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Quantifying alternate RNA decoding

 

This section organizes instructions and links to software and pipelines for identifying, validating, and quantifying amino acid substitutions from alternate RNA decoding.

 

Creating sample-specific protein databases from RNA-seq data

Pipeline for generating custom protein databases

This pipeline is used for generating sample-specific protein fasta databases from RNA-seq data.

 

Identifying, validating, and quantifying amino acid substitutions from alternate RNA decoding

Search LC-MS proteomics data with MaxQuant dependent peptide search against sample-specific protein databases to identify peptides with modifications which may represent amino acid substitutions.

Sample MaxQuant parameter file is provided in the decode GitHub repository.

A useful tutorial for running MaxQuant searches in Linux.

Identify candidate substituted amino acid peptides (SAAP)

Apply decode pipeline steps 1 and 2 to identify peptides with potential amino acid substitutions from alternate RNA decoding. See detailed pipeline instructions.

Validate SAAP

Decode pipeline step 3. Search LC-MS proteomics data with MaxQuant (or other search engine) standard database search against sample-specific protein databases appended with candidate SAAP.

Sample MaxQuant parameter file is provided in the decode GitHub repository.

Quantify SAAP

Apply decode pipeline steps 4 and 5 to quantify validated amino acid substitutions from alternate RNA decoding. See detailed pipeline instructions.

Results from running decode pipeline on TMT-labeled and label-free datasets, as described by Tsour et al are provided in decode data directory.

 

Detailed methods are found in the article, Tsour et al.

 

Data pipelines for reproducing decode data analysis

A pipeline for reproducing the analysis by Tsour et al is available at the decode GitHub repository.