Shotgun Protein Sequencing - usage

This page contain user instructions for running SPS/cSPS projects:

  • Running SPS projects
  • Running cSPS projects
  • Generating cSPS reports
  • Generating SPSplot reports

SPS / cSPS data processing on the Grid

After having unpacked the Matlab package mentioned below:

https://bix-lab.ucsd.edu/display/CCMStools/Installation

The entire data processing can be executed on the Grid with a simple script:

$ cd linux-g++-64/bin
$ ./grid.sh <PROJECT DIR> .. 128

Generating cSPS reports

This set of steps is illustrated for the Heavy Chain (HC); these should be repeated for the Light Chain (LC)

  • Create a new csps_MYAB_LC.xls Excel file
  • Load "<csps>/homology/main_homglue_overlaps.txt" into a new "Consensus sequence" Excel worksheet
    • Load as a text file with ";" (semicolon) separators
    • Change the font size to 8 and resize the columns to fit the text size (fits the coverage view to a typical screen width)
    • Change the fill color to white (hides Excel column/row lines)
    • Move worksheet to csps_MYAB_LC.xls
  • Load "<csps>/homology/homglue_ref.txt" into a new "All contigs" Excel worksheet
    • Load as a text file with ";" (semicolon) separators
    • Sort by "Protein index", "Match Start", "Match End"
    • Move worksheet to csps_MYAB_LC.xls
  • Load "<csps>/homology/ref_homglue_overlaps.txt" into a new "Mapped contigs" Excel worksheet
    • Load as a text file with ";" (semicolon) separators
    • Change the font size to 8 and resize the columns to fit the text size (fits the coverage view to a typical screen width)
    • Change the fill color to white (hides Excel column/row lines)
    • Move worksheet to csps_MYAB_LC.xls

Generating SPSplot reports

SPSplot statistics files are generated by passing in the required files and by using the '--stats' option.  The aforementioned files include the standard SPS and cSPS information:

FILE_STARS                    Stars spectrum data (pklbin format)
FILE_COMP                     Component file (bin format)
FILE_SEQS                     Consensus spectra file (plkbin format)
FILE_MS                       MS/MS spectrum data (pklbin format)
FILE_FASTA                    Proteins database file (txt format)

And also relies on PRM, Stars spectra, pairs and Inspect information:

FILE_PRM                      PRM spectrum data (pklbin format)
FILE_STARSINDEX               Identified spectra (bin format)
FILE_PAIRS                    Spectrum pairs (bin format)
FILE_MATCHPAIRS               Spectrum pairs data (pklbin format)
FILE_STARSPAIRS               Star pairs (bin format)
FILE_INSPECT                  Inpect data (txt format)

The call will generate the following files with their associated headers:

<outdir>/stats-ms.txt
<outdir>/stats-prm.txt
<outdir>/stats-stars.txt
* spectrum index
* % explained intensity
* % explained peaks
* % observed b-ions (charge 1 only)
* % observed y-ions (charge 1 only)
* % observed neutral-loss ions (-H2O, -NH3, a-ions, b-iso, y-iso, charge 1 only)
* % observed doubly-charged ions: b, b-iso, b-H2O, b-NH3, y, y-iso, y-H2O, y-NH3
* % intensity explained by b-ions (charge 1 only)
* % intensity explained by y-ions (charge 1 only)
* % intensity explained by neutral-loss ions (-H2O, -NH3, a-ions, b-iso, y-iso, charge 1 only)
* % intensity explained by doubly-charged ions: b, b-iso, b-H2O, b-NH3, y, y-iso, y-H2O, y-NH3
* total number of spectrum peaks
* parent mass error
* % observed "breaks": a break is a prefix mass supported by at least one b-ion or y-ion, in charge state 1 or 2
* Length of the longest series of consecutive breaks

<outdir>/stats-all.txt (per line)
* average of stats-ms.txt
* average of stats-prm.txt
* average of stats-stars.txt (%intensity in b-ions is greater or equal to that in y-ions)
* average of stats-stars.txt (%intensity in y-ions is greater or equal to that in b-ions)

<outdir>/stats-align.txt
* type of pair: -1 to 6
* correct shift: -1, 0, or 1
* # of matched peaks (minimum from both aligned spectra, if different)
* % matched intensity in spectrum 1
* % matched intensity in spectrum 2
* % mass overlap in spectrum 1
* % mass overlap in spectrum 2

<outdir>/stats-prm_pairs.txt
<outdir>/stats-stars_pairs.txt
* index of spectrum 1
* index of spectrum 2
* type of pair
* correct shift
* min % explained-intensity
* min % mass overlap

<outdir>/stats-prm_pairs_hist.txt
<outdir>/stats-stars_pairs_hist.txt
* x-axis: min(%explained-intensity(S1), %explained-intensity(S2))
* y-axis: min %mass-overlap between S1 and S2
* bin size 10%: 0-10, 11-20, etc.

<outdir>/stats-stars_seq.txt
* spectrum index
* % explained intensity
* % explained peaks
* % observed b-ions (charge 1 only)
* % observed y-ions (charge 1 only)
* % observed neutral-loss ions (-H2O, -NH3, a-ions, b-iso, y-iso, charge 1 only)
* % observed doubly-charged ions: b, b-iso, b-H2O, b-NH3, y, y-iso, y-H2O, y-NH3
* % intensity explained by b-ions (charge 1 only)
* % intensity explained by y-ions (charge 1 only)
* % intensity explained by neutral-loss ions (-H2O, -NH3, a-ions, b-iso, y-iso, charge 1 only)
* % intensity explained by doubly-charged ions: b, b-iso, b-H2O, b-NH3, y, y-iso, y-H2O, y-NH3
* total number of spectrum peaks
* parent mass error
* % observed "breaks": a break is a prefix mass supported by at least one b-ion or y-ion, in charge state 1 or 2
* Length of the longest series of consecutive breaks

<outdir>/stats-contig_seq.txt
* contig index
* # correct peaks
* # incorrect peaks
* # undefined peaks
* % intensity in correct peaks
* % intensity in incorrect peaks
* % intensity in undefined peaks
* average of the spectrum statistics for all identified spectra in each contig
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