Bowtie seems to be working fine (tested command '/gscratch/srlab/programs/bowtie2-2.3.5.1-linux-x86_64/bowtie2 --version' [2.3.5]) Output format is BAM (default) Alignments will be written out in BAM format. Samtools path provided as: '/gscratch/srlab/programs/samtools-1.9/samtools' Reference genome folder provided is /gscratch/srlab/strigg/data/Pgenr/OFS/ (absolute path is '/gscratch/srlab/strigg/data/Pgenr/OFS/)' FastQ format assumed (by default) Each Bowtie 2 instance is going to be run with 4 threads. Please monitor performance closely and tune down if needed! Input files to be analysed (in current folder '/gscratch/scrubbed/strigg/analyses/20200316/TG_EPI-Test2'): EPI-167_S10_L002_R1_001_val_1.fq.gz EPI-167_S10_L002_R2_001_val_2.fq.gz Library is assumed to be strand-specific (directional), alignments to strands complementary to the original top or bottom strands will be ignored (i.e. not performed!) Output will be written into the directory: /gscratch/scrubbed/strigg/analyses/20200316/TG_EPI-Test2/ Setting parallelization to single-threaded (default) Current working directory is: /gscratch/scrubbed/strigg/analyses/20200316/TG_EPI-Test2 Now reading in and storing sequence information of the genome specified in: /gscratch/srlab/strigg/data/Pgenr/OFS/ chr Scaffold_01 (89643857 bp) chr Scaffold_02 (69596280 bp) chr Scaffold_03 (57743597 bp) chr Scaffold_04 (65288255 bp) chr Scaffold_05 (67248332 bp) chr Scaffold_06 (61759565 bp) chr Scaffold_07 (43120122 bp) chr Scaffold_08 (61151155 bp) chr Scaffold_09 (38581958 bp) chr Scaffold_10 (53961475 bp) chr Scaffold_11 (51449921 bp) chr Scaffold_12 (50438331 bp) chr Scaffold_13 (44396874 bp) chr Scaffold_14 (45393038 bp) chr Scaffold_15 (47938513 bp) chr Scaffold_16 (31980953 bp) chr Scaffold_17 (34923512 bp) chr Scaffold_18 (27737463 bp) Single-core mode: setting pid to 1 Paired-end alignments will be performed ======================================= The provided filenames for paired-end alignments are EPI-167_S10_L002_R1_001_val_1.fq.gz and EPI-167_S10_L002_R2_001_val_2.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file EPI-167_S10_L002_R1_001_val_1.fq.gz to EPI-167_S10_L002_R1_001_val_1.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file EPI-167_S10_L002_R1_001_val_1.fq.gz (23436512 sequences in total) Writing a G -> A converted version of the input file EPI-167_S10_L002_R2_001_val_2.fq.gz to EPI-167_S10_L002_R2_001_val_2.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file EPI-167_S10_L002_R2_001_val_2.fq.gz (23436512 sequences in total) Input files are EPI-167_S10_L002_R1_001_val_1.fq.gz_C_to_T.fastq and EPI-167_S10_L002_R2_001_val_2.fq.gz_G_to_A.fastq (FastQ) Now running 2 instances of Bowtie 2 against the bisulfite genome of /gscratch/srlab/strigg/data/Pgenr/OFS/ with the specified options: -q --score-min L,0,-0.6 -p 4 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 Now starting a Bowtie 2 paired-end alignment for CTread1GAread2CTgenome (reading in sequences from EPI-167_S10_L002_R1_001_val_1.fq.gz_C_to_T.fastq and EPI-167_S10_L002_R2_001_val_2.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-0.6 -p 4 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: D00743:144:CAAWNANXX:2:2207:1666:2232_1:N:0:GATCAG/1 77 * 0 0 * * 0 0 TATGAATAATAAGTTTAATATTTAAGATGGAGAAGAAATAAGTGTAGAATTTTGGATAAAGGATAA FFBFFFFFFFFFFFFFFFFBFFFFFBFFF>> Writing bisulfite mapping results to EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.bam <<< Reading in the sequence files EPI-167_S10_L002_R1_001_val_1.fq.gz and EPI-167_S10_L002_R2_001_val_2.fq.gz Processed 1000000 sequence pairs so far Processed 2000000 sequence pairs so far Processed 3000000 sequence pairs so far Processed 4000000 sequence pairs so far Processed 5000000 sequence pairs so far Processed 6000000 sequence pairs so far Chromosomal sequence could not be extracted for D00743:144:CAAWNANXX:2:2114:10264:34490_1:N:0:GATCAG Scaffold_05 67248267 Processed 7000000 sequence pairs so far Processed 8000000 sequence pairs so far Processed 9000000 sequence pairs so far Processed 10000000 sequence pairs so far Processed 11000000 sequence pairs so far Processed 12000000 sequence pairs so far Processed 13000000 sequence pairs so far Processed 14000000 sequence pairs so far Processed 15000000 sequence pairs so far Processed 16000000 sequence pairs so far Processed 17000000 sequence pairs so far Processed 18000000 sequence pairs so far Processed 19000000 sequence pairs so far Processed 20000000 sequence pairs so far Processed 21000000 sequence pairs so far Processed 22000000 sequence pairs so far Processed 23000000 sequence pairs so far 23436512 reads; of these: 23436512 (100.00%) were paired; of these: 14949893 (63.79%) aligned concordantly 0 times 3525358 (15.04%) aligned concordantly exactly 1 time 4961261 (21.17%) aligned concordantly >1 times 36.21% overall alignment rate 23436512 reads; of these: 23436512 (100.00%) were paired; of these: 14882791 (63.50%) aligned concordantly 0 times 3530583 (15.06%) aligned concordantly exactly 1 time 5023138 (21.43%) aligned concordantly >1 times 36.50% overall alignment rate Processed 23436512 sequences in total Successfully deleted the temporary files EPI-167_S10_L002_R1_001_val_1.fq.gz_C_to_T.fastq and EPI-167_S10_L002_R2_001_val_2.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 23436512 Number of paired-end alignments with a unique best hit: 9588745 Mapping efficiency: 40.9% Sequence pairs with no alignments under any condition: 11082779 Sequence pairs did not map uniquely: 2764988 Sequence pairs which were discarded because genomic sequence could not be extracted: 1 Number of sequence pairs with unique best (first) alignment came from the bowtie output: CT/GA/CT: 4754776 ((converted) top strand) GA/CT/CT: 0 (complementary to (converted) top strand) GA/CT/GA: 0 (complementary to (converted) bottom strand) CT/GA/GA: 4833968 ((converted) bottom strand) Number of alignments to (merely theoretical) complementary strands being rejected in total: 0 Final Cytosine Methylation Report ================================= Total number of C's analysed: 199524252 Total methylated C's in CpG context: 6315385 Total methylated C's in CHG context: 695740 Total methylated C's in CHH context: 3620104 Total methylated C's in Unknown context: 18796 Total unmethylated C's in CpG context: 18667755 Total unmethylated C's in CHG context: 40114533 Total unmethylated C's in CHH context: 130110735 Total unmethylated C's in Unknown context: 361000 C methylated in CpG context: 25.3% C methylated in CHG context: 1.7% C methylated in CHH context: 2.7% C methylated in unknown context (CN or CHN): 4.9% Bismark completed in 0d 0h 38m 9s ==================== Bismark run complete ==================== *** Bismark methylation extractor version v0.19.0 *** Setting option '--no_overlap' since this is (normally) the right thing to do for paired-end data Summarising Bismark methylation extractor parameters: =============================================================== Bismark paired-end SAM format specified (default) Number of cores to be used: 14 Output will be written to the current directory ('/gscratch/scrubbed/strigg/analyses/20200316/TG_EPI-Test2') Summarising bedGraph parameters: =============================================================== Generating additional output in bedGraph and coverage format bedGraph format: coverage format: Using a cutoff of 1 read(s) to report cytosine positions Reporting and sorting cytosine methylation information in CpG context only (default) The bedGraph UNIX sort command will use the following memory setting: '75%'. Temporary directory used for sorting is the output directory Checking file >>EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.bam<< for signs of file truncation... Now testing Bismark result file EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.bam for positional sorting (which would be bad...) ...passed! Writing result file containing methylation information for C in CpG context from the original top strand to CpG_OT_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt Writing result file containing methylation information for C in CpG context from the complementary to original top strand to CpG_CTOT_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt Writing result file containing methylation information for C in CpG context from the complementary to original bottom strand to CpG_CTOB_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt Writing result file containing methylation information for C in CpG context from the original bottom strand to CpG_OB_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt Writing result file containing methylation information for C in CHG context from the original top strand to CHG_OT_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt Writing result file containing methylation information for C in CHG context from the complementary to original top strand to CHG_CTOT_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt Writing result file containing methylation information for C in CHG context from the complementary to original bottom strand to CHG_CTOB_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt Writing result file containing methylation information for C in CHG context from the original bottom strand to CHG_OB_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt Writing result file containing methylation information for C in CHH context from the original top strand to CHH_OT_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt Writing result file containing methylation information for C in CHH context from the complementary to original top strand to CHH_CTOT_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt Writing result file containing methylation information for C in CHH context from the complementary to original bottom strand to CHH_CTOB_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt Writing result file containing methylation information for C in CHH context from the original bottom strand to CHH_OB_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt Now reading in Bismark result file EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.bam Now reading in Bismark result file EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.bam Now reading in Bismark result file EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.bam Now reading in Bismark result file EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.bam Now reading in Bismark result file EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.bam Now reading in Bismark result file EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.bam Now reading in Bismark result file EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.bam Now reading in Bismark result file EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.bam Now reading in Bismark result file EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.bam Now reading in Bismark result file EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.bam Now reading in Bismark result file EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.bam Now reading in Bismark result file EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.bam Now reading in Bismark result file EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.bam Now reading in Bismark result file EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.bam skipping SAM header line: @HD VN:1.0 SO:unsorted skipping SAM header line: @SQ SN:Scaffold_01 LN:89643857 skipping SAM header line: @SQ SN:Scaffold_02 LN:69596280 skipping SAM header line: @SQ SN:Scaffold_03 LN:57743597 skipping SAM header line: @SQ SN:Scaffold_04 LN:65288255 skipping SAM header line: @SQ SN:Scaffold_05 LN:67248332 skipping SAM header line: @SQ SN:Scaffold_06 LN:61759565 skipping SAM header line: @SQ SN:Scaffold_07 LN:43120122 skipping SAM header line: @SQ SN:Scaffold_08 LN:61151155 skipping SAM header line: @SQ SN:Scaffold_09 LN:38581958 skipping SAM header line: @SQ SN:Scaffold_10 LN:53961475 skipping SAM header line: @SQ SN:Scaffold_11 LN:51449921 skipping SAM header line: @SQ SN:Scaffold_12 LN:50438331 skipping SAM header line: @SQ SN:Scaffold_13 LN:44396874 skipping SAM header line: @SQ SN:Scaffold_14 LN:45393038 skipping SAM header line: @SQ SN:Scaffold_15 LN:47938513 skipping SAM header line: @SQ SN:Scaffold_16 LN:31980953 skipping SAM header line: @SQ SN:Scaffold_17 LN:34923512 skipping SAM header line: @SQ SN:Scaffold_18 LN:27737463 skipping SAM header line: @PG ID:Bismark VN:v0.19.0 CL:"bismark --path_to_bowtie /gscratch/srlab/programs/bowtie2-2.3.5.1-linux-x86_64 --samtools_path /gscratch/srlab/programs/samtools-1.9/samtools -p 4 --score_min L,0,-0.6 --genome /gscratch/srlab/strigg/data/Pgenr/OFS/ -1 EPI-167_S10_L002_R1_001_val_1.fq.gz -2 EPI-167_S10_L002_R2_001_val_2.fq.gz -o /gscratch/scrubbed/strigg/analyses/20200316/TG_EPI-Test2" Processed lines: 500000 Processed lines: 500000 Processed lines: 500000 Processed lines: 500000 Processed lines: 500000 Processed lines: 500000 Processed lines: 500000 Processed lines: 500000 Processed lines: 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Merging individual splitting reports into overall report: 'EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt' Merging from these individual files: EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.1 EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.2 EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.3 EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.4 EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.5 EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.6 EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.7 EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.8 EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.9 EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.10 EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.11 EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.12 EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.13 EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.14 Processed 9588744 lines in total Total number of methylation call strings processed: 19177488 Final Cytosine Methylation Report ================================= Total number of C's analysed: 147637671 Total methylated C's in CpG context: 4621610 Total methylated C's in CHG context: 506131 Total methylated C's in CHH context: 2616476 Total C to T conversions in CpG context: 13741234 Total C to T conversions in CHG context: 29356812 Total C to T conversions in CHH context: 96795408 C methylated in CpG context: 25.2% C methylated in CHG context: 1.7% C methylated in CHH context: 2.6% Merging individual M-bias reports into overall M-bias statistics from these 14 individual files: EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.1.mbias EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.2.mbias EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.3.mbias EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.4.mbias EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.5.mbias EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.6.mbias EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.7.mbias EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.8.mbias EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.9.mbias EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.10.mbias EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.11.mbias EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.12.mbias EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.13.mbias EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt.14.mbias Determining maximum read lengths for M-Bias plots Maximum read length of Read 1: 66 Maximum read length of Read 2: 66 Perl module GD::Graph::lines is not installed, skipping drawing M-bias plots (only writing out M-bias plot table) Determining maximum read lengths for M-Bias plots Maximum read length of Read 1: 66 Maximum read length of Read 2: 66 Perl module GD::Graph::lines is not installed, skipping drawing M-bias plots (only writing out M-bias plot table) Deleting unused files ... CpG_OT_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt contains data -> kept CpG_CTOT_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt was empty -> deleted CpG_CTOB_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt was empty -> deleted CpG_OB_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt contains data -> kept CHG_OT_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt contains data -> kept CHG_CTOT_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt was empty -> deleted CHG_CTOB_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt was empty -> deleted CHG_OB_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt contains data -> kept CHH_OT_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt contains data -> kept CHH_CTOT_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt was empty -> deleted CHH_CTOB_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt was empty -> deleted CHH_OB_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt contains data -> kept Using these input files: CpG_OT_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt CpG_OB_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt CHG_OT_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt CHG_OB_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt CHH_OT_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt CHH_OB_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt Summary of parameters for bismark2bedGraph conversion: ====================================================== bedGraph output: EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.bedGraph.gz output directory: >< remove whitespaces: no CX context: no (CpG context only, default) No-header selected: no Sorting method: Unix sort-based (smaller memory footprint, but slower) Sort buffer size: 75% Coverage threshold: 1 ============================================================================= Methylation information will now be written into a bedGraph and coverage file ============================================================================= Using the following files as Input: /gscratch/scrubbed/strigg/analyses/20200316/TG_EPI-Test2/CpG_OT_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt /gscratch/scrubbed/strigg/analyses/20200316/TG_EPI-Test2/CpG_OB_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt Writing bedGraph to file: EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.bedGraph.gz Also writing out a coverage file including counts methylated and unmethylated residues to file: EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.bismark.cov.gz The genome of interest was specified to contain gazillions of chromosomes or scaffolds. Merging all input files and sorting everything in memory instead of writing out individual chromosome files... Writing all merged methylation calls to temp file EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.bedGraph.gz.methylation_calls.merged Finished writing methylation calls from CpG_OT_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt to merged temp file Finished writing methylation calls from CpG_OB_EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.txt to merged temp file Sorting input file EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.bedGraph.gz.methylation_calls.merged by positions (using -S of 75%) Successfully deleted the temporary input file EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.bedGraph.gz.methylation_calls.merged Finished BedGraph conversion ... Found 1 alignment reports in current directory. Now trying to figure out whether there are corresponding optional reports Writing Bismark HTML report to >> EPI-167_S10_L002_R1_001_val_1_bismark_bt2_PE_report.html << Redundant argument in sprintf at /gscratch/srlab/programs/Bismark-0.19.0/bismark2report line 130. ============================================================================================================== Using the following alignment report: > EPI-167_S10_L002_R1_001_val_1_bismark_bt2_PE_report.txt < Processing alignment report EPI-167_S10_L002_R1_001_val_1_bismark_bt2_PE_report.txt ... Complete No deduplication report file specified, skipping this step Using the following splitting report: > EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt < Processing splitting report EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe_splitting_report.txt ... Complete Using the following M-bias report: > EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.M-bias.txt < Processing M-bias report EPI-167_S10_L002_R1_001_val_1_bismark_bt2_pe.M-bias.txt ... Complete No nucleotide coverage report file specified, skipping this step ============================================================================================================== No Bismark/Bowtie2 single-end BAM files detected Found Bismark/Bowtie2 paired-end files No Bismark/Bowtie single-end BAM files detected No Bismark/Bowtie paired-end BAM files detected Generating Bismark summary report from 1 Bismark BAM file(s)... >> Reading from Bismark report: EPI-167_S10_L002_R1_001_val_1_bismark_bt2_PE_report.txt No deduplication report present, skipping... No methylation extractor report present, skipping... Wrote Bismark project summary to >> bismark_summary_report.html <<