## Description

This track displays multi-species conserved sequences (MCSs) derived from phastCons, binCons, and GERP (Genomic Evolutionary Rate Profiling), conservation scoring of human ENCODE genomic DNA alignments to 27 other vertebrates using the MLAGAN alignment package.

The multiple sequence alignments may be viewed in the MLAGAN Alignments track. Another related track, MLAGAN Cons, shows the conservation scoring. The descriptions accompanying these tracks detail the methods used to create the alignments and conservation.

## Display Conventions and Configuration

The locations of conserved elements are indicated by blocks in the graphical display. This composite annotation track consists of several subtracks that show conserved elements derived by the methods listed above. To view only selected subtracks, uncheck the boxes next to the tracks you wish to hide. The display may also be filtered to show only those items with unnormalized scores that meet or exceed a certain threshold. To set a threshold, type the minimum score into the text box at the top of the description page.

Display characteristics specific to certain subtracks are described in the respective Methods sections below.

## Methods

### PhastCons-based Elements

The predicted MCSs are segments of the alignment that are likely to have been "generated" by the conserved state of the phylo-HMM, i.e. maximal segments in which the maximum-likelihood (Viterbi) path remains in the conserved state.

### BinCons-based Elements

The binCons score is based on the cumulative binomial probability of detecting the observed number of identical bases (or greater) in sliding 25 bp windows (moving one bp at a time) between the reference sequence and each other species, given the neutral rate at four-fold degenerate sites. Neutral rates are calculated separately at each targeted region. For targets with no gene annotations, the average percent identity across all alignable sequence was instead used to weight the individual species binomial scores; this latter weighting scheme was found to closely match 4D weights.

The negative log of these p-values was then averaged across all human-referenced pairwise combinations, and the highest scoring overlapping 25 bp window for each base was the resulting score. This track shows the plotting of a ranked percentile score normalized between 0 and 1 across all ENCODE regions, such that the top 5% most conserved sequence across all ENCODE regions have a score of 0.95 or greater, the top 10% have a score of 0.9 or greater, and so on.

For each ENCODE target, a conservation score threshold was picked to match the number of conserved bases predicted by phastCons, an alternative method for measuring conservation. This latter method has been found slightly more reliable for predicting the expected fraction of conserved sequence in each target. Clusters of bases that exceeded the given conservation score threshold were designated as MCSs. The minimum length of an MCS is 25 bases. Strict cutoffs were used: if even one base fell below the conservation score threshold, it separated an MCS into two distinct regions.

More details on binCons can be found in Margulies et. al. (2003) cited below.

### GERP-based Elements

GERP constrained elements exhibit significant evidence of the effects of purifying selection. Elements are scored according to the inferred intensity of purifying selection and are measured as "rejected substitutions" (RSs). RSs capture the magnitude of difference between the number of "observed" substitutions (estimated using maximum likelihood) and the number that would be "expected" under a neutral model of evolution. The RS is displayed as part of the item name. Items with higher RSs are displayed in a darker shade of blue. The score shown on the details page, which has been scaled by 300 for display purposes, is generally not as accurate as the RS count that is part of the item name.

"Constrained elements" are identified as those groups of consecutive human bases that have an observed rate of evolution that is smaller than the expected rate. These groups of columns are merged if they are less than a few nucleotides apart and are scored according to the sum of the site-by-site difference between observed and expected rates (RS).

Permutations of the actual alignments were analyzed, and the "constrained elements" identified in these permuted alignments were treated as "false positives". Subsequently, an RS threshold was picked such that the total length of "false positive" constrained elements (identified in the permuted alignments) was less than 5% of the length of constrained elements identified in the actual alignment. Thus, all annotated constrained elements are significant at better than 95% confidence, and the total fraction of the ENCODE regions annotated as constrained is 5-7%.

## Credits

PhastCons was developed by Adam Siepel, Cornell University, while at the Haussler Lab at UCSC.

BinCons was developed by Elliott Margulies of NHGRI, while at the Eric Green lab. BinCons and phastCons MCS data were contributed by Elliott Margulies, with assistance from Adam Siepel of UCSC.

GERP was developed primarily by Greg Cooper in the lab of Arend Sidow at Stanford University (Depts of Pathology and Genetics), in close collaboration with Eric Stone (Biostatistics, NC State), and George Asimenos and Eugene Davydov in the lab of Serafim Batzoglou (Dept. of Computer Science, Stanford).

## References

See the MLAGAN Alignment and MLAGAN Cons tracks for references.