9Phylogenomic Resources at the UCSC Genome Browser
Kate Rosenbloom, James Taylor, Stephen Schaeffer, Jim Kent,
David Haussler, and Webb Miller

Summary
The UC Santa Cruz Genome Browser provides a number of resources that can beused for phylogenomic studies, including (1) whole-genome sequence data from anumber of vertebrate species, (2) pairwise alignments of the human genome sequence
to a number of other vertebrate genome, (3) a simultaneous alignment of 17 vertebrate
genomes (most of them incompletely sequenced) that covers all of the human sequence,(4) several independent sets of multiple alignments covering 1% of the human genome(ENCODE regions), (5) extensive sequence annotation for interpreting those sequences
and alignments, and (6) sequence, alignments, and annotations from certain other species,
including an alignment of nine insect genomes. We illustrate the use of these resources in
the context of assigning rare genomic changes to the branch of the phylogenetic tree where
they appear to have occurred, or of looking for evidence supporting a particular possible
tree topology. Sample source code for performing such studies is available.
Key Words: Evolutionary event; phylogenetic tree; interspersed repeat; chromosomal
break.

1. Introduction
Rare genomic changes (RGCs), such as retroposon integrations, indels(insertions/deletions) in protein-coding regions, and inversions, provide usefulalternatives to the use of nucleotide and amino-acid substitutions for determining
the evolutionary relationships among living organisms (1). If one knows wherein the genome to look, the UCSC Genome Browser (2,3) and the associateddatabase (4) make it easy to see evidence that an RGC occurred at a particularpoint in evolutionary history.
From: Methods in Molecular Biology: Phylogenomics
Edited by: W. J. Murphy © Humana Press Inc., Totowa, NJ

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Fig. 1. A proposed evolutionary tree for several mammals, as discussed in thismanuscript. Only the tree topology is sketched; branch lengths have no meaning.

Fig. 2. View in the UCSC Human Genome Browser of alignments of opossum,
mouse and dog to a region of human chromosome 13, showing a reversal that appears
to have occurred along branch A in the tree of Fig. 1.

For instance, it is widely held (5) that the human lineage diverged from dog(and other so-called laurasiatheres) before it diverged from mouse. Thus, we can
expect to observe evidence for genomic changes that occurred on the branchlabeled A in Fig. 1. Figure 2 shows the black-and-white version of a view in
the Browser of a 4500-bp interval of human chromosome 13 (positions relative
to the May 2004 human assembly). Dog shows a local inversion relative to humanPhylogenomic Resources at the UCSC Genome Browser

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and mouse, and the outgroup opossum indicates that dog is in the ancestralorientation. The most parsimonious explanation is that a single inversion event
occurred on branch A.
On the other hand, if one is not told where in the genome to look, a search
for RGCs that occurred along a hypothesized short or ancient branch cannot be
performed efficiently by eye; too many evolutionary events are recorded tomake it feasible to start looking through them one at a time. For a systematic
attack on the problem, it is often necessary to download large amounts of dataand process them with custom-built software. Fortunately, a graduate student or
talented undergraduate with a strong programming background can frequently
write such programs, provided they get over a few initial hurdles. This chapter
contains much of the necessary information.
A wealth of genomic data is available from the UCSC Browser, as well as
alternative data providers, such as Ensembl (6), NCBI (7), and Galaxy (8).
Sequence data, alignments, and annotations can be downloaded from the Table
Browser (9) or the UCSC “downloads” page, then searched in arbitrary ways.
The aim of this chapter is to illustrate how this can be done through three
examples. The general problem treated by the examples is assigning RGCs tobranches of the phylogenetic tree. Such an assignment is a natural part of an
attempt to reconstruct evolutionary histories, as has been proposed for eutherianmammals (10). The first example focuses on a search for informative retroposon integrations in the 1% of the human genome encompassed by the 44
regions chosen for extensive study by the ENCODE project (11). For the second
example, we look across the entire human genome for indels in protein-coding
regions that provide evidence concerning the earliest divergences among
eutherian mammals, e.g., perhaps occurring in the human lineage after divergencefrom elephants (afrotheres) but before divergence from armadillo (xenarthrans)(branch B in Fig. 1). In the third example, we look for particular kinds ofchromosomal breaks in an alignment of nine insect genomes. To help the reader
get started writing such programs, we make the source code used in thoseexamples freely available at http://bio.cse.psu.edu/miller_lab/ under the title“Phylogenomic Tools.” Although many biologists may hesitate to venture intosuch a project, we encourage them to consider hiring someone with solid
programming skills to perform whole-genome searches, helped by the clues weprovide here.
2. Methods
2.1. Interspersed Repeats in ENCODE Regions
As illustrated by Chapters 14 and 15 in this volume, interspersed repeat elements
can sometimes be used to answer phylogenomic questions. In particular, insertion
events that occurred very early in the mammalian radiation can provide evidence

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Fig. 3. Browser view of an interspersed repeat element in ENCODE region ENm001
that appears to have been inserted along branch A in Fig. 1.

to support certain hypotheses about phylogenetic relationships, such as that thehuman lineage diverged from dogs and cows before it diverged from mice (12),and that horses are more closely related to dogs than to cows (13).
A repetitive element that may have inserted on the branch labeled “A” in
Fig. 1 is shown in Fig. 3. Two important characteristics are that (i) a large fractionof the element aligns with mouse (for this to be possible, the element cannot becompletely masked e.g., by replacing each of its nucleotides by “N”), and (ii) noneof it aligns with dog. Those properties also hold of segments that were deleted
in the dog lineage after it diverged from human and mouse, but here we haveadditional information. Namely, we recognize the region as an insertion element;
for this to be a deletion in dog, the deleted interval would need to correspondto within a few nucleotides of the inserted element. (This can be seen by inspecting the alignment in detail, noting that the positions that align just to either sideof the repetitive element are only a few nucleotides apart in dog.) Moreover, theelement belongs to the family MLT1A0, which is known to have been transpositionally active early in the mammalian radiation. (For a list of such families, see
Table 6 of the mouse genome paper in ref. 14.) Finally, the percent identitywith the consensus sequence for the class, 20.8%, is consistent with what one
expects for an insertion that occurred early just before human–mouse divergence.

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For further evidence, one can hope to identify the flanking direct repeats atthe borders of the human element, and find a single copy of that sequence at theappropriate place in dog.
We wrote some simple computer programs to help search through large
genomic regions for retroposons that are likely candidates for having been
inserted at a particular branch of the phylogenetic tree. The program is essentiallythe same as what we had used earlier to find examples (12,13). These programs
are used on “soft-masked” sequence, i.e., where repetitive DNA is given withlower-case letters (“a”, “c”, “g”, and “t”), and the “.out” files produced by
RepeatMasker (a table of information about the identified repeat elements).
Both kinds of data can be downloaded from UCSC. The sequences arealigned with the blastz program (15), which can be freely obtained fromhttp://www.bx.psu.edu/ miller_lab/
Some small programs in the “Phylogenomic Tools” package (from the same
website as blastz) read the RepeatMasker “.out” files and the blastz alignments,
then report repeat elements with the desired property. When we downloaded
sequences and “.out” files for the ENCODE region ENm001, the tools identified
the repeat element shown in Fig. 3. The Phylogenomic Tools package contains
detailed instructions for how to proceed.
We ran our programs on all ENCODE regions, looking for repeats that
inserted on branch A. We ignored Alu repeats (because they inserted after
human–mouse divergence), as well as MIR and L2 repeats (because they arethought to have died out before the eutherian radiation). Four MLTA0 repeats
and two L1s were identified. As a check for specificity (i.e., lack of false
positives), we reversed the roles of mouse and dog; no repeats were predicted
to have inserted after the human lineage diverged from mouse but before it
diverged from dog.

2.2. Coding Indels
Indels (insertions/deletions) in protein-coding regions are another class of
RGC that has been utilized for phylogenetic analysis (16), including deletions
that apparently happened on branch A of Fig. 1 (17). We have written several
programs to automate most of the job of searching UCSC-generated alignments
for informative coding indels and tested them in a search for events on branch B
of Fig. 1. The first program reads a file of gene locations downloaded fromUCSC and produces a simplified list of coding exons (including the removalof redundant copies in the case of splice isoforms). The second program reads
the list of exons and three pairwise alignments of human, to, say, armadillo,elephant, and opossum, and looks for human exons that align without a gap toarmadillo, but align to elephant and opossum with gaps (lengths divisible by 3)
in precisely the same places. See Fig. 4 for one of the exons that it found.

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Fig. 4. Browser view of an alternatively spliced exon of the RTN3 gene, showing two codons in the human, mouse, dog,
and armadillo genes that are missing in elephant and opossum. This is a candidate for an insertion on branch B of Fig. 1.

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The most parsimonious explanation is that two codons were inserted in a single
event on branch B.

2.3. Chromosomal Breaks in Drosophila
One of the earliest uses of genetic data to reconstruct phylogenies was byDobzhansky and Sturtevant when they examined polymorphisms in the polytene
chromosomes isolated from Drosophila salivary gland cells (18). The polytene
chromosomes have reproducible banding and puffing patterns that reflect theorder of genes on chromosomes. Dobzhansky and Sturtevant showed that
Drosophila pseudoobscura had a wealth of gene order differences in natural
populations and inferred that the paracentric inversion mutations could relate
the different chromosomes to each other in an unrooted network (19). Remarkably,
all of the intermediate chromosomes in the network of gene arrangements havebeen collected from nature over the years with one exception, the Hypotheticalchromosome (20). It is now well documented that chromosomal inversions havebeen important in the evolution of Drosophila because many of the species arepolymorphic for gene order based on polytene chromosomal data (21). The
banding and puffing patterns of polytene chromosomes of different Drosophila
species are quite different and prevent direct comparison of gene order. With theadvent of complete genomic sequencing, we can now infer ancestral rearrangements between species by comparing inferred gene orders. Comparison of two
species of Drosophila has shown that at least 10 inversions occur per million years(22) and that in some cases repetitive elements may be responsible for the processof rearrangement. These analyses also show that some regions tend to resist
rearrangement because the size of conserved linkage blocks is too large given
the number of inversions that have occurred over esvolutionary time, suggestinga functional conservation of gene order to maintain coordinate regulation (23).
Our software toolkit contains programs to read multiway alignments downloaded from UC Santa Cruz and to look for places where one group of species
shows conservation of order and orientation of genomic features, but other species
have a chromosomal break. See Fig. 5 for an example.

2.4. Obtaining Sequences, Alignments, and Annotation from UCSCThe UCSC Genome Browser site provides two main facilities for obtaining
genomic sequence, alignments, and annotations:• a download server that supports bulk retrieval via FTP or HTTP access• the Table Browser data retrieval tool (8), useful for selective querying by region

Because this chapter is oriented toward high-throughput, whole-genome analysis,
this section focuses on the download server, although we will briefly describe
how to use the Table Browser to obtain alignments in selected regions.

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Fig. 5. Browser view of a region on Drosophila melanogaster chromosome 2R
where D. melanogaster, D. simulans, D. yakuba, and D. ananassae show colinearalignment, but where D. pseudoobscura, D. mojanavensis, D. virilis, and Anopheles
gambiae have a chromosomal break.

2.4.1. UCSC Download Server
The download server can be accessed from the “Downloads” menu link (http://hgdownload.cse.ucsc.edu) on the UCSC Browser main page or by anonymous
FTP (http://hgdownload.cse.ucsc.edu/goldenPath). The Downloads web page
provides a directory of links to the data files available, grouped by species
and assembly.
On the download server, each genome assembly is represented by a directorylabeled with the UCSC assembly release name. For example, files related to the
May 2004 human genome assembly are stored in the “hg17” directory. AssemblyPhylogenomic Resources at the UCSC Genome Browser

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releases and versions are described on the FAQ page (http://genome.ucsc.edu/
FAQ/FAQreleases).
The exact items vary by assembly, but the following data are typically available:
Link
Directory
Description
Full data set
bigZips
Misc assembly and annotation
Data set by chromosome chromosomes
Repeatmasked sequence
Annotation databasedatabase
Tab-delimited nightly database
dump
Multiple alignment(eight multiz8way Multiz alignments
species)(MAF format)
Conservation scores
phastCons
Per-base scores for multiplealignment
Mouse pairwise
vsMm7
Blastz/chain/net alignmentsalignments(AXT format)
Dog pairwise alignments vsCanFam2
Blastz/chain/net alignments(AXT format)
LiftOver files
liftOver
Conversion chains to otherassemblies
The section below details where to download the comparative genomics
resources used in this chapter. Note: all referenced human genome data are from
the May 2004 (UCSC release hg17) human genome assembly.
1. Human genome multiple alignments
Link: Multiple alignments of 16 vertebrate genomes to humanDirectory: hg17/multiz17way
2. Human alignments to opossum, armadillo, and elephant
Opossum Genome: Oct. 2004 assembly (UCSC release monDom1)
Link: Opossum/Human (hg17) alignments
Directory: monDom1/vsHg17
Armadillo Genome: May 2005 assembly (not released in UCSC browser)
Directory: dasNov1/vsHg17
Elephant Genome: May 2005 assembly (not released in UCSC browser)
Directory: loxAfr1/vsHg17
3. Human gene locations
Link: Annotation database
Directory: database
Tables: refFlat (RefSeq genes)
knownGene (UCSC Known genes)
encodeGencodeGeneKnown (ENCODE Gencode genes)
Files: *.txt.gz (data), *.sql (table schema)
4. Insect genome multiple alignment

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Assembly: Drosophila melanogaster Genome, April 2004 assembly(UCSC release dm2)
Link: Multiple alignment of eight insects with D. melanogaster
Directory: dm2/multiz9way

The ENCODE project has a project-specific downloads page, accessedfrom the “Downloads” menu link on the UCSC ENCODE portal page(http://genome.ucsc.edu/ENCODE). The following comparative genomics
resources in the ENCODE regions can be found on the May 2004 humangenome assembly (currently the ENCODE Reference Assembly):
1. ENCODE region sequence and RepeatMasker output for human genome
Link: Nucleotide sequences
Directory: hg17/encode/regions
2. ENCODE orthologous region sequence and RepeatMasker output for otherspecies, used to produce multiple alignments
Link: Multiple sequence alignments
Directory: hg17/encode/alignments/<freeze-data>/sequences
3. ENCODE region multiple alignments
Link: Multiple sequence alignments
Directory: hg17/encode/alignments/<freeze-date>/alignments

The alignment file formats are described in the following help files:
MAF: http://genome.ucsc.edu/goldenPath/help/maf.html
AXT: http://genome.ucsc.edu/goldenPath/help/axt.html
Chain: http://genome.ucsc.edu/goldenPath/help/chain.html

2.4.2. UCSC Table Browser
The Table Browser is accessed via a menu link on the UCSC Genome Browser
home page. Pull-down menus on the Table Browser allow selection of anorganism, assembly, genomic region, data type, and output format; options areprovided for filtering, intersecting, and correlating tables.
To obtain multiple alignments in a genomic region via the Table Browser,
select the following menu options:Group: Comparative Genomics
Track: Conservation
Table: multiz*
Output format: MAF

For ENCODE alignments, use:
Group: ENCODE Comparative Genomics
Tracks: TBA, MAVID, or MLAGAN Alignment
Tables: encodeTbaAlign, encodeMavidAlign, encodeMlaganAlign
Output format: MAF

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For compactness, select the gzip compression output option.

2.4.3. Access Guidelines
Data in the UCSC Genome database are generally freely available for public
use; any limitations are described in the Conditions of Use section of the UCSC
Browser home page. When downloading multiple files, we recommend using
the FTP site rather than the web access.
Postscript
Since this chapter was first written, several groups have employed RGCs topredict early mammalian divergences. Kriegs et al. (24) and Nishihara et al.
(25) used interspersed repeats, whereas Murphy et al. (26) employed insertions/deletions in protein-coding regions as well as interspersed repeats. The computer
programs used by Murphy et al. to find informative coding indels will be madeavailable along with the code described in this chapter.
Acknowledgments
Daryl Thomas and Elliott Margulies manage the ENCODE alignments andother resources, Brian Raney produced alignments to the low-redundancy genomes(including elephant and armadillo), and Angie Hinrichs generated the insect
multiple alignments.
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