amybingo/rpkm_rnaseq_count

RPKM for RNASeq counts

RPKM_normalization

RPKM for RNAseq V1.3

Usage for sample input provided:

perl rpkm_script_beta.pl sample_count_test.count 2:9 28 > sample_count_test.rpkm

Description

In above example 'sample_count_test.count' file has count data from 2 to 9th column;
28th column has length of each genes calculated from Gencode GTF (Note below).

General usage:

perl rpkm_script_beta.pl input_count_file.txt ActualColumnStart:ActualColumnEnd ColumnGeneLength > OUTPUT_RPKM_FILE 

ActualColumnStart = For example you have GeneID in first column and counts starts from second column. This should be '2'

ActualColumnEnd = Upto which column you need RPKM

ColumnGeneLength = Length of each gene (**NOTE below)

**NOTE: Steps to prepare your input

1. Length of the gene can be obtained from Gencode GTF by following command (Successfully tested upto Gencode V19)

cat gencode.vXX.annotation.gtf | awk -F'\t' '{if($3=="gene") {split($9,a,";"); print a[1]"\t"$5-$4};}' | sed 's/[gene_id |"|]//g' > YOUR_GENE_LENGTH_FILE

2. Combine input_count_file.txt and YOUR_GENE_LENGTH_FILE by GeneID or First column

join -j1  <(sort input_count_file.txt) <(sort YOUR_GENE_LENGTH_FILE) > OUTPUT_ANNOTATED_COUNT_FILE

3. Run the script over OUTPUT_ANNOTATED_COUNT_FILE

perl rpkm_script_beta.pl OUTPUT_ANNOTATED_COUNT_FILE ActualColumnStart:ActualColumnEnd ColumnGeneLength > OUTPUT_ANNOTATED_RPKM_FILE

Description

ActualColumnStart = For example you have GeneID in first column and counts starts from second column. This should be '2'

ActualColumnEnd = Upto which column you need RPKM

ColumnGeneLength = Length of each gene

RPKM calculation

RPKM = (10^9 * C)/(N * L), where

C = Number of reads mapped to a gene

N = Total mapped reads in the experiment

L = gene length in base-pairs for a gene

Author: Santhilal Subhash

Contact: [email protected]