Frequently asked questions

Check here to report Issues.

Check here for File Formats Guidelines.

Q. How can I run EncyclopeDIA with ToF instruments?

EncyclopeDIA is pre-configured for Orbitrap-based instruments. We recommend using the following additional commands either at the command line or in the "Additional Command Line Options:" box for ToF instruments:

-filterPeaklists true

We also recommend setting a minimum intensity threshold using an appropriate value like:

-minIntensity 125

Q. How can I run EncyclopeDIA with more RAM?

Many tasks in EncyclopeDIA, such as building large libraries or loading large mzMLs (>10GB), benefit from having access to more RAM. At the command line, you can specify this as:

java -Xmx12g -jar encyclopedia-1.2.2-executable.jar

Q. How can I look at EncyclopeDIA reports and ELIBs?

ELIBs are SQLite databases that can be opened and queried using a free browser. You can get more information on the file format and structure in the File Formats guide.

To get full quantitative tables in text format, you need to load multiple single files, and then click the "Save Quant Report" button. This will execute a "match-between-runs"-style quantitative algorithm to give you a full quantitative data matrix in TSV format at both the peptide- and protein-level for downstream statistical work.

Q. Why does "Save Quant Reports" not report a peptides.txt/proteins.txt report when quantifying a single sample? What does quantifying from a single sample even mean for DIA?

Quantitation with DIA is different than quant with DDA: with DDA you always measure precursor ions and you’re forced to use them for quant, even if they’re interfered with. As you’re always going to use these ions for quant, you can record their intensities and they stay meaningful when compared to other samples. With DIA, the quant engine can, and should, choose the best ions to quantify to optimally reduce the risk of interference. As a result, it doesn’t make sense for the engine to select quantified ions from only one sample, since in another sample the engine might want to select entirely different ions for quantification. To fit with this mentality, the peptide/protein quant reports are only generated when you have multiple samples that you want to compare. That said, you can extract out “quantitative” intensities for fragment ions using the ELIB in an SQLite Browser, and these measurements show up in the PeptideQuants table. However, we don’t recommend doing this unless you have a non-quantitative question (e.g. you DON’T want to compare between samples).

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-1.2.2-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-1.2.2-executable.jar -i $i -l library.dlib -f sequences.fasta; 
done

java -Xmx12g -jar encyclopedia-1.2.2-executable.jar -h

java -Xmx12g -jar encyclopedia-1.2.2-executable.jar -libexport -a true -l library.dlib -f sequences.fasta -i directory/to/my/files/ -o 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-1.2.2-executable.jar -walnut -i $i -f sequences.fasta; 
done
java -Xmx10g -jar encyclopedia-1.2.2-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-1.2.2-executable.jar -i $i -l chromatogram_library.elib -f sequences.fasta;
done
java -Xmx10g -jar encyclopedia-1.2.2-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-1.2.2-executable.jar -i $i -l prosit_library.dlib -f sequences.fasta; 
done
java -Xmx10g -jar encyclopedia-1.2.2-executable.jar -libexport -o chromatogram_library.elib -l prosit_library.dlib -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-1.2.2-executable.jar -i $i -l chromatogram_library.elib -f sequences.fasta;
done
java -Xmx10g -jar encyclopedia-1.2.2-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:

#!bash

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

#!bash

java -Xmx12g -Djava.awt.headless=true -jar encyclopedia-1.2.2-executable.jar -h

Q. How do I use the official EncyclopeDIA docker image?

First, you need to install docker (https://docs.docker.com/get-docker/). To pull the image locally, run docker pull searlelab/encyclopedia:<version_number>. The version number will always match the EncyclopeDIA executable jar inside the docker container. The excecutable jar lives in the /code directory within the docker container. It's path will be /code/encyclopedia-<version_number>-executable.jar. You'll need to use the headless environment to execute it. For that, see the question above Q. How do I run EncyclopeDIA in a headless environment?.

Here's a step by step example:

# Pull the image
docker pull searlelab/encyclopedia:1.12.31

# Run and enter the shell of the container
docker run -exec -it searlelab/encyclopedia:1.12.31 /bin/bash

# Show the help options
java -Djava.awt.headless=true -jar /code/encyclopedia-1.12.31-executable.jar -h

Q. Can I use the EncyclopeDIA source code or logo in print or on the web?

Yes! The source code is released under the Apache 2 licence, which allows redistribution and modification as long as you include the required notices.

The logo is Creative Commons licensed using the Attribution 4.0 International license (CC BY 4.0). You are free to use it, even in commercial instances, as long as you attribute back to this paper: Searle, B.C., Pino, L.K., Egertson, J.D. et al. Chromatogram libraries improve peptide detection and quantification by data independent acquisition mass spectrometry. Nat Commun 9, 5128 (2018). https://doi.org/10.1038/s41467-018-07454-w