Description

This track shows multiple alignments of 46 vertebrate species and measurements of evolutionary conservation using two methods (phastCons and phyloP) from the PHAST package, for all species (vertebrate) and two subsets (primate and placental mammal). The multiple alignments were generated using multiz and other tools in the UCSC/Penn State Bioinformatics comparative genomics alignment pipeline. Conserved elements identified by phastCons are also displayed in this track.

PhastCons (which has been used in previous Conservation tracks) is a hidden Markov model-based method that estimates the probability that each nucleotide belongs to a conserved element, based on the multiple alignment. It considers not just each individual alignment column, but also its flanking columns. By contrast, phyloP separately measures conservation at individual columns, ignoring the effects of their neighbors. As a consequence, the phyloP plots have a less smooth appearance than the phastCons plots, with more "texture" at individual sites. The two methods have different strengths and weaknesses. PhastCons is sensitive to "runs" of conserved sites, and is therefore effective for picking out conserved elements. PhyloP, on the other hand, is more appropriate for evaluating signatures of selection at particular nucleotides or classes of nucleotides (e.g., third codon positions, or first positions of miRNA target sites).

Another important difference is that phyloP can measure acceleration (faster evolution than expected under neutral drift) as well as conservation (slower than expected evolution). In the phyloP plots, sites predicted to be conserved are assigned positive scores (and shown in blue), while sites predicted to be fast-evolving are assigned negative scores (and shown in red). The absolute values of the scores represent -log p-values under a null hypothesis of neutral evolution. The phastCons scores, by contrast, represent probabilities of negative selection and range between 0 and 1.

Both phastCons and phyloP treat alignment gaps and unaligned nucleotides as missing data, and both were run with the same parameters for each species set (vertebrates, placental mammals, and primates). Thus, in regions in which only primates appear in the alignment, all three sets of scores will be the same, but in regions in which additional species are available, the mammalian and/or vertebrate scores may differ from the primate scores. The alternative plots help to identify sequences that are under different evolutionary pressures in, say, primates and non-primates, or mammals and non-mammals.

The species aligned for this track include the reptile, amphibian, bird, and fish clades, as well as marsupial, monotreme (platypus), and placental mammals. Compared to the previous 44-vertebrate alignment (hg18), this track includes 2 new species and 5 species with updated sequence assemblies (Table 1). The new species consist of two assemblies: baboon (papHam1) at 5.3X coverage and wallaby (macEug1) at 2X coverage. The elephant, opossum, rabbit, tetraodon, and zebrafish assemblies have been updated from those used in the previous 44-species alignment.

UCSC has repeatmasked and aligned the low-coverage genome assemblies, and provides the sequence for download; however, we do not construct genome browsers for them. Missing sequence in the low-coverage assemblies is highlighted in the track display by regions of yellow when zoomed out and Ns displayed at base level (see Gap Annotation, below).

Organism	Species	Release date	UCSC version	alignment type
Human	Homo sapiens	Feb. 2009	hg19/GRCh37	reference species
Alpaca	Vicugna pacos	Jul. 2008	vicPac1*	Reciprocal Best
Armadillo	Dasypus novemcinctus	Jul. 2008	dasNov2*	Reciprocal Best
Baboon	Papio hamadryas	Nov. 2008	papHam1*	Reciprocal Best
Bushbaby	Otolemur garnettii	Dec. 2006	otoGar1*	Reciprocal Best
Cat	Felis catus	Mar. 2006	felCat3	Reciprocal Best
Chicken	Gallus gallus	May 2006	galGal3	Syntenic Net
Chimp	Pan troglodytes	Mar. 2006	panTro2	Syntenic Net
Cow	Bos taurus	Oct. 2007	bosTau4	Syntenic Net
Dog	Canis lupus familiaris	May 2005	canFam2	Syntenic Net
Dolphin	Tursiops truncatus	Feb. 2008	turTru1*	Reciprocal Best
Elephant	Loxodonta africana	Jul. 2009	loxAfr3	Syntenic Net
Fugu	Takifugu rubripes	Oct. 2004	fr2	MAF Net
Gorilla	Gorilla gorilla gorilla	Oct. 2008	gorGor1*	Reciprocal Best
Guinea Pig	Cavia porcellus	Feb. 2008	cavPor3	Syntenic Net
Hedgehog	Erinaceus europaeus	June 2006	eriEur1*	Reciprocal Best
Horse	Equus caballus	Sep. 2007	equCab2	Syntenic Net
Kangaroo rat	Dipodomys ordii	Jul. 2008	dipOrd1*	Reciprocal Best
Lamprey	Petromyzon marinus	Mar. 2007	petMar1	MAF Net
Lizard	Anolis carolinensis	Feb. 2007	anoCar1	Syntenic Net
Marmoset	Callithrix jacchus	June 2007	calJac1	Reciprocal Best
Medaka	Oryzias latipes	Oct. 2005	oryLat2	MAF Net
Megabat	Pteropus vampyrus	Jul. 2008	pteVam1*	Reciprocal Best
Little brown bat	Myotis lucifugus	Mar. 2006	myoLuc1*	Reciprocal Best
Mouse	Mus musculus	July 2007	mm9	Syntenic Net
Mouse lemur	Microcebus murinus	Jul. 2007	micMur1*	Reciprocal Best
Opossum	Monodelphis domestica	Oct. 2006	monDom5	Syntenic Net
Orangutan	Pongo pygmaeus abelii	July 2007	ponAbe2	Syntenic Net
Pika	Ochotona princeps	Jul. 2008	ochPri2*	Reciprocal Best
Platypus	Ornithorhynchus anatinus	Mar. 2007	ornAna1	Reciprocal Best
Rabbit	Oryctolagus cuniculus	Apr. 2009	oryCun2	Syntenic Net
Rat	Rattus norvegicus	Nov. 2004	rn4	Syntenic Net
Rhesus	Macaca mulatta	Jan. 2006	rheMac2	Syntenic Net
Rock hyrax	Procavia capensis	Jul. 2008	proCap1*	Reciprocal Best
Shrew	Sorex araneus	June 2006	sorAra1*	Reciprocal Best
Sloth	Choloepus hoffmanni	Jul. 2008	choHof1*	Reciprocal Best
Squirrel	Spermophilus tridecemlineatus	Feb. 2008	speTri1*	Reciprocal Best
Stickleback	Gasterosteus aculeatus	Feb. 2006	gasAcu1	MAF Net
Tarsier	Tarsier syrichta	Aug. 2008	tarSyr1*	Reciprocal Best
Tenrec	Echinops telfairi	July 2005	echTel1*	Reciprocal Best
Tetraodon	Tetraodon nigroviridis	Mar. 2007	tetNig2	MAF Net
Tree Shrew	Tupaia belangeri	Dec. 2006	tupBel1*	Reciprocal Best
Wallaby	Macropus eugenii	Nov. 2007	macEug1*	Reciprocal Best
X. tropicalis	Xenopus tropicalis	Aug. 2005	xenTro2	MAF Net
Zebra finch	Taeniopygia guttata	Jul. 2008	taeGut1	Syntenic Net
Zebrafish	Danio rerio	Dec. 2008	danRer6	MAF Net

Table 1. Genome assemblies included in the 46-way Conservation track.
* Data download only, browser not available.

• Multiz alignments (MAF format), and phylogenetic trees
• PhyloP conservation (WIG format)
• PhastCons conservation (WIG format)

Display Conventions and Configuration

The track configuration options allow the user to display either the vertebrate or placental mammal conservation scores, or both simultaneously. In full and pack display modes, conservation scores are displayed as a wiggle track (histogram) in which the height reflects the size of the score. The conservation wiggles 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.

Pairwise alignments of each species to the human genome are displayed below the conservation histogram 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.

Checkboxes on the track configuration page allow selection of the species to include in the pairwise display. Configuration buttons are available to select all of the species (Set all), deselect all of the species (Clear all), or use the default settings (Set defaults). By default, the following 11 species are included in the pairwise display: rhesus, mouse, dog, horse, armadillo, opossum, platypus, lizard, chicken, X. tropicalis (frog), and stickleback. Note that excluding species from the pairwise display does not alter the the conservation score display.

To view detailed information about the alignments at a specific position, zoom the display in 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 human 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 human 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 human sequence at those alignment positions relative to the longest non-human sequence. If there is sufficient space in the display, the size of the gap is shown. If the space is insufficient and the gap size is a multiple of 3, a "*" is displayed; other gap sizes are indicated by "+".

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 (Table 2). Species listed in the row labeled "None" do not have species-specific reading frames for gene translation.

Gene Track	Species
Known Genes	human, mouse, rat
Ensembl Genes v55	alpaca, armadillo, bush baby, cat, chicken, chimp, cow, dog, dolphin, fugu, gorilla, guinea pig, hedgehog, horse, kangaroo rat, little brown bat, lizard, medaka, megabat, mouse, mouse lemur, opossum, orangutan, pika, platypus, rhesus, rock hyrax, shrew, sloth, squirrel, stickleback, tarsier, tenrec, tetraodon, tree shrew, X. tropicalis, zebra finch, zebrafish
Xeno Ref Genes	baboon, elephant, lamprey, marmoset, rabbit, wallaby

Table 2. Gene tracks used for codon translation.

Methods

Pairwise alignments with the human genome were generated for each species using blastz from repeat-masked genomic sequence. Pairwise alignments were then linked into chains using a dynamic programming algorithm that finds maximally scoring chains of gapless subsections of the alignments organized in a kd-tree. The scoring matrix and parameters for pairwise alignment and chaining were tuned for each species based on phylogenetic distance from the reference. High-scoring chains were then placed along the genome, with gaps filled by lower-scoring chains, to produce an alignment net. For more information about the chaining and netting process and parameters for each species, see the description pages for the Chain and Net tracks.

An additional filtering step was introduced in the generation of the 46-way conservation track to reduce the number of paralogs and pseudogenes from the high-quality assemblies and the suspect alignments from the low-quality assemblies: the pairwise alignments of high-quality mammalian sequences (placental and marsupial) were filtered based on synteny; those for 2X mammalian genomes were filtered to retain only alignments of best quality in both the target and query ("reciprocal best").

The resulting best-in-genome pairwise alignments were progressively aligned using multiz/autoMZ, following the tree topology diagrammed above, to produce multiple alignments. The multiple alignments were post-processed to add annotations indicating alignment gaps, genomic breaks, and base quality of the component sequences. The annotated multiple alignments, in MAF format, are available for bulk download. An alignment summary table containing an entry for each alignment block in each species was generated to improve track display performance at large scales. Framing tables were constructed to enable visualization of codons in the multiple alignment display.

Phylogenetic Tree Model

Both phastCons and phyloP are phylogenetic methods that rely on a tree model containing the tree topology, branch lengths representing evolutionary distance at neutrally evolving sites, the background distribution of nucleotides, and a substitution rate matrix. The vertebrate tree model for this track was generated using the phyloFit program from the PHAST package (REV model, EM algorithm, medium precision) using multiple alignments of 4-fold degenerate sites extracted from the 46way alignment (msa_view). The 4d sites were derived from the RefSeq (Reviewed+Coding) gene set, filtered to select single-coverage long transcripts. The placental mammal tree model and primate tree model were extracted from the vertebrate model.

PhastCons Conservation

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. Unlike many conservation-scoring programs, phastCons does not rely on a sliding window of fixed size; therefore, 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.

The phastCons parameters were tuned to produce 5% conserved elements in the genome for the vertebrate conservation measurement. This parameter set (expected-length=45, target-coverage=.3, rho=.31) was then used to generate the placental mammal and primate conservation scoring.

PhyloP Conservation

The phyloP program supports several different methods for computing p-values of conservation or acceleration, for individual nucleotides or larger elements (http://compgen.cshl.edu/phast/). Here it was used to produce separate scores at each base (--wig-scores option), considering all branches of the phylogeny rather than a particular subtree or lineage (i.e., the --subtree option was not used). The scores were computed by performing a likelihood ratio test at each alignment column (--method LRT), and scores for both conservation and acceleration were produced (--mode CONACC).

Conserved Elements

The conserved elements were predicted by running phastCons with the --viterbi option. The predicted elements are segments of the alignment that are likely to have been "generated" by the conserved state of the phylo-HMM. Each element is assigned a log-odds score equal to its log probability under the conserved model minus its log probability under the non-conserved model. The "score" field associated with this track contains transformed log-odds scores, taking values between 0 and 1000. (The scores are transformed using a monotonic function of the form a * log(x) + b.) The raw log odds scores are retained in the "name" field and can be seen on the details page or in the browser when the track's display mode is set to "pack" or "full".

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, phyloP, phyloFit, tree_doctor, msa_view and other programs in PHAST by Adam Siepel at Cold Spring Harbor Laboratory (original development done at the Haussler lab at UCSC).
• MAF Annotation tools: mafAddIRows by Brian Raney, UCSC; mafAddQRows by Richard Burhans, Penn State; 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 as of March 2007.

References

Phylo-HMMs, phastCons, and phyloP:

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

Pollard KS, Hubisz MJ, Rosenbloom KR, Siepel A. Detection of nonneutral substitution rates on mammalian phylogenies. Genome Res. 2010 Jan;20(1):110-21. PMID: 19858363; PMC: PMC2798823

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

Harris RS. Improved pairwise alignment of genomic DNA. Ph.D. Thesis. Pennsylvania State University, USA. 2007.

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