastCons can be found in Siepel et al. (2005).

PhastCons currently treats alignment gaps as missing data, which sometimes has the effect of producing undesirably high conservation scores in gappy regions of the alignment. We are looking at several possible ways of improving the handling of alignment gaps.

Credits

This track was created using the following programs:

• Alignment tools: blastz and multiz by Minmei Hou, Scott Schwartz and Webb Miller of the Penn State Bioinformatics Group
• Chaining and Netting: axtChain, chainNet by Jim Kent at UCSC
• Conservation scoring: PhastCons, phyloFit, tree_doctor, msa_view by Adam Siepel while at UCSC, now at Cold Spring Harbor Laboratory
• MAF Annotation tools: mafAddIRows by Brian Raney, UCSC; genePredToMafFrames by Mark Diekhans, UCSC
• Tree image generator: phyloPng by Galt Barber, UCSC
• Conservation track display: Kate Rosenbloom, Hiram Clawson (wiggle display), and Brian Raney (gap annotation and codon framing) at UCSC

The phylogenetic tree is based on Murphy et al. (2001) and general consensus in the vertebrate phylogeny community.

References

Phylo-HMMs and phastCons:

Felsenstein J, Churchill GA. A Hidden Markov Model approach to variation among sites in rate of evolution. Mol Biol Evol. 1996 Jan;13(1):93-104. PMID: 8583911

Siepel A, Bejerano G, Pedersen JS, Hinrichs AS, Hou M, Rosenbloom K, Clawson H, Spieth J, Hillier LW, Richards S, et al. Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res. 2005 Aug;15(8):1034-50. PMID: 16024819; PMC: PMC1182216

Siepel A, Haussler D. Phylogenetic Hidden Markov Models. In: Nielsen R, editor. Statistical Methods in Molecular Evolution. New York: Springer; 2005. pp. 325-351.

Yang Z. A space-time process model for the evolution of DNA sequences. Genetics. 1995 Feb;139(2):993-1005. PMID: 7713447; PMC: PMC1206396

Chain/Net:

Kent WJ, Baertsch R, Hinrichs A, Miller W, Haussler D. Evolution's cauldron: duplication, deletion, and rearrangement in the mouse and human genomes. Proc Natl Acad Sci U S A. 2003 Sep 30;100(20):11484-9. PMID: 14500911; PMC: PMC208784

Multiz:

Blanchette M, Kent WJ, Riemer C, Elnitski L, Smit AF, Roskin KM, Baertsch R, Rosenbloom K, Clawson H, Green ED, et al. Aligning multiple genomic sequences with the threaded blockset aligner. Genome Res. 2004 Apr;14(4):708-15. PMID: 15060014; PMC: PMC383317

Blastz:

Chiaromonte F, Yap VB, Miller W. Scoring pairwise genomic sequence alignments. Pac Symp Biocomput. 2002:115-26. PMID: 11928468

Schwartz S, Kent WJ, Smit A, Zhang Z, Baertsch R, Hardison RC, Haussler D, Miller W. Human-mouse alignments with BLASTZ. Genome Res. 2003 Jan;13(1):103-7. PMID: 12529312; PMC: PMC430961

Phylogenetic Tree:

Murphy WJ, Eizirik E, O'Brien SJ, Madsen O, Scally M, Douady CJ, Teeling E, Ryder OA, Stanhope MJ, de Jong WW, Springer MS. Resolution of the early placental mammal radiation using Bayesian phylogenetics. Science. 2001 Dec 14;294(5550):2348-51. PMID: 11743200