----------------------- Need shift 5 command (have more than 9 arguments): ----------------------- ----------------------- Start of variable list: ----------------------- SCRIPT_DIR: /unprotected/projects/waxmanlab/kkarri/G160_Samples/Scripts/09c_DiffExp_4_lncRNA_featureCounts Dataset_DIR: /restricted/projectnb/waxmanlab/kkarri/G160_Samples Dataset_Label: G160 ANNOTATION_FILE_DIR: /unprotected/projects/waxmanlab/routines/GTF_Files ANNOTATION_FILE: /unprotected/projects/waxmanlab/routines/GTF_Files/ncRNA_output_filtered_final_gene.txt CONDITION_1_NAME: F_Nuclear CONDITION_2_NAME: M_Nuclear Lengths_DIR: /unprotected/projects/waxmanlab/routines/GTF_Files/lengths GENE_LENGTHS_FILE: intronic_only_gene_models_ncRNA_for_counting_lengths.txt COUNT_DIR: LncRNA_Intronic_Only_GTF OUTPUT_PREFIX: DiffExp_v2_LncRNA_Intronic_Only DiffExp_Index: DiffExp_4g COL_SUFFIX: LncRNA_Intronic_Only COUNT_PROGRAM: featureCounts ----------------------- End of variable list ----------------------- ========================================================== Starting on : Thu Aug 24 13:37:22 EDT 2017 Running on node : scc-wa1 Current directory : /restricted/projectnb/waxmanlab/kkarri/G160_Samples/Scripts/09c_DiffExp_4_lncRNA_featureCounts Current job ID : 390598 Current job name : Step_09c_DiffExp_4g Task index number : undefined Parameter for multiple cores : 1 ========================================================== Change dir to scratch directory Print scratch directory location: /scratch/390598.1.w Loading required modules... ------------------------------------------ Sample_DIR: G160_M3 Sample_ID: G160_M3 Description: Female M_Num: M3 Copy Condition_1 sample count files to Condition_1 folder Copy Condition_1 sample count summary files to Condition_1 folder M_Num_Cond1_List: M3 ------------------------------------------ ------------------------------------------ Sample_DIR: G160_M5 Sample_ID: G160_M5 Description: Male M_Num: M5 Copy Condition_2 sample count files to Condition_2 folder Copy Condition_2 sample count summary files to Condition_2 folder M_Num_Cond2_List: M5 ------------------------------------------ ========================================================== Number of replicates in each condition: NUM_REP_CONDITION1: 1 NUM_REP_CONDITION1: 1 WARNING: Either your Condition_1 or Condition_2 has only 1 sample (no replicates) The differential expression analysis is designed for cases where there are at least 2 replicates/biological condition. The resulting differential expression analysis will be converted to a stringent version due to absence of replicate samples. Samples from this comparison will be treated as "replicates" to estimate the gene level variance in edgeR and, separately in, DESeq2. Expect very few to zero DE genes from DESeq2, EdgeR should yield a DE gene count. Interpreting the results: use the edgeR fold change or DESeq ratio (not DESeq fold change) Note regarding the SEGEX upload file(s): Since DESeq2 fold changes are not the same as the ratio of the normalized counts between the conditions, so the output of the segex upload file will be different between "DESeq ratio" and "DESeq foldChange". The former is truly the ratio between the normalized counts between the two conditions (plus pseudocounts) whereas the latter is the fold change estimated by DESeq. ========================================================== Renaming input count files number of mapped reads (feature count summary) Status G160_M3_sorted.bam Assigned 14760214 Unassigned_Ambiguity 524736 Unassigned_MultiMapping 0 Unassigned_NoFeatures 8499638 Unassigned_Unmapped 0 Unassigned_MappingQuality 0 Unassigned_FragmentLength 0 Unassigned_Chimera 0 Unassigned_Secondary 0 Unassigned_Nonjunction 0 Unassigned_Duplicate 0 Status G160_M5_sorted.bam Assigned 10266584 Unassigned_Ambiguity 402214 Unassigned_MultiMapping 0 Unassigned_NoFeatures 10603117 Unassigned_Unmapped 0 Unassigned_MappingQuality 0 Unassigned_FragmentLength 0 Unassigned_Chimera 0 Unassigned_Secondary 0 Unassigned_Nonjunction 0 Unassigned_Duplicate 0 ========================================================== List files in Input -rw-r--r-- 1 kkarri pulmseq 359173 Aug 24 13:37 /scratch/390598.1.w/Input/F_Nuclear0.out -rw-r--r-- 1 kkarri pulmseq 298 Aug 24 13:37 /scratch/390598.1.w/Input/F_Nuclear0.summary -rw-r--r-- 1 kkarri pulmseq 355636 Aug 24 13:37 /scratch/390598.1.w/Input/M_Nuclear0.out -rw-r--r-- 1 kkarri pulmseq 299 Aug 24 13:37 /scratch/390598.1.w/Input/M_Nuclear0.summary -rw-r--r-- 1 kkarri pulmseq 414633 Aug 24 13:37 /scratch/390598.1.w/Input/intronic_only_gene_models_ncRNA_for_counting_lengths.txt /scratch/390598.1.w/Input/F_Nuclear: total 8 drwxr-xr-x 2 kkarri pulmseq 4096 Aug 24 13:37 . drwxr-xr-x 4 kkarri pulmseq 4096 Aug 24 13:37 .. /scratch/390598.1.w/Input/M_Nuclear: total 8 drwxr-xr-x 2 kkarri pulmseq 4096 Aug 24 13:37 . drwxr-xr-x 4 kkarri pulmseq 4096 Aug 24 13:37 .. ========================================================== Starting to run my commands Printing Rscript command: Rscript differentialAnalysis.R F_Nuclear M_Nuclear 1 1 /unprotected/projects/waxmanlab/routines/GTF_Files/ncRNA_output_filtered_final_gene.txt /scratch/390598.1.w/Input DiffExp_v2_LncRNA_Intronic_Only intronic_only_gene_models_ncRNA_for_counting_lengths.txt [1] "Arguments for differentialAnalysisDESeq.R:" [1] "F_Nuclear" [1] "M_Nuclear" [1] 1 [1] 1 [1] "/unprotected/projects/waxmanlab/routines/GTF_Files/ncRNA_output_filtered_final_gene.txt" [1] "/scratch/390598.1.w/Input" [1] "DiffExp_v2_LncRNA_Intronic_Only" [1] "intronic_only_gene_models_ncRNA_for_counting_lengths.txt" ========================================================== Create SEGEX formatted file: Printing Rscript command: Rscript formatForSegex_ver3.R DiffExp_v2_LncRNA_Intronic_Only_F_Nuclear_M_Nuclear.txt M_Nuclear_G160_M5_vs_F_Nuclear_G160_M3_DiffExp_v2_LncRNA_Intronic_Only_forSEGEXUpload 1 LncRNA_Intronic_Only ========================================================== Comparison_Info: M_Nuclear_G160_M5_F_Nuclear_G160_M3 #---------------------------------------------------------------------------------- Running Diff_Genes.R #---------------------------------------------------------------------------------- Printing Rscript command: Rscript Diff_Genes.R M_Nuclear_G160_M5_vs_F_Nuclear_G160_M3_DiffExp_v2_LncRNA_Intronic_Only_forSEGEXUpload_DESeq.txt 2 0.05 DESeq LncRNA_Intronic_Only'_'M_Nuclear_G160_M5_F_Nuclear_G160_M3 [1] "Print arguments:" [1] "-----------------" [1] "Differential expression_Output:" [1] "M_Nuclear_G160_M5_vs_F_Nuclear_G160_M3_DiffExp_v2_LncRNA_Intronic_Only_forSEGEXUpload_DESeq.txt" [1] "fold_change_cutoff:" [1] "2" [1] "padj_cutoff:" [1] "0.05" [1] "postfix:" [1] "DESeq" [1] "count_method:" [1] "LncRNA_Intronic_Only_M_Nuclear_G160_M5_F_Nuclear_G160_M3" [1] "-----------------" [1] "DESeq or EdgeR job most likely failed." [1] "Quitting R now." Rscript Diff_Genes.R M_Nuclear_G160_M5_vs_F_Nuclear_G160_M3_DiffExp_v2_LncRNA_Intronic_Only_forSEGEXUpload_EdgeR.txt 2 0.05 EdgeR LncRNA_Intronic_Only'_'M_Nuclear_G160_M5_F_Nuclear_G160_M3 [1] "Print arguments:" [1] "-----------------" [1] "Differential expression_Output:" [1] "M_Nuclear_G160_M5_vs_F_Nuclear_G160_M3_DiffExp_v2_LncRNA_Intronic_Only_forSEGEXUpload_EdgeR.txt" [1] "fold_change_cutoff:" [1] "2" [1] "padj_cutoff:" [1] "0.05" [1] "postfix:" [1] "EdgeR" [1] "count_method:" [1] "LncRNA_Intronic_Only_M_Nuclear_G160_M5_F_Nuclear_G160_M3" [1] "-----------------" [1] "DESeq or EdgeR job most likely failed." [1] "Quitting R now." #---------------------------------------------------------------------------------- Running Venn_Diff_Genes.R #---------------------------------------------------------------------------------- Printing Rscript command: #---------------------------------------------------------------------------------- Rscript Venn_Diff_Genes.R Down_Genes_DESeq_LncRNA_Intronic_Only_M_Nuclear_G160_M5_F_Nuclear_G160_M3.txt Down_Genes_EdgeR_LncRNA_Intronic_Only_M_Nuclear_G160_M5_F_Nuclear_G160_M3.txt LncRNA_Intronic_Only_Counting DiffExp_4g [1] "Print arguments:" [1] "-----------------" [1] "File1:" [1] "Down_Genes_DESeq_LncRNA_Intronic_Only_M_Nuclear_G160_M5_F_Nuclear_G160_M3.txt" [1] "File2:" [1] "Down_Genes_EdgeR_LncRNA_Intronic_Only_M_Nuclear_G160_M5_F_Nuclear_G160_M3.txt" [1] "Subtitle:" [1] "LncRNA_Intronic_Only_Counting" [1] "DiffExp_Index:" [1] "DiffExp_4g" [1] "-----------------" #---------------------------------------------------------------------------------- Rscript Venn_Diff_Genes.R Up_Genes_DESeq_LncRNA_Intronic_Only_M_Nuclear_G160_M5_F_Nuclear_G160_M3.txt Up_Genes_EdgeR_LncRNA_Intronic_Only_M_Nuclear_G160_M5_F_Nuclear_G160_M3.txt LncRNA_Intronic_Only_Counting DiffExp_4g [1] "Print arguments:" [1] "-----------------" [1] "File1:" [1] "Up_Genes_DESeq_LncRNA_Intronic_Only_M_Nuclear_G160_M5_F_Nuclear_G160_M3.txt" [1] "File2:" [1] "Up_Genes_EdgeR_LncRNA_Intronic_Only_M_Nuclear_G160_M5_F_Nuclear_G160_M3.txt" [1] "Subtitle:" [1] "LncRNA_Intronic_Only_Counting" [1] "DiffExp_Index:" [1] "DiffExp_4g" [1] "-----------------" #---------------------------------------------------------------------------------- #---------------------------------------------------------------------------------- Merging Count.Table(s) into one text file #---------------------------------------------------------------------------------- Comparison_Info: M_Nuclear_G160_M5_F_Nuclear_G160_M3 #---------------------------------------------------------------------------------- ========================================================== Renaming the Differential_Expression_File ========================================================== List files in Input -rw-r--r-- 1 kkarri pulmseq 0 Aug 24 13:37 /scratch/390598.1.w/Input/DiffExp_4g_Venn_Tables_M_Nuclear_G160_M5_F_Nuclear_G160_M3.txt -rwxr-xr-x 1 kkarri pulmseq 7706 Aug 24 13:37 /scratch/390598.1.w/Input/Diff_Genes.R -rw-r--r-- 1 kkarri pulmseq 359173 Aug 24 13:37 /scratch/390598.1.w/Input/F_Nuclear0.out -rw-r--r-- 1 kkarri pulmseq 298 Aug 24 13:37 /scratch/390598.1.w/Input/F_Nuclear0.summary -rw-r--r-- 1 kkarri pulmseq 355636 Aug 24 13:37 /scratch/390598.1.w/Input/M_Nuclear0.out -rw-r--r-- 1 kkarri pulmseq 299 Aug 24 13:37 /scratch/390598.1.w/Input/M_Nuclear0.summary -rwxr-xr-x 1 kkarri pulmseq 9993 Aug 24 13:37 /scratch/390598.1.w/Input/Venn_Diff_Genes.R -rwxr-xr-x 1 kkarri pulmseq 4968 Aug 24 13:37 /scratch/390598.1.w/Input/formatForSegex_ver3.R -rw-r--r-- 1 kkarri pulmseq 414633 Aug 24 13:37 /scratch/390598.1.w/Input/intronic_only_gene_models_ncRNA_for_counting_lengths.txt /scratch/390598.1.w/Input/F_Nuclear: total 8 drwxr-xr-x 2 kkarri pulmseq 4096 Aug 24 13:37 . drwxr-xr-x 4 kkarri pulmseq 4096 Aug 24 13:37 .. /scratch/390598.1.w/Input/M_Nuclear: total 8 drwxr-xr-x 2 kkarri pulmseq 4096 Aug 24 13:37 . drwxr-xr-x 4 kkarri pulmseq 4096 Aug 24 13:37 .. ========================================================== List files in scratch total 3.4M drwxr-xr-x 3 kkarri pulmseq 4.0K Aug 24 13:37 . drwxrwxrwt. 47 root root 24K Aug 24 13:37 .. -rw-r--r-- 1 kkarri pulmseq 56 Aug 24 13:37 Condition_1.txt -rw-r--r-- 1 kkarri pulmseq 54 Aug 24 13:37 Condition_2.txt drwxr-xr-x 4 kkarri pulmseq 4.0K Aug 24 13:37 Input -rwxr-xr-x 1 kkarri pulmseq 13K Aug 24 13:37 differentialAnalysis.R -rw-r--r-- 1 kkarri pulmseq 3.3M Aug 24 13:37 ncRNA_output_filtered_final_gene.txt ========================================================== Re-naming files in OUTPUT_DIR Need to append the COUNT_PROGRAM name to all output files List files in OUTPUT_DIR total 32K drwxr-sr-x 2 kkarri waxmanlab 512 Aug 24 13:37 . drwxr-sr-x 7 kkarri waxmanlab 32K Aug 24 13:37 .. -rw-r--r-- 1 kkarri waxmanlab 0 Aug 24 13:37 DiffExp_4g_Venn_Tables_M_Nuclear_G160_M5_F_Nuclear_G160_M3_featureCounts.txt ========================================================== ========================================================== Finished on : Thu Aug 24 13:37:37 EDT 2017 0 minutes and 15 seconds elapsed. ==========================================================