Downloads for data in this track are available:

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

Description

This track shows multiple alignments of 44 virus sequences, aligned to the SARS-CoV-2 reference sequence NC_045512.2, genome assembly GCF_009858895.2. It also includes measurements of evolutionary conservation using two methods (phastCons and phyloP) from the PHAST package, for all 44 virus sequences. 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.

In the track display, the sequence is labeled using its NCBI Nucleotide accession number.

The mapping between sequence accession identifiers and more descriptive names is provided via a text file on our download server.

Display Conventions and Configuration

Pairwise alignments of each species to the SARS-CoV-2 genome are displayed as a series of colored blocks indicating the functional effect of polymorphisms (in pack mode), or as a wiggle (in full mode) that indicates alignment quality. In dense display mode, percent identity of the whole alignments is shown in grayscale using darker values to indicate higher levels of identity.

In pack mode, regions that align with 100% identity are not shown. When there is not 100% percent identity, blocks of four colors are drawn.

• Red blocks are drawn when a polymorphism in a coding region results in a change in the amino acid that is generated.
• Green blocks are drawn when a polymorphism in a coding region results in no change to the amino acid that is generated.
• Blue blocks are drawn when a polymorphism is outside a coding region.
• Pale yellow blocks are drawn when there are no aligning bases to that region in the reference genome.

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).

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.

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 SARS-CoV-2 sequence at those alignment positions relative to the longest non-SARS-CoV-2 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.

Methods

Pairwise alignments with the reference sequence were generated for each sequence using lastz version 1.04.00. Parameters used for each lastz alignment:

# hsp_threshold      = 2200
# gapped_threshold   = 4000 = L
# x_drop             = 910
# y_drop             = 3400 = Y
# gap_open_penalty   = 400
# gap_extend_penalty = 30
#        A    C    G    T
#   A   91  -90  -25 -100
#   C  -90  100 -100  -25
#   G  -25 -100  100  -90
#   T -100  -25  -90   91
# seed=1110100110010101111 w/transition
# step=1

High-scoring chains were then placed along the genome, with gaps filled by lower-scoring chains, to produce an alignment net.

The multiple alignment was constructed from the resulting pairwise alignments progressively aligned using multiz/autoMZ. The phylogenetic tree was calculated on 31mer frequency similarity and neighbor joining that distance matrix with the phylip toolset command: neighbor. The reference sequence NC_045512v2 is at the top of the tree:

(((NC_045512v2 MN996532v1) ((((DQ022305v2 GQ153547v1) GQ153542v1)
(MG772933v1 MG772934v1)) ((((((DQ071615v1 KJ473815v1)
((((FJ588686v1 KY770858v1) ((((((KF367457v1 KY417144v1)
(KY417151v1 KY417152v1)) ((KY417142v1 MK211377v1) (MK211376v1 MK211378v1)))
((((KT444582v1 KY417143v1) KY417149v1) KY417146v1) (KY417147v1 KY417148v1)))
(KJ473816v1 KY417145v1)) MK211375v1)) NC_004718v3) KP886808v1)) MK211374v1)
(KF569996v1 KU973692v1)) JX993988v1) ((((DQ412042v1 DQ648856v1) (KJ473812v1
KY770860v1)) KY938558v1) ((DQ412043v1 KJ473814v1) JX993987v1)))))
(KY352407v1 NC_014470v1))

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 all-species 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 44-way alignment (msa_view). The 4d sites were derived from the NCBI gene set, filtered to select single-coverage long transcripts.

This same tree model was used in the phyloP calculations; however, the background frequencies were modified to maintain reversibility. The resulting tree model: all species.

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 used were: expected-length=45, target-coverage=0.3, rho=0.3.

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 --most-conserved 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: lastz (formerly blastz) and multiz by Minmei Hou, Scott Schwartz, Robert Harris, and Webb Miller of the Penn State Bioinformatics Group
• 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).
• Chaining and Netting: axtChain, chainNet by Jim Kent 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

References

Gire SK, Goba A, Andersen KG, Sealfon RS, Park DJ, Kanneh L, Jalloh S, Momoh M, Fullah M, Dudas G et al. Genomic surveillance elucidates Ebola virus origin and transmission during the 2014 outbreak. Science 2014 Sep 12;345(6202):1369-72. PMID: 25214632; Supplemental Materials and Methods

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

Lastz (formerly Blastz):

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

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

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

Funding

This annotation track in the UCSC SARS-CoV-2 genome browser is funded by generous private donors to the UC Santa Cruz Genomics Institute.