TPMCalculator quantifies mRNA abundance directly from the alignments by parsing BAM files. The input parameters are the same GTF files used to generate the alignments, and one or multiple input BAM file(s) containing either single-end or paired-end sequencing reads. The TPMCalculator output is comprised of four files per sample reporting the TPM values and raw read counts for genes, transcripts, exons and introns respectively.
- Roberto Vera Alvarez, Lorinc Sandor Pongor, Leonardo Mariño-Ramírez, David Landsman; TPMCalculator: one-step software to quantify mRNA abundance of genomic features, Bioinformatics, , bty896, https://doi.org/10.1093/bioinformatics/bty896
TPMCalculator is available on Bioconda: https://bioconda.github.io/recipes/tpmcalculator/README.html
NIH Biowulf users can load TPMcalculator as a module: https://hpc.nih.gov/apps/TPMCalculator.html
Clone the BAMTools repository from GitHub: https://github.com/pezmaster31/bamtools
Compile it on this way and set the environment variables for TPMCalculator:
cd bamtools
mkdir build
cd build
cmake -DCMAKE_INSTALL_PREFIX=../ ..
make
make install
cd ..
export BAMTOOLS_DIR=`pwd`
export CPPFLAGS="-I $BAMTOOLS_DIR/include/bamtools/"
export LDFLAGS="-L $BAMTOOLS_DIR/lib64 -Wl,-rpath,$BAMTOOLS_DIR/lib64"
That's it. BAMTools was compiled and the env variables were set for compiling TPMCalculator.
After the installation of BAMTools go to the TPMCalculator folder and do make:
make
A bin folder will be created with the TPMCalculator executable.
Use provided Dockerfile based on the BioContainers base image.
docker build -t biocontainers/tpmcalculator:0.0.1 https://raw.githubusercontent.com/ncbi/TPMCalculator/master/Dockerfile
docker run -v /path_to_data:/data --user=yourUID:your:GID biocontainers/tpmcalculator:0.0.1 TPMCalculator -g /data/path_to_GTF/genes.gtf -b /data/path_to_bam/sample1.bam
A CWL tool definition is also provided tpmcalculator.cwl
Use it like this:
cwl-runner tpmcalculator.cwl --out_stderr=test.stderr --out_stdout=test.stdout -g genes.gtf -b sample_1.bam
Usage: ./bin/TPMCalculator -g GTF_file [-d BAM_files_directory|-b BAM_file]
./bin/TPMCalculator options:
-v Print info
-h Display this usage information.
-g GTF file
-d Directory with the BAM files
-b BAM file
-k Gene key to use from GTF file. Default: gene_id
-t Transcript key to use from GTF file. Default: transcript_id
-c Smaller size allowed for an intron created for genes. Default: 16. We recommend to use the reads length
-p Use only properly paired reads. Default: No. Recommended for paired-end reads.
-q Minimum MAPQ value to filter out reads. Default: 0. This value depends on the aligner MAPQ value.
-o Minimum overlap between a reads and a feature. Default: 8.
-e Extended output. This will include transcript level TPM values. Default: No.
-a Print out all features with read counts equal to zero. Default: No.
The model to describe the genomic features used for a gene is created from the GTF provided by the user. TPMCalculator performs two transformations which are executed on the genomic coordinates generating regions for the genes that include the exons and “pure” intron regions as shown in Figure S1. The first transformation creates overlapped exons for all alternative spliced forms of the genes. A single gene model is generated with unique exons and introns which includes the sequence of all exonic regions. The second transformation process creates a list of pure intron regions that replace those generated by the first transformation. We should indicate that only the intron regions are modified to generate regions not overlapped by exons of other genes. Reporting TPM values for these unique introns allows further identification of alternative splicing events like intron retention. Additionally, a set of non-overlapped gene features (exons and introns) are generated and used for TPM calculation.
For more detailed description and instalation guide lines see https://github.com/ncbi/TPMCalculator/wiki/
Roberto Vera Alvarez Email: veraalva@ncbi.nlm.nih.gov
Lorinc Pongor Email: pongorlorinc@gmail.com
Leonardo Mariño-Ramírez Email: marino@ncbi.nlm.nih.gov
David Landsman Email: landsman@ncbi.nlm.nih.gov
This software is a "United States Government Work" under the terms of the United States Copyright Act. It was written as part of the authors' official duties as United States Government employees and thus cannot be copyrighted. This software is freely available to the public for use. The National Library of Medicine and the U.S. Government have not placed any restriction on its use or reproduction.
Although all reasonable efforts have been taken to ensure the accuracy and reliability of the software and data, the NLM and the U.S. Government do not and cannot warrant the performance or results that may be obtained by using this software or data. The NLM and the U.S. Government disclaim all warranties, express or implied, including warranties of performance, merchantability or fitness for any particular purpose.
Please cite NCBI in any work or product based on this material.
