Load Blast

Create the FASTA files

Run Blast or Diamond

Load Blast files

Add to existing hits

Example and details

This page describes using Blast or Diamond for computing the hits for SyMAP.

The steps are very easy:

• Use the SyMAP Export to create a FASTA file of the protein, CDS or Exons.
• Run Blast or Diamond on them and put the results in data/seq_results/<project1>_to_<project2>/blast.
• Set Load Blast files from Parameters, then run Selected Pairs or Selected Redo (add to existing hits).

Please read the following for the details.

Create the FASTA files

When you load the project, you may request that either the Exons or CDSs be loaded; see Parameters.

Select View for the project, then Export. →If you have loaded the CDSs, then you can create a file of Proteins and/or Spliced CDSs. →If you have loaded the Exons , you can create a file of Spliced Exons. Likewise, export the proteins or spliced transcripts from the second project.

The export can take awhile, e.g. for the demoArab 13,476 genes:

• It took 13 minutes on my old MacOS x86_64
• It took 3 minutes on my newer MacOS M4
• It uses a single CPU and does not use much memory.

By default, the exported files will be in the SyMAP /export directory.
The FASTA header line uses the ID= value from the GFF file attribute; see Annotation file.

Run Blast or Diamond

SyMAP does not run Blast or Diamond for you. You must download Blast or Diamond.
For protein sequences, use Blastp or Diamond.
For nucleotide sequences, use Blastn.

The output file must be the tabular format (-outfmt 6).

It is recommended that the results only show the top 5 hits per gene (-max_target_seqs 5).

For the Demo, you can copy and paste the commands from Example below.

Load Blast files

Start SyMAP and select Pair Parameters. Select the Load Blast files option. If protein files are being compared, select Proteins, else select Nucleotides. For protein files, the length of the hits is multiplied by 3 to better approximate its alignment. When you select Save, the directory data/<project1>_to_<project2>/blast will be created.
Exit SyMAP and move the blast tab file(s) to data/<project1>_to_<project2>/blast.
Restart SyMAP and select Selected Pair; a popup (as shown on right) will confirm your parameters; select Confirm. SyMAP will load the blast hits, then execute the Collinear algorithm and Synteny algorithm.

SyMAP will load ALL files in the /blast directory.

• The hits coordinates are set to start at the beginning of the first CDS or exon. That is, the hit does not align correctly with the gene (MUMmer hits do align correctly).

• Only one hit per gene pair from a file will be loaded unless there are hits that do not overlap.
  The blast hits are not clustered like the MUMmer hits are.

• If a hit between a gene pair already exists in the database, only the Hit Type column will be updated to indicate that the hit was found in multiple files (see Query table).

Add to existing hits

• In order to load both MUMmer and Blast hits, first run SyMAP with MUMmer. Then add the Blast hits.

• The Load&Synteny can be executed with any number of blast files.
  If multiple files are being loaded, make sure they are all from protein sequences or all from nucleotide sequences, and have the parameter correctly set to Protein or Nucleotide, respectively.
  SyMAP does NOT check if a file is already loaded.

• MUMmer, Blast and Diamond produce different subsets of gene pair hits. Moreover, Blast and Diamond produce different subsets depending one whether project1 is the blast database or project2. By loading all variations, the maximum number of gene pair hits will be in the SyMAP database.

Remove hits added by a given file: There is no way to do this. Clear Pair then reload the desired files.

Example and Details

Load MUMmer + Blast Hits

Query Table

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By default, the exported files are written to /export. I generally create a directory
   data/seq_results/<project1>_to_<project2>/export
and move the FASTA files here, and run Blast/Diamond from this directory.
Then I copy the desired *.tsv files to ../blast.
Note: Clear Pair only removes the /align and /blast directories.

The following filenames are suggested naming; SyMAP does not care as long as they are outfmt 6.

Diamond:

diamond makedb --in demoBrapProt.faa -d brapdb
diamond blastp -q demoArabProt.faa -d brapdb  -o diamBrapProt.tsv -f 6 -k 5 -p 4

Blastp:

makeblastdb -in demoBrapProt.faa -dbtype prot -out brapAAdb
blastp -query demoArabProt.faa -db brapAAdb -out bltBrapProt.tsv -outfmt 6 -max_target_seqs 5 -num_threads 4

Blastn

makeblastdb -in demoBrapExon.fna -dbtype nucl -out brapNTdb
blastn -query demoArabExon.fna -db brapNTdb -out bltBrapExon.tsv -outfmt 6 -max_target_seqs 5 -num_threads 4

or

makeblastdb -in demoBrapCDS.fna -dbtype nucl -out brapNTdb
blastn -query demoArabCDS.fna -db brapNTdb -out bltBrapCDS.tsv -outfmt 6 -max_target_seqs 5 -num_threads 4

MUMmer + Blast hits

Mask vs Blast shows timing comparison results between using mask MUMmer and Blast.
The following shows adding multiple files of blast hits.

1. MUMmer hits were loaded and clustered.

   >> Summary for demoArab to demoBrap
        8,507 hits
           44 synteny blocks
        8,143 gene hits
        5,789 synteny hits
   

2. Two files of diamond protein results were added, one with demoArab as the blast database, the other with demoBrap as the blast database.

   Load Blast for demoArab and demoBrap
        7,701 gene pair hits in database
      --- Read File /diamBrapProt.tsv ---
          Projects Gene IDs in correct order
       29,448 Hits from Blast file
        8,202 Failed %identity cutoff of 40
          626 Failed E-value cutoff of 1.0E-10
        7,481 Update existing hits - file #1
       13,139 Saved new hits
      --- Read File /diamArabProt.tsv ---
          Projects Gene IDs in reverse order - swapping
       22,864 Hits from Blast file
        4,725 Failed %identity cutoff of 40
          390 Failed E-value cutoff of 1.0E-10
       15,687 Update existing hits - file #2
        2,062 Saved new hits
       22,902 Total hits in database
   Finish Load Blast                    12s
   
   >> Summary for demoArab to demoBrap
       23,708 hits
           76 synteny blocks
       23,344 gene hits
        8,023 synteny hits
   Complete processing                  51s
   

The bottom of the Summary shows:

Query table

EE indicates a MUMmer hit. The Bp12 means it was also found in both blast files. A Bp1 indicates it was only found in the 1st blast file, A Bp2 indicates it was only found in the 2nd blast file. Load Blast will add numbers for up to 9 blast files.

All hit values are from the FIRST hit entered into the database, e.g. the %Olap and %Id from the first row are from the EE hit.

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Email Comments To: cas1@arizona.edu