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5_quant_express.txt
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5_quant_express.txt
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#warming_AK
#Adriana L. Romero-Olivares' protocol for processing RNAseq data of soil
#Section 5
#File name: 5_quant_express
#Step: Quantification of reads
#This protocol uses Express to quantify the abundance of contigs from control and treatment reads
#for control
module load express/1.5.1
express --rf-stranded --no-bias-correct -o express_CALL Trinity_ALL_f.Trinity.fasta bowtie2.CALL_sorted_rf.bam
#for treatment
module load express/1.5.1
express --rf-stranded --no-bias-correct -o express_WALL Trinity_ALL_f.Trinity.fasta bowtie2.WALL_sorted_rf.bam
#Next, using Trinity's toolkit quantify transcript abundance using the script "align_and_estimate_abundance.pl"
#parameters are specific for the data used in this experiment
module load trinity/r2016-2.3.2
module load R/3.3.2
module load bowtie2/2.2.7
module load express/1.5.1
/data/apps/trinity/r2016-2.3.2/util/align_and_estimate_abundance.pl --transcripts Trinity_ALL_f.Trinity.fasta --prep_reference --seqType fq --SS_lib_type RF --samples_file reps.txt --est_method eXpress --aln_method bowtie2 --trinity_mode --output_dir estimate_abundance --thread_count 4
#you will need a .txt file indicating replicates, in the script above the file is "reps.txt"
#Example:
# cond_A cond_A_rep1 Trinity_input_files/all_C1-5.r1.fq Trinity_input_files/all_C1-5.r2.fq
# cond_A cond_A_rep2 Trinity_input_files/all_C3-2.r1.fq Trinity_input_files/all_C3-2.r2.fq
# cond_A cond_A_rep3 Trinity_input_files/C4-5_repeat.r1.fq Trinity_input_files/C4-5_repeat.r2.fq
# cond_A cond_A_rep4 Trinity_input_files/all_C5-5.r1.fq Trinity_input_files/all_C5-5.r2.fq
# cond_B cond_B_rep1 Trinity_input_files/all_W1-3.r1.fq Trinity_input_files/all_W1-3.r2.fq
# cond_B cond_B_rep2 Trinity_input_files/all_W3-1.r1.fq Trinity_input_files/all_W3-1.r2.fq
# cond_B cond_B_rep3 Trinity_input_files/all_W4-3.r1.fq Trinity_input_files/all_W4-3.r2.fq
# cond_B cond_B_rep4 Trinity_input_files/all_W5-5.r1.fq Trinity_input_files/all_W5-5.r2.fq
#using Trinity's toolkit build matrix from above results (files results.xprs) with script "abundance_estimates_to_matrix.pl"
#parameters are specific for the data used in this experiment
module load trinity/r2016-2.3.2
module load R/3.3.2
module load bowtie2/2.2.7
module load express/1.5.1
/data/apps/trinity/r2016-2.3.2/util/abundance_estimates_to_matrix.pl --est_method eXpress cond_A_rep1/results.xprs cond_A_rep2/results.xprs cond_A_rep3/results.xprs cond_A_rep4/results.xprs cond_B_rep1/results.xprs cond_B_rep2/results.xprs cond_B_rep3/results.xprs cond_B_rep4/results.xprs --name_sample_by_basedir --out_prefix express_matrix
#files generated in express_matrix will be used for downstream analyses