Downloading genomic sequence alignments from UCSC in maf format

Step 1 : Connect to UCSC Genome home page.

Click on "Genome Browser" at left of page.

Step 2 : Select reference genome and assembly, and specify the genome position of interest.

After entering specifications then click 'Submit'.

Step 3 : Click 'Tables' at top of the browser display.

Step 4 : Enter specifications in Table Browser.

• Group : Comparative Genomics
• Table : multiz28way
• Output format : MAF
• Output file : something.maf
• File type returned : plain text

After entering specifications then click 'get output'. Save this file to your computer as a text file (not html).

Step 5 : You are now ready to upload the maf genomic alignment file to the Uprobe website for the custom design of universal probes.

Help for Custom Probe Design

Step 1 : Upload your aligned sequence file.

• Sequence Alignment File - select the file of aligned sequences from which we will design universal probes. Alignment files for custom probe design may not be larger than 50 megabytes in size. If your file is larger, please subdivide it before uploading for probe design.
• Sequence Alignment Format - tell us the file format (must be one of maf, fasta, blastz, clustal, phylip ( interleaved or sequential), or lagan ( pairwise or multiple)). Note that the length of species names must not exceed 10 characters.

Step 2 : Choose reference species and enter phylogeny (optional)

• Reference Species - from the display of all species in the uploaded file, select one species from which the probe sequence should be designed (the 'reference species').
• Phylogeny - you may enter a phylogeny of the species that will be analyzed during probe design. This is optional. If left empty, we will create a phylogeny for you from the the aligned sequences. Phylogeny must be in Newick format. Include branch lengths if you know them, otherwise we will calculate them based on the sequences. If we create the phylogeny or add branch lengths, you will be able to edit the phylogeny in a later step.

If you enter a phylogeny, it should include only the species that you want to be considered for scoring and design of universal probes. If unwanted species are present in the alignment file, do not include these species in the phylogeny.

Phylogeny for custom probe design must be rooted (not unrooted) and must be bifurcating at all levels (not multifurcating).

Step 3 : Choose the species to include, and designate one as outgroup.

This step is included only if you did not enter a phylogeny for the aligned species.

• Species to include - from the display of all species in the uploaded file, select all the species for which the aligned sequences should be included in scoring to design universal probes. (This selection allows the exclusion of unwanted species that may be present in the alignment file.)
• Outgroup Species - from the display of all species in the uploaded file, select one species as the 'outgroup'. If the alignment file includes species that you did not 'include' (above), one of these excluded species may be suitable for outgroup. The ideal outgroup species is 'outside' of the group ('clade') for which you are designing probes, but is no farther 'outside' than necessary. If your alignment file does not contain 'excluded' species, then you should choose for 'outgroup' the included species that is phylogenetically most distant from the reference species.

Step 4 : Final edit of phylogeny

This step is included only if you did not enter a phylogeny for the aligned species, or your phylogeny did not include branch lengths.

• Phylogeny - you may edit branch lengths of the phylogeny, if desired. Relative branch lengths of the phylogeny are the basis of scoring by which universal probes are selected. If you are confident that branch lengths we derived are wrong, change them here. Do not change them without reason. You should understand the Newick format before making changes.

Step 5 : Choose probe spacing, and genome for megablast (optional)

• Spacing - Specify physical spacing of probes. The minimum distance between select probes will be one-half the distance specified.
• Megablast Genome (Optional) - If you specify a megablast genome, the probes will be classified as 'unique' (occurring only once in that genome) or 'non-unique'. Select from the currently available genomes in the drop-down list.

Step 6 : Choose megablast classification type

This step is included only if you chose a megablast genome.

• Probe Megablast Type - specify 'unique' or 'non-unique' probes, with respect to the megablast genome chosen in the previous step.

Step 7 : Enter your email address, and choose file formats for probes

• Email address - the address to receive probes and a summary of the design process.
• Probe File Formats - choose the forms of file content produced from the Custom Probe Design process. Three forms of results files are produced by default :
  • csv 'comma separated values'    Example
  • fasta, standard for use in Blast queries    Example
  • aligned sequences of species used in probe design    Example