Using safeQuant.R					Updated 2015-03-13

Author: erikahrne
Contact: erik.ahrne@unibas.ch

### Basic usage common to all workflows ###

1) Navigate to the folder where the safeQuant.R script is installed.
mymac:exec erikahrne$ cd /Users/erikahrne/dev/R/workspace/SafeQuant/exec

2) Display help
mymac:exec erikahrne$ Rscript safeQuant.R -h

3) Specify input file
mymac:exec erikahrne$ Rscript safeQuant.R -i  ../inst/testData/QI_2.0/peptide_measurements1.csv

4) Define experimental design

2 Conditions. Control Samples: 1,2,3. Case 1: Samples 4,5
mymac:exec erikahrne$ Rscript safeQuant.R -i  ../inst/testData/QI_2.0/peptide_measurements2.csv  --EX 1,2,3:4,5

3 Conditions. Control Samples: 4,5. Case 1: Samples 1,2. Case 2: Sample 3
mymac:exec erikahrne$ Rscript safeQuant.R -i  ../inst/testData/QI_2.0/peptide_measurements2.csv  --EX 4,5:1,2:3

2 Conditions. Control Samples: 4,5. Case 1: Sample 1
mymac:exec erikahrne$ Rscript safeQuant.R -i  ../inst/testData/QI_2.0/peptide_measurements2.csv  --EX 4,5:1

5) Require at least X peptide per protein
mymac:exec erikahrne$ Rscript safeQuant.R -i  ../inst/testData/QI_2.0/peptide_measurements2.csv  --FN 2

LFQ Protein Relative Quantification

### TO BE REVIEWED
Compatible with Feature, Protein and Peptide Measurement Progenesis .csv exports. If using Progenesis QI 2.0 or later we recommend using Peptide Measurement Progenesis exports. 
To export:
- File -> Export Peptide Measurements.  This option is available once you have reached the "Resolve Conflicts" Step in Progenesis QI.
    - When choosing properties to be included in the exported file check the "Grouped accessions (for this sequence)" check box.


### LFQ Phospho Relative Quantification ###

mymac:exec erikahrne$ Rscript safeQuant.R -i  ../inst/testData/2014/peptides1_FILTERED.csv -f ../inst/testData/2014/sp_hum_160512.decoy.fasta --FM phospho Ð-EP ÐFS 2

-f ../inst/testData/2014/sp_hum_160512.decoy.fasta
Supply protein (.fasta) database so that all phospho sites will be annotated with their protein position (amino acid number starting from n-term) as well as sequence motifs (motif-X input format http://motif-x.med.harvard.edu/).

--FM phospho
Filter data to only include phospho peptides

--EP
Do not carry out Protein level quantification

--FS 2
Filter data to only include peptides carrying maximum 2 variable PTMs. 

### TMT Protein Relative Quantification ###

mymac:exec erikahrne$ Rscript safeQuant.R -i  ../inst/testData/2014/TMT_10-Plex_Scaffold_Raw_Export_Example.xls

Make sure to specifiy the experimental design if needed using (--EX flag).

TMT 6-plex default experimental design is 1,2,3:4,5,6
TMT 10-plex default experimental design is 1,2,3,4:5,6,7:8,9,10

### LFQ Protein Absolute Abundance Estimation ###

Calculate intensity-based absolute-protein-quantification (iBAQ) metric per protein.
Reference: Global quantification of mammalian gene expression control, Schwanhausser (2011), \url{http://www.ncbi.nlm.nih.gov/pubmed/21593866}

mymac:exec erikahrne$ Rscript safeQuant.R -i  ../inst/testData/2014/peptides2_FILTERED.csv -f ../inst/testData/2014/sp_mouse_160512.decoy.fasta ÐAI

Calculate Top3 (Mean of X most intense features)
Reference: Absolute quantification of proteins by LCMSE: A virtue of parallel MS acquisition, Silva (2006), \url{http://www.ncbi.nlm.nih.gov/pubmed/16219938}

mymac:exec erikahrne$ Rscript safeQuant.R -i  ../inst/testData/2014/peptides2_FILTERED.csv --AT