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Pamgene phosphorylation chip analysis

Custom homebrew solution to analyse data from Pamgene kinase activity profiling system, created due to issues with the original Bionavigator software. Workflow was reverse engineered and approximated from various presentations and posters found online and as such is less complex and complete then the one offered commercially.

Example usage

pamgene.R -i raw_data_stk.csv --pamgene pamgene.csv --phosphonet phosphonet.csv --ctrl A1 --exp A2 --kinexus_score 300 --batch_correction

Commandline arguments

-i, --file_in input file containg data from pamgene kinase activity profiling system. See preview of input formats for more information.

-o, --dir_out output directory

--pamgene csv file containing information about peptides on the pamgene chip

--phosphonet csv file containing information about kinases phosphorylating specific protein residues of proteins included im pamgene csv file

-c, --ctrl control group name as a single string or comma-separated indices of alphabetically arranged <group_name>_<chip_id> denominators

-e, --exp experimental group name as a single string or comma-separated indices of alphabetically arranged <group_name>_<chip_id> denominators

--top_kinases top kinases from phosphonet file to include in calculations, if NULL the number will be estimated from data

--kinexus_score threshold kinexus version 2 score that determines whether the residue is considered as being phosphorylated by a kinase. All phosphorylations below threshold are omitted.

--no_perm number of permutations used to calculate specificity and selectivity scores

-b, --batch_correction whether to remove batch effect from peptide kinetics matrix. Uses sva::ComBat for calculations.

Example output

Peptide statistics

id mean_ctrl mean_exp statistic pvalue A1_710250713 A1_710250715 A2_710250713 A2_710250715
ACM1_421_433 6.79 6.38 0.65 0.55 6.54 6.32 6.54 6.76
ACM1_444_456 6.70 6.46 0.37 0.74 6.53 6.42 6.85 7.02
ACM4_456_468 6.44 6.03 0.69 0.52 6.21 5.87 6.40 6.31
ACM5_494_506 11.02 10.70 0.22 0.84 11.39 10.67 11.06 11.62

Kinase scores

kinase_id kinase_name specificity selectivity total sum_score mean_score sd_score
Q9UBS0 p70S6Kb (RPS6KB2) 0.70 1.00 1.70 -0.28 -0.01 0.03
P41743 PKCi (PRKCI) 0.36 1.00 1.36 -0.66 -0.33 0.48
P22694 PKACb PRKACB 1.00 0.34 1.34 -0.30 -0.01 0.02
P25098 BARK1 (ADRBK1; GRK2) 0.20 1.00 1.20 -0.45 -0.23 0.42

Preview of input formats

Raw data

Csv file with one of the following structures:

ID UniprotAccession <group1>_<chip_id1>_<time1> <group1>_<chip_id1>_<time2> etc.

for all groups, chips and time points, or same data transformed into long format with exactly 6 columns:

ID UniprotAccession group chip_id time value

Pamgene

File describing the peptides included on the pamgene chip. Information can be inferred from the peptides reported in the raw data file by aligning the peptide protein sequences and adding proteins that pass similarity threshold and contain phosphoryltable amino acids in the corresponding frequences. Following columns are required in any order.

id uniprot_id phosphosite
ATF2_47_59 P15336 69
ATF2_47_59 P15336 71
ATF2_47_59 P15336 73
ATF2_47_59 P17544 51

Phosphonet

With the knowledge of peptides included in the chip, phosphonet file can be generated by Phosphonet scraper. Following columns are required in any order.

substrate site kinase_rank kinase_name kinase_id kinexus_score_v2
P15336 69 1 JNK1 (MAPK8) P45983 633
P15336 69 2 JNK3 (MAPK10) P53779 633
P15336 69 3 ERK2 (MAPK1) P28482 615
P15336 69 4 ERK1 P27361 611

Known Issues and limitations

  • Script is only able to calculate kinase activity comparing two groups (control vs. experimental)
  • Pamgene and Phosphonet files are not supplied, users are required to generate their own
  • In current release the peptide kinetics matrix is automatically scaled and centered, if other beheviour is desired, it has to be changed in the code directly