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Frequently asked questions

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Q. How can I use EncyclopeDIA in a non-English speaking environment?

Sorry, EncyclopeDIA is not currently internationalized for different languages. Aside from translation issues, EncyclopeDIA also assumes a decimal point separator (common to North America and most of Asia), rather than a decimal comma separator (common to mainland Europe). You can change the default settings for Java to force your computer to operate in this manner by specifying "user.language" and "user.region" to "US". For example, you can start EncyclopeDIA using the command:

java -Xmx12g -jar encyclopedia-0.9.0-executable.jar -Duser.language=en-US -Duser.region=US

Q. How do I run EncyclopeDIA or Walnut at the command line?

EncyclopeDIA can also be used at the command line. For example, to run EncyclopeDIA across all mzMLs in a directory, you can execute the shell command:

for i in directory/to/my/files/*.mzML; do 
    java -Xmx12g -jar encyclopedia-0.9.0-executable.jar -i $i -l library.dlib -f sequences.fasta; 
done

Here, Java is run with 12 GB of RAM with the flag "-Xmx12g". Additional flags are "-i" to specify the input mzML, "-l" to specify the library, and "-f" to specify the FASTA database. You can run the help function to get additional command line options:

java -Xmx12g -jar encyclopedia-0.9.0-executable.jar -h

The "-libexport" function can be used to generate global quantitative results.

java -Xmx12g -jar encyclopedia-0.9.0-executable.jar -libexport -a true -l library.dlib -f sequences.fasta -i directory/to/my/files/ -o output.elib

With the "-a true" flag, this command performs retention time alignment (match-between-runs) and global FDR estimation. After you run the -libexport function, all of the results are filtered to a 1% FDR with Percolator at the peptide (in the .peptides.txt table) and protein level (in the .proteins.txt table). With the "-a false" flag, the -libexport function can be used to generate a chromatogram library as "output.elib".


Q. How do I mimic the EncyclopeDIA GUI to generate chromatogram libraries with the command line?

Here is an example Walnut workflow for using GPF-DIA to generate a chromatogram library, and using that library to search single-injection DIA data:

# generate a Walnut CLIB from GPF-DIA and a FASTA
for i in library/*.mzML; do 
    java -Xmx10g -jar encyclopedia-0.9.0-executable.jar -walnut -i $i -f sequences.fasta; 
done
java -Xmx10g -jar encyclopedia-0.9.0-executable.jar -libexport -walnut -o chromatogram_library.elib -i library -f sequences.fasta -a false 

# process single-injection DIA with the CLIB
for i in quant/*.mzML; do 
    java -Xmx10g -jar encyclopedia-0.9.0-executable.jar -i $i -l chromatogram_library.elib -f sequences.fasta;
done
java -Xmx10g -jar encyclopedia-0.9.0-executable.jar -libexport -o report.elib -l chromatogram_library.elib -i library -f sequences.fasta -a true 

And here is the same process using a Prosit-predicted library or a DDA library:

# generate an EncyclopeDIA CLIB from GPF-DIA and a Prosit Library
for i in library/*.mzML; do 
    java -Xmx10g -jar encyclopedia-0.9.0-executable.jar -i $i -l prosit_library.dlib -f sequences.fasta; 
done
java -Xmx10g -jar encyclopedia-0.9.0-executable.jar -libexport -o chromatogram_library.elib -i library -f sequences.fasta -a false 

# process single-injection DIA with the CLIB (exactly the same as with Walnut)
for i in quant/*.mzML; do 
    java -Xmx10g -jar encyclopedia-0.9.0-executable.jar -i $i -l chromatogram_library.elib -f sequences.fasta;
done
java -Xmx10g -jar encyclopedia-0.9.0-executable.jar -libexport -o report.elib -l chromatogram_library.elib -i library -f sequences.fasta -a true 

Q. How do I search for PTMs with Walnut?

In general, we do not suggest you search for PTMs unless you are enriching specifically for them. You can specify PTMs at the command line as "-variable M=15.9949" or "-fixed C=57.0214635,K=8.014199,R=10.008269". PTM codes are comma deliminated so you can specify multiple PTMs, but beware that Walnut does not accept multiple fixed or multiple variable PTMs on the same amino acids.


Q. How do I run EncyclopeDIA in a headless environment?

In some "headless" unix environments at the command line, you might get an error that starts:

java.awt.AWTError: Can’t connect to X11 window server

In these cases you need to tell Java to not connect to a windowing server with "-Djava.awt.headless=true":

java -Xmx12g -Djava.awt.headless=true -jar encyclopedia-0.9.0-executable.jar -h

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