With eXpress
# Download and unzip the latest binary files for eXpress software:
wget https://pachterlab.github.io/eXpress/downloads/express-1.5.1/express-1.5.1-linux_x86_64.tgz
tar -xvzf express-1.5.1-linux_x86_64.tgz
Copy binaries to your $PATH (e.g. /usr/local/bin):
cd express-1.5.1-linux_x86_64
sudo cp express /usr/local/bin/
# Calculate raw reads count using BAM file and a corresponding reference transcriptome file:
## For read1 of a stranded library (accept only forward single-end alignments):
express --f-stranded --no-bias-correct reference.fa INPUT_over_reference.bam
mv results.xprs INPUT_over_reference.xprs
## For read2 of a stranded library (accept only reverse single-end alignments):
express --r-stranded --no-bias-correct reference.fa INPUT.bam
mv results.xprs INPUT_over_reference.xprs
Important notes:
- eXpress works equally well on sorted BAM files generated by most aligners
- eXpress will not work on sorted BAM files generated by bowtie or bowtie2 aligner if the -k option was used for mapping
# Combine separate XPRS files into counts matrices:
sort -k2,2 INPUT1_over_reference.xprs > INPUT1_over_reference_sorted.xprs
sort -k2,2 INPUT2_over_reference.xprs > INPUT2_over_reference_sorted.xprs
sort -k2,2 INPUT3_over_reference.xprs > INPUT3_over_reference_sorted.xprs
sort -k2,2 INPUT4_over_reference.xprs > INPUT4_over_reference_sorted.xprs
sort -k2,2 INPUTN_over_reference.xprs > INPUTN_over_reference_sorted.xprs
cut -f2,3,5 INPUT1_over_reference_sorted.xprs > INPUT1_over_reference_sorted_ss.xprs
cut -f2,3,5 INPUT2_over_reference_sorted.xprs > INPUT2_over_reference_sorted_ss.xprs
cut -f2,3,5 INPUT3_over_reference_sorted.xprs > INPUT3_over_reference_sorted_ss.xprs
cut -f2,3,5 INPUT4_over_reference_sorted.xprs > INPUT4_over_reference_sorted_ss.xprs
cut -f2,3,5 INPUTN_over_reference_sorted.xprs > INPUTN_over_reference_sorted_ss.xprs
paste INPUT1_over_reference_sorted_ss.xprs INPUT2_over_reference_sorted_ss.xprs INPUT3_over_reference_sorted_ss.xprs INPUT4_over_reference_sorted_ss.xprs INPUTN_over_reference_sorted_ss.xprs > INPUT1234N_over_reference_temp.xprs
cut -f1,2,3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,N+3 INPUT1234N_over_reference_temp.xprs > INPUT1234N_over_reference.xprs
With RSEM
# Download the latest source files for RSEM software, unzip, compile and install:
wget https://github.com/deweylab/RSEM/archive/v1.3.1.tar.gz
gunzip RSEM-1.3.1.tar.gz
cd RSEM-1.3.1
make
sudo make install #RSEM executables will be installed to /usr/local/bin
# Preparing transcriptome reference sequences
## For transcriptome reference
rsem-prepare-reference reference.fa reference.fa
## For genome reference
rsem-prepare-reference --gtf path_to_gtf_file/genome_reference.gtf path_to_fasta_file/genome_reference.fa genome_reference.fa
# Perform reads counting
## For strand-specific reads mapped to transcriptome reference
rsem-calculate-expression --bam --no-bam-output -p [insert number of threads] --strand-specific INPUT_over_reference.bam path_to_RSEM_refs/RSEM_refs
## For strand-specific reads mapped to genome reference
rsem-calculate-expression --bam --no-bam-output -p [insert number of threads] --strand-specific INPUT_over_reference.bam path_to_RSEM_refs/RSEM_refs outputfilename
