Part 1 of a series in the directory [paper-pacific.oyster-larvae/tree/master/protocols](https://github.com/grace-ac/paper-pacific.oyster-larvae/tree/master/protocols). This protocol outlines the method of creating chromatogram libraries from raw data for data visualization purposes. # DIA analysis Performed on Woodpecker in FTR 209, Roberts' Lab. Modified from a DIA Analysis from MacCoss lab: [data-analysis](https://docs.google.com/document/d/1Vr3wE7Z8eJVenUWgbxJ3CmXxCoNiba_HYQXh7sNce_k/edit) [EncyclopeDIA manual](https://bitbucket.org/searleb/encyclopedia/downloads/EncyclopeDIA%20manual.pdf) ### You will need: - MSConvert (_Windows only!_) [http://proteowizard.sourceforge.net/download.html](http://proteowizard.sourceforge.net/download.html) - EncyclopeDIA suite (*.jar file): [https://bitbucket.org/searleb/encyclopedia/downloads/](https://bitbucket.org/searleb/encyclopedia/downloads/) - Raw files: [2015Oysterseed *.raw](http://owl.fish.washington.edu/phainopepla/C_gigas/2015-12-30/) ### Main steps: 1. Convert .raw to .mzML using MSConvert 2. Build library using Walnut in EncyclopeDIA suite 3. Use SkylineDaily (separate markdown file) ## Step 1: Convert .raw to .mzML using MSConvert Download .raw files from [/phainopepla/C_gigas/2015-12-30/](http://owl.fish.washington.edu/phainopepla/C_gigas/2015-12-30/) ### 1.1 Open MSConvert and set filters: - Peak picking - Click "Add" ### 1.2 In top left, click "Browse" and navigate to directory where you put the .raw files, set output directory for .mzML files, and select all and click "Start" ![img](../images/EncyclopeDIA-images/01-MSconvert.PNG) ### 1.3 Monitor progress in new window that pops up ![img](../images/EncyclopeDIA-images/02-converting-to-mzML.PNG) ## Step 2: Build library using Walnut in EncyclopeDIA suite ### 2.1 Set background and target as your fasta of interest ![img](../images/EncyclopeDIA-images/03-walnut-add-background.PNG) This fasta came from (included in original [DIA protocol](https://github.com/RobertsLab/resources/blob/master/protocols/DIA-data-Analyses.md), end of Step 2): ``` srlab@swan MINGW64 ~ $ cd Desktop/ srlab@swan MINGW64 ~/Desktop $ cd grace/ srlab@swan MINGW64 ~/Desktop/grace $ head Cg_Giga_cont_prtc_AA_digested_Mass400to6000.txt Protein_Name Sequence Unique_ID Monoisotopic_Mass Predicte d_NET Tryptic_Name CHOYP_043R.5.5|m.64252 SPSEDPDAPIENILQTNSVYKPK 1 2541.2598016 0.3655t2 .1 CHOYP_043R.5.5|m.64252 SPSEDPDAPIENILQTNSVYKPKK 2 2669.35475980.34 14 t2.2 CHOYP_043R.5.5|m.64252 SPSEDPDAPIENILQTNSVYKPKKEPTYDENVVVK 3 3942.973 762 0.3449 t2.3 CHOYP_043R.5.5|m.64252 SPSEDPDAPIENILQTNSVYKPKKEPTYDENVVVKIISQDTPTILR 45180.67 6764 0.5144 t2.4 CHOYP_043R.5.5|m.64252 KEPTYDENVVVK 5 1419.7245246 0.2186 t3.2 CHOYP_043R.5.5|m.64252 KEPTYDENVVVKIISQDTPTILR 6 2657.4275266 0.4593t3 .3 CHOYP_043R.5.5|m.64252 KEPTYDENVVVKIISQDTPTILRVSFTVNR 7 3460.85649280.56 58 t3.4 CHOYP_043R.5.5|m.64252 EPTYDENVVVK 8 1291.6295664 0.2301 t4.1 CHOYP_043R.5.5|m.64252 EPTYDENVVVKIISQDTPTILR 9 2529.3325684 0.4402t4 .2 srlab@swan MINGW64 ~/Desktop/grace $ awk '{print $1,$2}' Cg_Giga_cont_prtc_AA_digested_Mass400to6000.txt | head Protein_Name Sequence CHOYP_043R.5.5|m.64252 SPSEDPDAPIENILQTNSVYKPK CHOYP_043R.5.5|m.64252 SPSEDPDAPIENILQTNSVYKPKK CHOYP_043R.5.5|m.64252 SPSEDPDAPIENILQTNSVYKPKKEPTYDENVVVK CHOYP_043R.5.5|m.64252 SPSEDPDAPIENILQTNSVYKPKKEPTYDENVVVKIISQDTPTILR CHOYP_043R.5.5|m.64252 KEPTYDENVVVK CHOYP_043R.5.5|m.64252 KEPTYDENVVVKIISQDTPTILR CHOYP_043R.5.5|m.64252 KEPTYDENVVVKIISQDTPTILRVSFTVNR CHOYP_043R.5.5|m.64252 EPTYDENVVVK CHOYP_043R.5.5|m.64252 EPTYDENVVVKIISQDTPTILR srlab@swan MINGW64 ~/Desktop/grace $ awk '{print $1,$2}' Cg_Giga_cont_prtc_AA_digested_Mass400to6000.txt \ > > Cg_Giga_cont_prtc_AA_M400-6000-2c.txt ``` And was converted to .fasta ### 2.2 Add .mzML files that were converted in Step 1: ![img](../images/EncyclopeDIA-images/04-walnut-add-mzml.PNG) And you can monitor progress of reading .mzML files: ![img](../images/EncyclopeDIA-images/05-read-mzml-progres.PNG) **Note:** this process creates a lot of files with different extensions (i.e., `*.dia`; `*.mzML.delta_rt.pdf`; `*.mzML.pecan.decoy.txt`; etc), but you don't need to worry about those. ### 2.3 Save Chromatogram Library (.elib) ![img](../images/EncyclopeDIA-images/06-save-chroma-lib.PNG) ## Step 3: Save quant files and BLIB in EncyclopeDIA ### 3.1 Close and re-open EncylopeDIA GUI. Select .elib saved in Step 2.3 for the library ![img](../images/EncyclopeDIA-images/07-select-elib.PNG) ### 3.2 Select background: same .fasta used in Step 2.1 ![img](../images/EncyclopeDIA-images/08-select-background-fasta.PNG) ### 3.3 Add .mzML files: same as those used in Step 2.2 ![img](../images/EncyclopeDIA-images/09-add-mzML.PNG) ### 3.4 Save Quant Reports: ![img](../images/EncyclopeDIA-images/11-save-quant-reports.PNG) ### 3.5 Save BLIB ![img](../images/EncyclopeDIA-images/13-save-blib.PNG) ### 3.6 Save new chromatogram Library ![img](../images/EncyclopeDIA-images/15-saving-chrom-lib2.PNG) ### Here is what my directory contents look like with all the libraries and saved reports: ![img](../images/EncyclopeDIA-images/17-final-contents.PNG)