----------------------- Need shift 5 command (have more than 9 arguments): ----------------------- ----------------------- Start of variable list: ----------------------- SCRIPT_DIR: /restricted/projectnb/waxmanlab/kkarri/G160_Samples/Scripts/09a_DiffExp_3_HTSeq Dataset_DIR: /restricted/projectnb/waxmanlab/kkarri/G160_Samples Dataset_Label: G160 GTF_Files_DIR: /unprotected/projects/waxmanlab/routines/GTF_Files ANNOTATION_FILE: RefSeq_GeneBody.gtf CONDITION_1_NAME: M_Nuclear CONDITION_2_NAME: M_Cytoplasmic Lengths_DIR: /unprotected/projects/waxmanlab/routines/GTF_Files/lengths GENE_LENGTHS_FILE: Intron_Only_Regions_Lengths.txt COUNT_DIR: RefSeq_Intron_GTF OUTPUT_PREFIX: DiffExp_v2_Intronic_Only DiffExp_Index: DiffExp_3c COL_SUFFIX: Intronic_Only COUNT_PROGRAM: HTSeq ----------------------- End of variable list ----------------------- ========================================================== Starting on : Thu Aug 24 04:34:19 EDT 2017 Running on node : scc-kb2 Current directory : /restricted/projectnb/waxmanlab/kkarri/G160_Samples/Scripts/09a_DiffExp_3_HTSeq Current job ID : 383329 Current job name : Step_09a_DiffExp_3c Task index number : undefined Parameter for multiple cores : 1 ========================================================== Change dir to scratch directory Print scratch directory location: /scratch/383329.1.linga Loading required modules... ------------------------------------------ Sample_DIR: G160_M5 Sample_ID: G160_M5 Description: Male M_Num: M5 Copy Condition_1 sample count files to Condition_1 folder calculate mapped reads: G160_M5 M_Num_Cond1_List: M5 ------------------------------------------ ------------------------------------------ Sample_DIR: G160_M6 Sample_ID: G160_M6 Description: Male M_Num: M6 Copy Condition_2 sample count files to Condition_2 folder calculate mapped reads: G160_M6 M_Num_Cond2_List: M6 ------------------------------------------ ========================================================== 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 Counting program: HTSeq Removing last 5 lines (remove special counters) Counting program: HTSeq Removing last 5 lines (remove special counters) number of mapped reads 30291227 40878015 ========================================================== List files in Input -rw-r--r-- 1 kkarri pulmseq 300238 Aug 24 04:34 /scratch/383329.1.linga/Input/Intron_Only_Regions_Lengths.txt -rw-r--r-- 1 kkarri pulmseq 229213 Aug 24 04:34 /scratch/383329.1.linga/Input/M_Cytoplasmic0.out -rw-r--r-- 1 kkarri pulmseq 9 Aug 24 04:34 /scratch/383329.1.linga/Input/M_Cytoplasmic0_num_mapped_reads.txt -rw-r--r-- 1 kkarri pulmseq 246277 Aug 24 04:34 /scratch/383329.1.linga/Input/M_Nuclear0.out -rw-r--r-- 1 kkarri pulmseq 9 Aug 24 04:34 /scratch/383329.1.linga/Input/M_Nuclear0_num_mapped_reads.txt /scratch/383329.1.linga/Input/M_Cytoplasmic: total 8 drwxr-xr-x 2 kkarri pulmseq 4096 Aug 24 04:34 . drwxr-xr-x 4 kkarri pulmseq 4096 Aug 24 04:34 .. /scratch/383329.1.linga/Input/M_Nuclear: total 8 drwxr-xr-x 2 kkarri pulmseq 4096 Aug 24 04:34 . drwxr-xr-x 4 kkarri pulmseq 4096 Aug 24 04:34 .. ========================================================== Starting to run my commands Printing Rscript command: Rscript differentialAnalysis.R M_Nuclear M_Cytoplasmic 1 1 RefSeq_GeneBody.gtf /scratch/383329.1.linga/Input DiffExp_v2_Intronic_Only Intron_Only_Regions_Lengths.txt [1] "Arguments for differentialAnalysisDESeq.R:" [1] "M_Nuclear" [1] "M_Cytoplasmic" [1] 1 [1] 1 [1] "RefSeq_GeneBody.gtf" [1] "/scratch/383329.1.linga/Input" [1] "DiffExp_v2_Intronic_Only" [1] "Intron_Only_Regions_Lengths.txt" load GTF file ... parse attributes ... [1] "output file is in: /scratch/383329.1.linga/Input/DiffExp_v2_Intronic_Only_M_Nuclear_M_Cytoplasmic.txt" ========================================================== Create SEGEX formatted file: Printing Rscript command: Rscript formatForSegex_ver3.R DiffExp_v2_Intronic_Only_M_Nuclear_M_Cytoplasmic.txt M_Cytoplasmic_G160_M6_vs_M_Nuclear_G160_M5_DiffExp_v2_Intronic_Only_forSEGEXUpload 1 Intronic_Only ========================================================== Comparison_Info: M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5 #---------------------------------------------------------------------------------- Running Diff_Genes.R #---------------------------------------------------------------------------------- Printing Rscript command: Rscript Diff_Genes.R M_Cytoplasmic_G160_M6_vs_M_Nuclear_G160_M5_DiffExp_v2_Intronic_Only_forSEGEXUpload_DESeq.txt 2 0.05 DESeq Intronic_Only'_'M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5 [1] "Print arguments:" [1] "-----------------" [1] "Differential expression_Output:" [1] "M_Cytoplasmic_G160_M6_vs_M_Nuclear_G160_M5_DiffExp_v2_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] "Intronic_Only_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5" [1] "-----------------" [1] "Differential Gene Counts" [1] "Number of significant differential genes with positive fold change (Up Genes):" [1] 0 7 [1] "Number of significant differential genes with negative fold change (Down Genes):" [1] 0 7 [1] "Check out Up_Genes_DESeq_Intronic_Only_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5.txt!" [1] "Check out Down_Genes_DESeq_Intronic_Only_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5.txt!" Rscript Diff_Genes.R M_Cytoplasmic_G160_M6_vs_M_Nuclear_G160_M5_DiffExp_v2_Intronic_Only_forSEGEXUpload_EdgeR.txt 2 0.05 EdgeR Intronic_Only'_'M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5 [1] "Print arguments:" [1] "-----------------" [1] "Differential expression_Output:" [1] "M_Cytoplasmic_G160_M6_vs_M_Nuclear_G160_M5_DiffExp_v2_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] "Intronic_Only_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5" [1] "-----------------" [1] "Differential Gene Counts" [1] "Number of significant differential genes with positive fold change (Up Genes):" [1] 47 7 [1] "Number of significant differential genes with negative fold change (Down Genes):" [1] 1 7 [1] "Check out Up_Genes_EdgeR_Intronic_Only_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5.txt!" [1] "Check out Down_Genes_EdgeR_Intronic_Only_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5.txt!" #---------------------------------------------------------------------------------- Running Venn_Diff_Genes.R #---------------------------------------------------------------------------------- Printing Rscript command: #---------------------------------------------------------------------------------- Rscript Venn_Diff_Genes.R Down_Genes_DESeq_Intronic_Only_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5.txt Down_Genes_EdgeR_Intronic_Only_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5.txt Intronic_Only_Counting DiffExp_3c [1] "Print arguments:" [1] "-----------------" [1] "File1:" [1] "Down_Genes_DESeq_Intronic_Only_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5.txt" [1] "File2:" [1] "Down_Genes_EdgeR_Intronic_Only_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5.txt" [1] "Subtitle:" [1] "Intronic_Only_Counting" [1] "DiffExp_Index:" [1] "DiffExp_3c" [1] "-----------------" [1] "Down.DESeq.Intronic_Only.HTSeq" [1] "Down.EdgeR.Intronic_Only.HTSeq" null device 1 [1] "Removing VennDiagram*.log files" [1] "Check out Venn diagram and Count.Table!" #---------------------------------------------------------------------------------- Rscript Venn_Diff_Genes.R Up_Genes_DESeq_Intronic_Only_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5.txt Up_Genes_EdgeR_Intronic_Only_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5.txt Intronic_Only_Counting DiffExp_3c [1] "Print arguments:" [1] "-----------------" [1] "File1:" [1] "Up_Genes_DESeq_Intronic_Only_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5.txt" [1] "File2:" [1] "Up_Genes_EdgeR_Intronic_Only_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5.txt" [1] "Subtitle:" [1] "Intronic_Only_Counting" [1] "DiffExp_Index:" [1] "DiffExp_3c" [1] "-----------------" [1] "Up.DESeq.Intronic_Only.HTSeq" [1] "Up.EdgeR.Intronic_Only.HTSeq" null device 1 [1] "Removing VennDiagram*.log files" [1] "Check out Venn diagram and Count.Table!" #---------------------------------------------------------------------------------- #---------------------------------------------------------------------------------- Merging Count.Table(s) into one text file #---------------------------------------------------------------------------------- ========================================================== Renaming the Differential_Expression_File ========================================================== List files in Input -rw-r--r-- 1 kkarri pulmseq 316 Aug 24 04:37 /scratch/383329.1.linga/Input/DiffExp_3c_Venn_Tables_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5.txt -rw-r--r-- 1 kkarri pulmseq 4782300 Aug 24 04:37 /scratch/383329.1.linga/Input/DiffExp_v2_Intronic_Only_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5.txt -rwxr-xr-x 1 kkarri pulmseq 7394 Aug 24 04:34 /scratch/383329.1.linga/Input/Diff_Genes.R -rw-r--r-- 1 kkarri pulmseq 182 Aug 24 04:37 /scratch/383329.1.linga/Input/Down_Genes_DESeq_Intronic_Only_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5.txt -rw-r--r-- 1 kkarri pulmseq 238 Aug 24 04:37 /scratch/383329.1.linga/Input/Down_Genes_EdgeR_Intronic_Only_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5.txt -rw-r--r-- 1 kkarri pulmseq 300238 Aug 24 04:34 /scratch/383329.1.linga/Input/Intron_Only_Regions_Lengths.txt -rw-r--r-- 1 kkarri pulmseq 229213 Aug 24 04:34 /scratch/383329.1.linga/Input/M_Cytoplasmic0.out -rw-r--r-- 1 kkarri pulmseq 9 Aug 24 04:34 /scratch/383329.1.linga/Input/M_Cytoplasmic0_num_mapped_reads.txt -rw-r--r-- 1 kkarri pulmseq 2160625 Aug 24 04:37 /scratch/383329.1.linga/Input/M_Cytoplasmic_G160_M6_vs_M_Nuclear_G160_M5_DiffExp_v2_Intronic_Only_forSEGEXUpload_DESeq.txt -rw-r--r-- 1 kkarri pulmseq 3273687 Aug 24 04:37 /scratch/383329.1.linga/Input/M_Cytoplasmic_G160_M6_vs_M_Nuclear_G160_M5_DiffExp_v2_Intronic_Only_forSEGEXUpload_EdgeR.txt -rw-r--r-- 1 kkarri pulmseq 246277 Aug 24 04:34 /scratch/383329.1.linga/Input/M_Nuclear0.out -rw-r--r-- 1 kkarri pulmseq 9 Aug 24 04:34 /scratch/383329.1.linga/Input/M_Nuclear0_num_mapped_reads.txt -rw-r--r-- 1 kkarri pulmseq 182 Aug 24 04:37 /scratch/383329.1.linga/Input/Up_Genes_DESeq_Intronic_Only_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5.txt -rw-r--r-- 1 kkarri pulmseq 5211 Aug 24 04:37 /scratch/383329.1.linga/Input/Up_Genes_EdgeR_Intronic_Only_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5.txt -rwxr-xr-x 1 kkarri pulmseq 9553 Aug 24 04:34 /scratch/383329.1.linga/Input/Venn_Diff_Genes.R -rw-r--r-- 1 kkarri pulmseq 30877 Aug 24 04:37 /scratch/383329.1.linga/Input/Venn_Down.DESeq.Intronic_Only.HTSeq.Down.EdgeR.Intronic_Only.HTSeq.png -rw-r--r-- 1 kkarri pulmseq 30274 Aug 24 04:37 /scratch/383329.1.linga/Input/Venn_Up.DESeq.Intronic_Only.HTSeq.Up.EdgeR.Intronic_Only.HTSeq.png -rwxr-xr-x 1 kkarri pulmseq 3925 Aug 24 04:34 /scratch/383329.1.linga/Input/formatForSegex_ver3.R /scratch/383329.1.linga/Input/M_Cytoplasmic: total 8 drwxr-xr-x 2 kkarri pulmseq 4096 Aug 24 04:34 . drwxr-xr-x 4 kkarri pulmseq 4096 Aug 24 04:37 .. /scratch/383329.1.linga/Input/M_Nuclear: total 8 drwxr-xr-x 2 kkarri pulmseq 4096 Aug 24 04:34 . drwxr-xr-x 4 kkarri pulmseq 4096 Aug 24 04:37 .. ========================================================== List files in scratch total 72M drwxr-xr-x 3 kkarri pulmseq 4.0K Aug 24 04:37 . drwxrwxrwt. 66 root root 92K Aug 24 04:37 .. -rw-r--r-- 1 kkarri pulmseq 54 Aug 24 04:34 Condition_1.txt -rw-r--r-- 1 kkarri pulmseq 54 Aug 24 04:34 Condition_2.txt drwxr-xr-x 4 kkarri pulmseq 4.0K Aug 24 04:37 Input -rw-r--r-- 1 kkarri pulmseq 72M Aug 24 04:34 RefSeq_GeneBody.gtf -rwxr-xr-x 1 kkarri pulmseq 16K Aug 24 04:34 differentialAnalysis.R ========================================================== Re-naming files in OUTPUT_DIR Need to append the COUNT_PROGRAM name to all output files List files in OUTPUT_DIR total 9.1M drwxr-sr-x 2 kkarri waxmanlab 32K Aug 24 04:37 . drwxr-sr-x 6 kkarri waxmanlab 32K Aug 24 04:34 .. -rw-r--r-- 1 kkarri waxmanlab 316 Aug 24 04:37 DiffExp_3c_Venn_Tables_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5_HTSeq.txt -rw-r--r-- 1 kkarri waxmanlab 4.6M Aug 24 04:37 DiffExp_v2_Intronic_Only_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5_HTSeq.txt -rw-r--r-- 1 kkarri waxmanlab 182 Aug 24 04:37 Down_Genes_DESeq_Intronic_Only_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5_HTSeq.txt -rw-r--r-- 1 kkarri waxmanlab 238 Aug 24 04:37 Down_Genes_EdgeR_Intronic_Only_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5_HTSeq.txt -rw-r--r-- 1 kkarri waxmanlab 2.1M Aug 24 04:37 M_Cytoplasmic_G160_M6_vs_M_Nuclear_G160_M5_DiffExp_v2_Intronic_Only_forSEGEXUpload_DESeq_HTSeq.txt -rw-r--r-- 1 kkarri waxmanlab 3.2M Aug 24 04:37 M_Cytoplasmic_G160_M6_vs_M_Nuclear_G160_M5_DiffExp_v2_Intronic_Only_forSEGEXUpload_EdgeR_HTSeq.txt -rw-r--r-- 1 kkarri waxmanlab 182 Aug 24 04:37 Up_Genes_DESeq_Intronic_Only_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5_HTSeq.txt -rw-r--r-- 1 kkarri waxmanlab 5.1K Aug 24 04:37 Up_Genes_EdgeR_Intronic_Only_M_Cytoplasmic_G160_M6_M_Nuclear_G160_M5_HTSeq.txt -rw-r--r-- 1 kkarri waxmanlab 31K Aug 24 04:37 Venn_Down.DESeq.Intronic_Only.HTSeq.Down.EdgeR.Intronic_Only.HTSeq_HTSeq.png -rw-r--r-- 1 kkarri waxmanlab 30K Aug 24 04:37 Venn_Up.DESeq.Intronic_Only.HTSeq.Up.EdgeR.Intronic_Only.HTSeq_HTSeq.png ========================================================== ========================================================== Finished on : Thu Aug 24 04:37:24 EDT 2017 3 minutes and 5 seconds elapsed. ==========================================================