/var/spool/slurm/d/job2025665/slurm_script: line 20: fg: no job control No quality encoding type selected. Assuming that the data provided uses Sanger encoded Phred scores (default) Please provide an even number of input files for paired-end FastQ trimming! Aborting ... No quality encoding type selected. Assuming that the data provided uses Sanger encoded Phred scores (default) Path to Cutadapt set as: '/gscratch/srlab/programs/miniconda3/bin/cutadapt' (user defined) 2.4 Cutadapt seems to be working fine (tested command '/gscratch/srlab/programs/miniconda3/bin/cutadapt --version') AUTO-DETECTING ADAPTER TYPE =========================== Attempting to auto-detect adapter type from the first 1 million sequences of the first file (>> /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth13_R1_001.fastq.gz <<) Found perfect matches for the following adapter sequences: Adapter type Count Sequence Sequences analysed Percentage Illumina 457885 AGATCGGAAGAGC 1000000 45.79 smallRNA 0 TGGAATTCTCGG 1000000 0.00 Nextera 0 CTGTCTCTTATA 1000000 0.00 Using Illumina adapter for trimming (count: 457885). Second best hit was smallRNA (count: 0) Writing report to '/gscratch/scrubbed/strigg/analyses/20200306/Meth13_R1_001.fastq.gz_trimming_report.txt' SUMMARISING RUN PARAMETERS ========================== Input filename: /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth13_R1_001.fastq.gz Trimming mode: paired-end Trim Galore version: 0.4.4_dev Cutadapt version: 2.4 Quality Phred score cutoff: 20 Quality encoding type selected: ASCII+33 Adapter sequence: 'AGATCGGAAGAGC' (Illumina TruSeq, Sanger iPCR; auto-detected) Maximum trimming error rate: 0.1 (default) Minimum required adapter overlap (stringency): 1 bp Minimum required sequence length for both reads before a sequence pair gets removed: 20 bp File was specified to be an MspI-digested RRBS sample. Read 1 sequences with adapter contamination will be trimmed a further 2 bp from their 3' end, and Read 2 sequences will be trimmed by 2 bp from their 5' end to remove potential methylation-biased bases from the end-repair reaction File was specified to be a non-directional MspI-digested RRBS sample. Sequences starting with either 'CAA' or 'CGA' will have the first 2 bp trimmed off to remove potential methylation-biased bases from the end-repair reaction Running FastQC on the data once trimming has completed Running FastQC with the following extra arguments: '--outdir /gscratch/scrubbed/strigg/analyses/20200306/FASTQC' Output file(s) will be GZIP compressed >>> Now performing adaptive quality trimming with a Phred-score cutoff of: 20 <<< This is cutadapt 2.4 with Python 3.7.3 Command line parameters: -f fastq -e 0.1 -q 20 -a X /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth13_R1_001.fastq.gz WARNING: Option --format is deprecated and ignored because the input file format is always auto-detected Processing reads on 1 core in single-end mode ... 10000000 sequences processed 20000000 sequences processed 30000000 sequences processed 40000000 sequences processed 50000000 sequences processed 60000000 sequences processed 70000000 sequences processed 80000000 sequences processed 90000000 sequences processed 100000000 sequences processed Finished in 935.51 s (9 us/read; 6.48 M reads/minute). === Summary === Total reads processed: 100,994,125 Reads with adapters: 0 (0.0%) Reads written (passing filters): 100,994,125 (100.0%) Total basepairs processed: 15,149,118,750 bp Quality-trimmed: 15,277,665 bp (0.1%) Total written (filtered): 15,133,841,085 bp (99.9%) === Adapter 1 === Sequence: X; Type: regular 3'; Length: 1; Trimmed: 0 times. >>> Quality trimming completed <<< 100994125 sequences processed in total Writing final adapter and quality trimmed output to Meth13_R1_001_trimmed.fq.gz >>> Now performing adapter trimming for the adapter sequence: 'AGATCGGAAGAGC' from file Meth13_R1_001.fastq.gz_qual_trimmed.fastq <<< 10000000 sequences processed 20000000 sequences processed 30000000 sequences processed 40000000 sequences processed 50000000 sequences processed 60000000 sequences processed 70000000 sequences processed 80000000 sequences processed 90000000 sequences processed 100000000 sequences processed This is cutadapt 2.4 with Python 3.7.3 Command line parameters: -f fastq -e 0.1 -O 1 -a AGATCGGAAGAGC /gscratch/scrubbed/strigg/analyses/20200306/Meth13_R1_001.fastq.gz_qual_trimmed.fastq WARNING: Option --format is deprecated and ignored because the input file format is always auto-detected Processing reads on 1 core in single-end mode ... Finished in 2557.47 s (25 us/read; 2.37 M reads/minute). === Summary === Total reads processed: 100,994,125 Reads with adapters: 75,452,441 (74.7%) Reads written (passing filters): 100,994,125 (100.0%) Total basepairs processed: 15,133,841,085 bp Total written (filtered): 11,286,581,533 bp (74.6%) === Adapter 1 === Sequence: AGATCGGAAGAGC; Type: regular 3'; Length: 13; Trimmed: 75452441 times. No. of allowed errors: 0-9 bp: 0; 10-13 bp: 1 Bases preceding removed adapters: A: 46.8% C: 3.5% G: 42.0% T: 7.8% none/other: 0.0% Overview of removed sequences length count expect max.err error counts 1 16889854 25248531.2 0 16889854 2 1387092 6312132.8 0 1387092 3 993122 1578033.2 0 993122 4 431060 394508.3 0 431060 5 275682 98627.1 0 275682 6 233401 24656.8 0 233401 7 293154 6164.2 0 293154 8 291627 1541.0 0 291627 9 282078 385.3 0 278508 3570 10 329147 96.3 1 300652 28495 11 581523 24.1 1 522909 58614 12 349882 6.0 1 315325 34557 13 313904 1.5 1 280320 33584 14 311573 1.5 1 277270 34303 15 260519 1.5 1 232317 28202 16 280060 1.5 1 248629 31431 17 309633 1.5 1 275006 34627 18 299523 1.5 1 268511 31012 19 308381 1.5 1 275762 32619 20 319544 1.5 1 285436 34108 21 307838 1.5 1 274585 33253 22 346274 1.5 1 309836 36438 23 436976 1.5 1 390674 46302 24 385479 1.5 1 342645 42834 25 336519 1.5 1 300157 36362 26 286169 1.5 1 255735 30434 27 302039 1.5 1 269115 32924 28 505249 1.5 1 451790 53459 29 356797 1.5 1 316417 40380 30 333714 1.5 1 298431 35283 31 348698 1.5 1 311129 37569 32 367127 1.5 1 327760 39367 33 317415 1.5 1 282607 34808 34 352429 1.5 1 314640 37789 35 326336 1.5 1 290643 35693 36 339179 1.5 1 301577 37602 37 481410 1.5 1 429087 52323 38 448729 1.5 1 395717 53012 39 414453 1.5 1 370021 44432 40 544488 1.5 1 486617 57871 41 545768 1.5 1 486251 59517 42 474348 1.5 1 425187 49161 43 335612 1.5 1 295805 39807 44 363737 1.5 1 321199 42538 45 612969 1.5 1 550568 62401 46 236931 1.5 1 210315 26616 47 340836 1.5 1 303534 37302 48 428571 1.5 1 381101 47470 49 789485 1.5 1 705703 83782 50 421164 1.5 1 373382 47782 51 489216 1.5 1 432238 56978 52 533011 1.5 1 475013 57998 53 470642 1.5 1 418275 52367 54 823609 1.5 1 732350 91259 55 528241 1.5 1 470022 58219 56 413780 1.5 1 367680 46100 57 485033 1.5 1 430576 54457 58 739339 1.5 1 657723 81616 59 419372 1.5 1 371464 47908 60 857360 1.5 1 765572 91788 61 470376 1.5 1 417741 52635 62 433854 1.5 1 384546 49308 63 401851 1.5 1 356696 45155 64 433832 1.5 1 385879 47953 65 358350 1.5 1 317317 41033 66 444050 1.5 1 393209 50841 67 582832 1.5 1 515092 67740 68 746325 1.5 1 660769 85556 69 670332 1.5 1 580864 89468 70 2073202 1.5 1 1871175 202027 71 121451 1.5 1 105945 15506 72 52792 1.5 1 43475 9317 73 538152 1.5 1 472151 66001 74 1756813 1.5 1 1560951 195862 75 441617 1.5 1 389006 52611 76 504090 1.5 1 445913 58177 77 565547 1.5 1 500475 65072 78 426572 1.5 1 377709 48863 79 456994 1.5 1 404267 52727 80 462729 1.5 1 408983 53746 81 405931 1.5 1 358406 47525 82 437959 1.5 1 387478 50481 83 408216 1.5 1 360003 48213 84 562518 1.5 1 497661 64857 85 514509 1.5 1 454185 60324 86 424696 1.5 1 374471 50225 87 445051 1.5 1 391943 53108 88 443537 1.5 1 390586 52951 89 546419 1.5 1 481197 65222 90 499277 1.5 1 439779 59498 91 566915 1.5 1 498750 68165 92 375931 1.5 1 329894 46037 93 500854 1.5 1 440092 60762 94 547235 1.5 1 480575 66660 95 391331 1.5 1 343323 48008 96 422734 1.5 1 370502 52232 97 536091 1.5 1 469971 66120 98 532528 1.5 1 466445 66083 99 488932 1.5 1 428065 60867 100 500021 1.5 1 436747 63274 101 389481 1.5 1 340084 49397 102 384198 1.5 1 336082 48116 103 467887 1.5 1 408747 59140 104 410457 1.5 1 357935 52522 105 398892 1.5 1 347934 50958 106 628657 1.5 1 548475 80182 107 497594 1.5 1 432357 65237 108 499014 1.5 1 433694 65320 109 553229 1.5 1 481173 72056 110 382989 1.5 1 332050 50939 111 364439 1.5 1 316458 47981 112 405699 1.5 1 351377 54322 113 356009 1.5 1 308400 47609 114 619887 1.5 1 538428 81459 115 1146598 1.5 1 996001 150597 116 330227 1.5 1 283825 46402 117 447731 1.5 1 387042 60689 118 395667 1.5 1 341652 54015 119 446331 1.5 1 385057 61274 120 254368 1.5 1 218804 35564 121 257448 1.5 1 221667 35781 122 238783 1.5 1 205563 33220 123 250549 1.5 1 215898 34651 124 240416 1.5 1 206474 33942 125 321804 1.5 1 276819 44985 126 183366 1.5 1 157670 25696 127 148680 1.5 1 127230 21450 128 93397 1.5 1 80218 13179 129 76162 1.5 1 65344 10818 130 57264 1.5 1 49107 8157 131 74918 1.5 1 64528 10390 132 32450 1.5 1 27800 4650 133 11091 1.5 1 9620 1471 134 4995 1.5 1 4285 710 135 1217 1.5 1 1029 188 136 490 1.5 1 426 64 137 53 1.5 1 49 4 138 8 1.5 1 7 1 139 9 1.5 1 9 140 9 1.5 1 6 3 141 6 1.5 1 4 2 142 1 1.5 1 1 145 1 1.5 1 1 146 1 1.5 1 0 1 147 2 1.5 1 0 2 148 86 1.5 1 67 19 149 7 1.5 1 6 1 150 327 1.5 1 286 41 Successfully deleted temporary file Meth13_R1_001.fastq.gz_qual_trimmed.fastq RUN STATISTICS FOR INPUT FILE: /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth13_R1_001.fastq.gz ============================================= 100994125 sequences processed in total Sequences were truncated to a varying degree because of deteriorating qualities (Phred score quality cutoff: 20): 5451098 (5.4%) The length threshold of paired-end sequences gets evaluated later on (in the validation step) RRBS reads trimmed by additional 2 bp when adapter contamination was detected: 36165151 (35.8%) RRBS reads trimmed by 2 bp at the start when read started with CAA (1139376) or CGA (49790870) in total: 50930246 (50.4%) Writing report to '/gscratch/scrubbed/strigg/analyses/20200306/Meth13_R2_001.fastq.gz_trimming_report.txt' SUMMARISING RUN PARAMETERS ========================== Input filename: /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth13_R2_001.fastq.gz Trimming mode: paired-end Trim Galore version: 0.4.4_dev Cutadapt version: 2.4 Quality Phred score cutoff: 20 Quality encoding type selected: ASCII+33 Adapter sequence: 'AGATCGGAAGAGC' (Illumina TruSeq, Sanger iPCR; auto-detected) Maximum trimming error rate: 0.1 (default) Minimum required adapter overlap (stringency): 1 bp Minimum required sequence length for both reads before a sequence pair gets removed: 20 bp File was specified to be an MspI-digested RRBS sample. Read 1 sequences with adapter contamination will be trimmed a further 2 bp from their 3' end, and Read 2 sequences will be trimmed by 2 bp from their 5' end to remove potential methylation-biased bases from the end-repair reaction File was specified to be a non-directional MspI-digested RRBS sample. Sequences starting with either 'CAA' or 'CGA' will have the first 2 bp trimmed off to remove potential methylation-biased bases from the end-repair reaction Running FastQC on the data once trimming has completed Running FastQC with the following extra arguments: '--outdir /gscratch/scrubbed/strigg/analyses/20200306/FASTQC' Output file(s) will be GZIP compressed >>> Now performing adaptive quality trimming with a Phred-score cutoff of: 20 <<< This is cutadapt 2.4 with Python 3.7.3 Command line parameters: -f fastq -e 0.1 -q 20 -a X /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth13_R2_001.fastq.gz WARNING: Option --format is deprecated and ignored because the input file format is always auto-detected Processing reads on 1 core in single-end mode ... 10000000 sequences processed 20000000 sequences processed 30000000 sequences processed 40000000 sequences processed 50000000 sequences processed 60000000 sequences processed 70000000 sequences processed 80000000 sequences processed 90000000 sequences processed 100000000 sequences processed Finished in 1043.37 s (10 us/read; 5.81 M reads/minute). === Summary === Total reads processed: 100,994,125 Reads with adapters: 0 (0.0%) Reads written (passing filters): 100,994,125 (100.0%) Total basepairs processed: 15,149,118,750 bp Quality-trimmed: 116,375,081 bp (0.8%) Total written (filtered): 15,032,743,669 bp (99.2%) === Adapter 1 === Sequence: X; Type: regular 3'; Length: 1; Trimmed: 0 times. >>> Quality trimming completed <<< 100994125 sequences processed in total Writing final adapter and quality trimmed output to Meth13_R2_001_trimmed.fq.gz >>> Now performing adapter trimming for the adapter sequence: 'AGATCGGAAGAGC' from file Meth13_R2_001.fastq.gz_qual_trimmed.fastq <<< 10000000 sequences processed 20000000 sequences processed 30000000 sequences processed 40000000 sequences processed 50000000 sequences processed 60000000 sequences processed 70000000 sequences processed 80000000 sequences processed 90000000 sequences processed 100000000 sequences processed This is cutadapt 2.4 with Python 3.7.3 Command line parameters: -f fastq -e 0.1 -O 1 -a AGATCGGAAGAGC /gscratch/scrubbed/strigg/analyses/20200306/Meth13_R2_001.fastq.gz_qual_trimmed.fastq WARNING: Option --format is deprecated and ignored because the input file format is always auto-detected Processing reads on 1 core in single-end mode ... Finished in 2649.32 s (26 us/read; 2.29 M reads/minute). === Summary === Total reads processed: 100,994,125 Reads with adapters: 74,282,317 (73.6%) Reads written (passing filters): 100,994,125 (100.0%) Total basepairs processed: 15,032,743,669 bp Total written (filtered): 11,294,465,516 bp (75.1%) === Adapter 1 === Sequence: AGATCGGAAGAGC; Type: regular 3'; Length: 13; Trimmed: 74282317 times. No. of allowed errors: 0-9 bp: 0; 10-13 bp: 1 Bases preceding removed adapters: A: 46.4% C: 3.1% G: 42.4% T: 8.1% none/other: 0.0% Overview of removed sequences length count expect max.err error counts 1 16858905 25248531.2 0 16858905 2 1409178 6312132.8 0 1409178 3 1022247 1578033.2 0 1022247 4 449685 394508.3 0 449685 5 282584 98627.1 0 282584 6 231575 24656.8 0 231575 7 275852 6164.2 0 275852 8 305134 1541.0 0 305134 9 261671 385.3 0 259774 1897 10 318513 96.3 1 293638 24875 11 567085 24.1 1 515255 51830 12 364336 6.0 1 330294 34042 13 290786 1.5 1 258428 32358 14 303138 1.5 1 270634 32504 15 246093 1.5 1 220948 25145 16 285123 1.5 1 253722 31401 17 284664 1.5 1 255137 29527 18 286469 1.5 1 258114 28355 19 308210 1.5 1 277962 30248 20 324953 1.5 1 291571 33382 21 303171 1.5 1 270805 32366 22 350374 1.5 1 316862 33512 23 396907 1.5 1 358066 38841 24 381124 1.5 1 343563 37561 25 381056 1.5 1 343740 37316 26 245479 1.5 1 220057 25422 27 291422 1.5 1 259208 32214 28 487231 1.5 1 443161 44070 29 347094 1.5 1 312544 34550 30 331761 1.5 1 300206 31555 31 338587 1.5 1 306037 32550 32 358592 1.5 1 324285 34307 33 314593 1.5 1 283789 30804 34 342874 1.5 1 309361 33513 35 336170 1.5 1 304544 31626 36 344386 1.5 1 308783 35603 37 415065 1.5 1 375228 39837 38 459882 1.5 1 415569 44313 39 404887 1.5 1 365056 39831 40 571974 1.5 1 518878 53096 41 505738 1.5 1 457619 48119 42 365004 1.5 1 331197 33807 43 413898 1.5 1 374763 39135 44 357178 1.5 1 323405 33773 45 457513 1.5 1 412411 45102 46 435158 1.5 1 391388 43770 47 457055 1.5 1 415593 41462 48 395224 1.5 1 357064 38160 49 674244 1.5 1 611485 62759 50 434007 1.5 1 393088 40919 51 427500 1.5 1 385958 41542 52 527271 1.5 1 477474 49797 53 471108 1.5 1 426085 45023 54 923952 1.5 1 841152 82800 55 367371 1.5 1 332096 35275 56 430114 1.5 1 387358 42756 57 935761 1.5 1 849893 85868 58 412305 1.5 1 370915 41390 59 386265 1.5 1 348305 37960 60 1040895 1.5 1 947342 93553 61 455365 1.5 1 412374 42991 62 358198 1.5 1 316355 41843 63 1602264 1.5 1 1461599 140665 64 380614 1.5 1 342988 37626 65 211938 1.5 1 190908 21030 66 250630 1.5 1 223979 26651 67 816496 1.5 1 742960 73536 68 606413 1.5 1 549483 56930 69 513297 1.5 1 464468 48829 70 778232 1.5 1 706072 72160 71 638686 1.5 1 578942 59744 72 481606 1.5 1 435034 46572 73 985078 1.5 1 894565 90513 74 1294627 1.5 1 1176076 118551 75 402361 1.5 1 363629 38732 76 367554 1.5 1 332914 34640 77 298809 1.5 1 267530 31279 78 347369 1.5 1 313518 33851 79 412815 1.5 1 372637 40178 80 429933 1.5 1 387774 42159 81 384917 1.5 1 347266 37651 82 421423 1.5 1 380392 41031 83 424394 1.5 1 382335 42059 84 551836 1.5 1 497555 54281 85 529303 1.5 1 476883 52420 86 424156 1.5 1 381288 42868 87 432689 1.5 1 389037 43652 88 428451 1.5 1 385513 42938 89 518280 1.5 1 466734 51546 90 480143 1.5 1 432447 47696 91 534963 1.5 1 481319 53644 92 359938 1.5 1 323524 36414 93 478512 1.5 1 430743 47769 94 513875 1.5 1 462209 51666 95 371059 1.5 1 333246 37813 96 402062 1.5 1 361080 40982 97 514433 1.5 1 461861 52572 98 516253 1.5 1 463634 52619 99 484360 1.5 1 434891 49469 100 492619 1.5 1 440850 51769 101 379216 1.5 1 339118 40098 102 367605 1.5 1 329106 38499 103 441408 1.5 1 394943 46465 104 386261 1.5 1 345006 41255 105 380001 1.5 1 339706 40295 106 591703 1.5 1 528612 63091 107 472566 1.5 1 421694 50872 108 474327 1.5 1 422729 51598 109 522002 1.5 1 466969 55033 110 362933 1.5 1 323370 39563 111 347657 1.5 1 309474 38183 112 388527 1.5 1 345990 42537 113 339670 1.5 1 302340 37330 114 591254 1.5 1 526319 64935 115 1088418 1.5 1 968434 119984 116 319102 1.5 1 283133 35969 117 434552 1.5 1 386087 48465 118 390585 1.5 1 345582 45003 119 434222 1.5 1 384929 49293 120 249611 1.5 1 221221 28390 121 250773 1.5 1 222295 28478 122 229186 1.5 1 202720 26466 123 241833 1.5 1 213756 28077 124 232011 1.5 1 205157 26854 125 312472 1.5 1 276186 36286 126 178172 1.5 1 157076 21096 127 143853 1.5 1 126656 17197 128 91075 1.5 1 80088 10987 129 73099 1.5 1 64502 8597 130 55464 1.5 1 48732 6732 131 71783 1.5 1 63274 8509 132 30986 1.5 1 27253 3733 133 10709 1.5 1 9385 1324 134 4811 1.5 1 4208 603 135 1166 1.5 1 1010 156 136 476 1.5 1 425 51 137 53 1.5 1 50 3 138 11 1.5 1 8 3 139 8 1.5 1 8 140 6 1.5 1 6 141 6 1.5 1 5 1 142 1 1.5 1 1 143 1 1.5 1 1 145 1 1.5 1 1 146 1 1.5 1 1 148 83 1.5 1 78 5 149 11 1.5 1 10 1 150 304 1.5 1 276 28 Successfully deleted temporary file Meth13_R2_001.fastq.gz_qual_trimmed.fastq RUN STATISTICS FOR INPUT FILE: /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth13_R2_001.fastq.gz ============================================= 100994125 sequences processed in total Sequences were truncated to a varying degree because of deteriorating qualities (Phred score quality cutoff: 20): 11269639 (11.2%) The length threshold of paired-end sequences gets evaluated later on (in the validation step) RRBS reads trimmed by additional 2 bp when adapter contamination was detected: 0 (0.0%) RRBS reads trimmed by 2 bp at the start when read started with CAA (1082919) or CGA (46567288) in total: 47650207 (47.2%) Validate paired-end files Meth13_R1_001_trimmed.fq.gz and Meth13_R2_001_trimmed.fq.gz file_1: Meth13_R1_001_trimmed.fq.gz, file_2: Meth13_R2_001_trimmed.fq.gz >>>>> Now validing the length of the 2 paired-end infiles: Meth13_R1_001_trimmed.fq.gz and Meth13_R2_001_trimmed.fq.gz <<<<< Writing validated paired-end read 1 reads to Meth13_R1_001_val_1.fq.gz Writing validated paired-end read 2 reads to Meth13_R2_001_val_2.fq.gz Total number of sequences analysed: 100994125 Number of sequence pairs removed because at least one read was shorter than the length cutoff (20 bp): 386198 (0.38%) >>> Now running FastQC on the validated data Meth13_R1_001_val_1.fq.gz<<< Can't exec "fastqc": No such file or directory at /gscratch/srlab/programs/TrimGalore-0.4.5/trim_galore line 821, line 403976500. >>> Now running FastQC on the validated data Meth13_R2_001_val_2.fq.gz<<< Can't exec "fastqc": No such file or directory at /gscratch/srlab/programs/TrimGalore-0.4.5/trim_galore line 831, line 403976500. Deleting both intermediate output files Meth13_R1_001_trimmed.fq.gz and Meth13_R2_001_trimmed.fq.gz ==================================================================================================== Writing report to '/gscratch/scrubbed/strigg/analyses/20200306/Meth14_R1_001.fastq.gz_trimming_report.txt' SUMMARISING RUN PARAMETERS ========================== Input filename: /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth14_R1_001.fastq.gz Trimming mode: paired-end Trim Galore version: 0.4.4_dev Cutadapt version: 2.4 Quality Phred score cutoff: 20 Quality encoding type selected: ASCII+33 Adapter sequence: 'AGATCGGAAGAGC' (Illumina TruSeq, Sanger iPCR; auto-detected) Maximum trimming error rate: 0.1 (default) Minimum required adapter overlap (stringency): 1 bp Minimum required sequence length for both reads before a sequence pair gets removed: 20 bp File was specified to be an MspI-digested RRBS sample. Read 1 sequences with adapter contamination will be trimmed a further 2 bp from their 3' end, and Read 2 sequences will be trimmed by 2 bp from their 5' end to remove potential methylation-biased bases from the end-repair reaction File was specified to be a non-directional MspI-digested RRBS sample. Sequences starting with either 'CAA' or 'CGA' will have the first 2 bp trimmed off to remove potential methylation-biased bases from the end-repair reaction Running FastQC on the data once trimming has completed Running FastQC with the following extra arguments: '--outdir /gscratch/scrubbed/strigg/analyses/20200306/FASTQC' Output file(s) will be GZIP compressed >>> Now performing adaptive quality trimming with a Phred-score cutoff of: 20 <<< This is cutadapt 2.4 with Python 3.7.3 Command line parameters: -f fastq -e 0.1 -q 20 -a X /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth14_R1_001.fastq.gz WARNING: Option --format is deprecated and ignored because the input file format is always auto-detected Processing reads on 1 core in single-end mode ... 10000000 sequences processed 20000000 sequences processed 30000000 sequences processed 40000000 sequences processed 50000000 sequences processed 60000000 sequences processed Finished in 756.95 s (11 us/read; 5.51 M reads/minute). === Summary === Total reads processed: 69,473,196 Reads with adapters: 0 (0.0%) Reads written (passing filters): 69,473,196 (100.0%) Total basepairs processed: 10,420,979,400 bp Quality-trimmed: 11,201,680 bp (0.1%) Total written (filtered): 10,409,777,720 bp (99.9%) === Adapter 1 === Sequence: X; Type: regular 3'; Length: 1; Trimmed: 0 times. >>> Quality trimming completed <<< 69473196 sequences processed in total Writing final adapter and quality trimmed output to Meth14_R1_001_trimmed.fq.gz >>> Now performing adapter trimming for the adapter sequence: 'AGATCGGAAGAGC' from file Meth14_R1_001.fastq.gz_qual_trimmed.fastq <<< 10000000 sequences processed 20000000 sequences processed 30000000 sequences processed 40000000 sequences processed 50000000 sequences processed 60000000 sequences processed This is cutadapt 2.4 with Python 3.7.3 Command line parameters: -f fastq -e 0.1 -O 1 -a AGATCGGAAGAGC /gscratch/scrubbed/strigg/analyses/20200306/Meth14_R1_001.fastq.gz_qual_trimmed.fastq WARNING: Option --format is deprecated and ignored because the input file format is always auto-detected Processing reads on 1 core in single-end mode ... Finished in 1633.77 s (24 us/read; 2.55 M reads/minute). === Summary === Total reads processed: 69,473,196 Reads with adapters: 55,400,990 (79.7%) Reads written (passing filters): 69,473,196 (100.0%) Total basepairs processed: 10,409,777,720 bp Total written (filtered): 7,291,489,780 bp (70.0%) === Adapter 1 === Sequence: AGATCGGAAGAGC; Type: regular 3'; Length: 13; Trimmed: 55400990 times. No. of allowed errors: 0-9 bp: 0; 10-13 bp: 1 Bases preceding removed adapters: A: 40.9% C: 2.5% G: 51.1% T: 5.5% none/other: 0.0% Overview of removed sequences length count expect max.err error counts 1 8782866 17368299.0 0 8782866 2 857608 4342074.8 0 857608 3 658045 1085518.7 0 658045 4 300067 271379.7 0 300067 5 207375 67844.9 0 207375 6 181801 16961.2 0 181801 7 219369 4240.3 0 219369 8 219799 1060.1 0 219799 9 205689 265.0 0 202777 2912 10 246677 66.3 1 226049 20628 11 418235 16.6 1 378095 40140 12 254220 4.1 1 229651 24569 13 229447 1.0 1 205325 24122 14 231819 1.0 1 206513 25306 15 197934 1.0 1 176411 21523 16 207582 1.0 1 184590 22992 17 239494 1.0 1 213264 26230 18 227732 1.0 1 204314 23418 19 240456 1.0 1 215059 25397 20 243286 1.0 1 218005 25281 21 270542 1.0 1 241962 28580 22 261114 1.0 1 233681 27433 23 324289 1.0 1 290172 34117 24 304424 1.0 1 270683 33741 25 268262 1.0 1 239785 28477 26 230253 1.0 1 205804 24449 27 266605 1.0 1 238152 28453 28 422673 1.0 1 378173 44500 29 273666 1.0 1 242704 30962 30 258507 1.0 1 230975 27532 31 260642 1.0 1 232758 27884 32 282796 1.0 1 252895 29901 33 251359 1.0 1 223613 27746 34 270491 1.0 1 241227 29264 35 261946 1.0 1 233346 28600 36 264319 1.0 1 234923 29396 37 363860 1.0 1 324492 39368 38 330615 1.0 1 294792 35823 39 317729 1.0 1 281947 35782 40 443709 1.0 1 396553 47156 41 439031 1.0 1 392495 46536 42 293226 1.0 1 261246 31980 43 349316 1.0 1 310324 38992 44 278944 1.0 1 247166 31778 45 430265 1.0 1 386344 43921 46 209795 1.0 1 186554 23241 47 270492 1.0 1 241475 29017 48 338914 1.0 1 301468 37446 49 608396 1.0 1 543874 64522 50 345036 1.0 1 305958 39078 51 393371 1.0 1 346977 46394 52 434189 1.0 1 387030 47159 53 375166 1.0 1 333040 42126 54 677937 1.0 1 602728 75209 55 438277 1.0 1 389961 48316 56 336391 1.0 1 299219 37172 57 381521 1.0 1 338996 42525 58 578958 1.0 1 514594 64364 59 348832 1.0 1 309050 39782 60 690597 1.0 1 616628 73969 61 378363 1.0 1 335286 43077 62 371312 1.0 1 328574 42738 63 325780 1.0 1 288761 37019 64 360043 1.0 1 320109 39934 65 299565 1.0 1 265521 34044 66 351273 1.0 1 310428 40845 67 446381 1.0 1 393754 52627 68 581169 1.0 1 513322 67847 69 555370 1.0 1 477063 78307 70 1821958 1.0 1 1645415 176543 71 159722 1.0 1 141530 18192 72 66696 1.0 1 57291 9405 73 388987 1.0 1 339110 49877 74 1374358 1.0 1 1216794 157564 75 332841 1.0 1 292206 40635 76 416417 1.0 1 367866 48551 77 460145 1.0 1 406050 54095 78 331342 1.0 1 291983 39359 79 366128 1.0 1 323010 43118 80 382456 1.0 1 336936 45520 81 326902 1.0 1 287716 39186 82 348100 1.0 1 306813 41287 83 332056 1.0 1 292244 39812 84 475457 1.0 1 419007 56450 85 420225 1.0 1 369669 50556 86 345642 1.0 1 303706 41936 87 426380 1.0 1 375165 51215 88 363082 1.0 1 318909 44173 89 441663 1.0 1 388095 53568 90 420556 1.0 1 368610 51946 91 461064 1.0 1 404128 56936 92 336462 1.0 1 294241 42221 93 393138 1.0 1 344342 48796 94 443807 1.0 1 388607 55200 95 316764 1.0 1 276823 39941 96 338920 1.0 1 295107 43813 97 449770 1.0 1 391931 57839 98 424585 1.0 1 369668 54917 99 375734 1.0 1 327187 48547 100 399744 1.0 1 347485 52259 101 306235 1.0 1 266586 39649 102 303444 1.0 1 263487 39957 103 381845 1.0 1 331309 50536 104 325653 1.0 1 282316 43337 105 312837 1.0 1 271156 41681 106 518166 1.0 1 449072 69094 107 394290 1.0 1 340403 53887 108 400937 1.0 1 346365 54572 109 457671 1.0 1 394132 63539 110 296032 1.0 1 255379 40653 111 290983 1.0 1 250446 40537 112 324856 1.0 1 279357 45499 113 280038 1.0 1 240483 39555 114 482507 1.0 1 414788 67719 115 943892 1.0 1 812500 131392 116 271430 1.0 1 232083 39347 117 382741 1.0 1 327790 54951 118 350788 1.0 1 300238 50550 119 368835 1.0 1 315411 53424 120 207719 1.0 1 177597 30122 121 211750 1.0 1 180751 30999 122 191203 1.0 1 162999 28204 123 206942 1.0 1 176595 30347 124 200886 1.0 1 171101 29785 125 263147 1.0 1 224479 38668 126 157461 1.0 1 133795 23666 127 132546 1.0 1 112871 19675 128 82021 1.0 1 69657 12364 129 66010 1.0 1 56158 9852 130 47701 1.0 1 40590 7111 131 65077 1.0 1 55570 9507 132 29191 1.0 1 24701 4490 133 10959 1.0 1 9307 1652 134 6055 1.0 1 5140 915 135 1656 1.0 1 1395 261 136 705 1.0 1 608 97 137 41 1.0 1 32 9 138 10 1.0 1 10 139 6 1.0 1 6 140 9 1.0 1 9 141 6 1.0 1 4 2 142 2 1.0 1 2 144 1 1.0 1 0 1 145 2 1.0 1 1 1 148 59 1.0 1 52 7 149 26 1.0 1 17 9 150 306 1.0 1 266 40 Successfully deleted temporary file Meth14_R1_001.fastq.gz_qual_trimmed.fastq RUN STATISTICS FOR INPUT FILE: /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth14_R1_001.fastq.gz ============================================= 69473196 sequences processed in total Sequences were truncated to a varying degree because of deteriorating qualities (Phred score quality cutoff: 20): 3888847 (5.6%) The length threshold of paired-end sequences gets evaluated later on (in the validation step) RRBS reads trimmed by additional 2 bp when adapter contamination was detected: 30667943 (44.1%) RRBS reads trimmed by 2 bp at the start when read started with CAA (641373) or CGA (30028291) in total: 30669664 (44.1%) Writing report to '/gscratch/scrubbed/strigg/analyses/20200306/Meth14_R2_001.fastq.gz_trimming_report.txt' SUMMARISING RUN PARAMETERS ========================== Input filename: /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth14_R2_001.fastq.gz Trimming mode: paired-end Trim Galore version: 0.4.4_dev Cutadapt version: 2.4 Quality Phred score cutoff: 20 Quality encoding type selected: ASCII+33 Adapter sequence: 'AGATCGGAAGAGC' (Illumina TruSeq, Sanger iPCR; auto-detected) Maximum trimming error rate: 0.1 (default) Minimum required adapter overlap (stringency): 1 bp Minimum required sequence length for both reads before a sequence pair gets removed: 20 bp File was specified to be an MspI-digested RRBS sample. Read 1 sequences with adapter contamination will be trimmed a further 2 bp from their 3' end, and Read 2 sequences will be trimmed by 2 bp from their 5' end to remove potential methylation-biased bases from the end-repair reaction File was specified to be a non-directional MspI-digested RRBS sample. Sequences starting with either 'CAA' or 'CGA' will have the first 2 bp trimmed off to remove potential methylation-biased bases from the end-repair reaction Running FastQC on the data once trimming has completed Running FastQC with the following extra arguments: '--outdir /gscratch/scrubbed/strigg/analyses/20200306/FASTQC' Output file(s) will be GZIP compressed >>> Now performing adaptive quality trimming with a Phred-score cutoff of: 20 <<< This is cutadapt 2.4 with Python 3.7.3 Command line parameters: -f fastq -e 0.1 -q 20 -a X /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth14_R2_001.fastq.gz WARNING: Option --format is deprecated and ignored because the input file format is always auto-detected Processing reads on 1 core in single-end mode ... 10000000 sequences processed 20000000 sequences processed 30000000 sequences processed 40000000 sequences processed 50000000 sequences processed 60000000 sequences processed Finished in 646.15 s (9 us/read; 6.45 M reads/minute). === Summary === Total reads processed: 69,473,196 Reads with adapters: 0 (0.0%) Reads written (passing filters): 69,473,196 (100.0%) Total basepairs processed: 10,420,979,400 bp Quality-trimmed: 78,623,751 bp (0.8%) Total written (filtered): 10,342,355,649 bp (99.2%) === Adapter 1 === Sequence: X; Type: regular 3'; Length: 1; Trimmed: 0 times. >>> Quality trimming completed <<< 69473196 sequences processed in total Writing final adapter and quality trimmed output to Meth14_R2_001_trimmed.fq.gz >>> Now performing adapter trimming for the adapter sequence: 'AGATCGGAAGAGC' from file Meth14_R2_001.fastq.gz_qual_trimmed.fastq <<< 10000000 sequences processed 20000000 sequences processed 30000000 sequences processed 40000000 sequences processed 50000000 sequences processed 60000000 sequences processed This is cutadapt 2.4 with Python 3.7.3 Command line parameters: -f fastq -e 0.1 -O 1 -a AGATCGGAAGAGC /gscratch/scrubbed/strigg/analyses/20200306/Meth14_R2_001.fastq.gz_qual_trimmed.fastq WARNING: Option --format is deprecated and ignored because the input file format is always auto-detected Processing reads on 1 core in single-end mode ... Finished in 1669.09 s (24 us/read; 2.50 M reads/minute). === Summary === Total reads processed: 69,473,196 Reads with adapters: 54,757,002 (78.8%) Reads written (passing filters): 69,473,196 (100.0%) Total basepairs processed: 10,342,355,649 bp Total written (filtered): 7,306,131,783 bp (70.6%) === Adapter 1 === Sequence: AGATCGGAAGAGC; Type: regular 3'; Length: 13; Trimmed: 54757002 times. No. of allowed errors: 0-9 bp: 0; 10-13 bp: 1 Bases preceding removed adapters: A: 52.5% C: 2.6% G: 39.4% T: 5.6% none/other: 0.0% Overview of removed sequences length count expect max.err error counts 1 9197747 17368299.0 0 9197747 2 791126 4342074.8 0 791126 3 650060 1085518.7 0 650060 4 300051 271379.7 0 300051 5 207094 67844.9 0 207094 6 183231 16961.2 0 183231 7 206636 4240.3 0 206636 8 226217 1060.1 0 226217 9 194194 265.0 0 192768 1426 10 239058 66.3 1 221959 17099 11 408625 16.6 1 373369 35256 12 262789 4.1 1 239771 23018 13 214488 1.0 1 191823 22665 14 227214 1.0 1 204150 23064 15 186533 1.0 1 168602 17931 16 210213 1.0 1 188151 22062 17 224535 1.0 1 202585 21950 18 215457 1.0 1 195058 20399 19 241405 1.0 1 218468 22937 20 243924 1.0 1 220366 23558 21 264163 1.0 1 237649 26514 22 264559 1.0 1 239756 24803 23 297505 1.0 1 269472 28033 24 302339 1.0 1 273883 28456 25 298219 1.0 1 270303 27916 26 200126 1.0 1 179911 20215 27 255485 1.0 1 228796 26689 28 406873 1.0 1 371566 35307 29 269701 1.0 1 243338 26363 30 252209 1.0 1 229366 22843 31 259037 1.0 1 234311 24726 32 273918 1.0 1 248477 25441 33 248703 1.0 1 224614 24089 34 260017 1.0 1 236461 23556 35 257033 1.0 1 232060 24973 36 268162 1.0 1 243710 24452 37 339901 1.0 1 308266 31635 38 334547 1.0 1 303634 30913 39 314407 1.0 1 284548 29859 40 443344 1.0 1 403840 39504 41 422616 1.0 1 382612 40004 42 288615 1.0 1 262857 25758 43 319929 1.0 1 290681 29248 44 277603 1.0 1 252069 25534 45 317264 1.0 1 287321 29943 46 342879 1.0 1 309141 33738 47 299956 1.0 1 273661 26295 48 336404 1.0 1 305257 31147 49 553560 1.0 1 503398 50162 50 348651 1.0 1 316232 32419 51 349468 1.0 1 316200 33268 52 436733 1.0 1 395379 41354 53 379569 1.0 1 344088 35481 54 721200 1.0 1 657042 64158 55 342173 1.0 1 309916 32257 56 347373 1.0 1 314411 32962 57 608317 1.0 1 552920 55397 58 367949 1.0 1 332225 35724 59 324979 1.0 1 294257 30722 60 740386 1.0 1 674689 65697 61 382326 1.0 1 346166 36160 62 332142 1.0 1 295077 37065 63 951147 1.0 1 867402 83745 64 272348 1.0 1 245701 26647 65 198937 1.0 1 179459 19478 66 215348 1.0 1 193519 21829 67 572565 1.0 1 521252 51313 68 542463 1.0 1 491631 50832 69 427048 1.0 1 386599 40449 70 618005 1.0 1 560912 57093 71 569054 1.0 1 515913 53141 72 462342 1.0 1 417497 44845 73 941079 1.0 1 855358 85721 74 1301136 1.0 1 1181408 119728 75 406521 1.0 1 367659 38862 76 338564 1.0 1 306385 32179 77 230982 1.0 1 206795 24187 78 267339 1.0 1 241279 26060 79 331027 1.0 1 298842 32185 80 355168 1.0 1 319944 35224 81 309160 1.0 1 278700 30460 82 335140 1.0 1 302340 32800 83 345577 1.0 1 311091 34486 84 464235 1.0 1 418098 46137 85 430740 1.0 1 387769 42971 86 344274 1.0 1 309038 35236 87 409694 1.0 1 368415 41279 88 347765 1.0 1 312880 34885 89 419319 1.0 1 376833 42486 90 402929 1.0 1 362455 40474 91 435170 1.0 1 390938 44232 92 321501 1.0 1 288699 32802 93 375027 1.0 1 336301 38726 94 417103 1.0 1 374154 42949 95 299840 1.0 1 268676 31164 96 322439 1.0 1 288700 33739 97 430709 1.0 1 385800 44909 98 410208 1.0 1 367535 42673 99 371684 1.0 1 332048 39636 100 391758 1.0 1 349967 41791 101 297443 1.0 1 265087 32356 102 290284 1.0 1 258921 31363 103 359954 1.0 1 321158 38796 104 306323 1.0 1 272755 33568 105 298940 1.0 1 266054 32886 106 486818 1.0 1 433188 53630 107 373478 1.0 1 331629 41849 108 383489 1.0 1 341083 42406 109 430389 1.0 1 382372 48017 110 284755 1.0 1 252983 31772 111 284268 1.0 1 252483 31785 112 320100 1.0 1 283685 36415 113 283802 1.0 1 251693 32109 114 472995 1.0 1 418840 54155 115 891460 1.0 1 789541 101919 116 259122 1.0 1 228258 30864 117 361635 1.0 1 318519 43116 118 335624 1.0 1 295770 39854 119 352655 1.0 1 310802 41853 120 201209 1.0 1 176857 24352 121 204289 1.0 1 179728 24561 122 183152 1.0 1 160739 22413 123 199205 1.0 1 174646 24559 124 193647 1.0 1 169805 23842 125 255331 1.0 1 223703 31628 126 153044 1.0 1 133926 19118 127 128172 1.0 1 112200 15972 128 79879 1.0 1 69697 10182 129 63552 1.0 1 55540 8012 130 46254 1.0 1 40233 6021 131 62315 1.0 1 54565 7750 132 28013 1.0 1 24322 3691 133 10585 1.0 1 9135 1450 134 5808 1.0 1 4947 861 135 1598 1.0 1 1368 230 136 684 1.0 1 601 83 137 36 1.0 1 33 3 138 11 1.0 1 10 1 139 7 1.0 1 6 1 140 8 1.0 1 8 141 8 1.0 1 6 2 142 2 1.0 1 1 1 144 2 1.0 1 1 1 145 2 1.0 1 2 147 2 1.0 1 2 148 58 1.0 1 51 7 149 24 1.0 1 20 4 150 289 1.0 1 264 25 Successfully deleted temporary file Meth14_R2_001.fastq.gz_qual_trimmed.fastq RUN STATISTICS FOR INPUT FILE: /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth14_R2_001.fastq.gz ============================================= 69473196 sequences processed in total Sequences were truncated to a varying degree because of deteriorating qualities (Phred score quality cutoff: 20): 7124230 (10.3%) The length threshold of paired-end sequences gets evaluated later on (in the validation step) RRBS reads trimmed by additional 2 bp when adapter contamination was detected: 0 (0.0%) RRBS reads trimmed by 2 bp at the start when read started with CAA (771107) or CGA (36511320) in total: 37282427 (53.7%) Validate paired-end files Meth14_R1_001_trimmed.fq.gz and Meth14_R2_001_trimmed.fq.gz file_1: Meth14_R1_001_trimmed.fq.gz, file_2: Meth14_R2_001_trimmed.fq.gz >>>>> Now validing the length of the 2 paired-end infiles: Meth14_R1_001_trimmed.fq.gz and Meth14_R2_001_trimmed.fq.gz <<<<< Writing validated paired-end read 1 reads to Meth14_R1_001_val_1.fq.gz Writing validated paired-end read 2 reads to Meth14_R2_001_val_2.fq.gz Total number of sequences analysed: 69473196 Number of sequence pairs removed because at least one read was shorter than the length cutoff (20 bp): 313337 (0.45%) >>> Now running FastQC on the validated data Meth14_R1_001_val_1.fq.gz<<< Can't exec "fastqc": No such file or directory at /gscratch/srlab/programs/TrimGalore-0.4.5/trim_galore line 821, line 681869284. >>> Now running FastQC on the validated data Meth14_R2_001_val_2.fq.gz<<< Can't exec "fastqc": No such file or directory at /gscratch/srlab/programs/TrimGalore-0.4.5/trim_galore line 831, line 681869284. Deleting both intermediate output files Meth14_R1_001_trimmed.fq.gz and Meth14_R2_001_trimmed.fq.gz ==================================================================================================== Writing report to '/gscratch/scrubbed/strigg/analyses/20200306/Meth15_R1_001.fastq.gz_trimming_report.txt' SUMMARISING RUN PARAMETERS ========================== Input filename: /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth15_R1_001.fastq.gz Trimming mode: paired-end Trim Galore version: 0.4.4_dev Cutadapt version: 2.4 Quality Phred score cutoff: 20 Quality encoding type selected: ASCII+33 Adapter sequence: 'AGATCGGAAGAGC' (Illumina TruSeq, Sanger iPCR; auto-detected) Maximum trimming error rate: 0.1 (default) Minimum required adapter overlap (stringency): 1 bp Minimum required sequence length for both reads before a sequence pair gets removed: 20 bp File was specified to be an MspI-digested RRBS sample. Read 1 sequences with adapter contamination will be trimmed a further 2 bp from their 3' end, and Read 2 sequences will be trimmed by 2 bp from their 5' end to remove potential methylation-biased bases from the end-repair reaction File was specified to be a non-directional MspI-digested RRBS sample. Sequences starting with either 'CAA' or 'CGA' will have the first 2 bp trimmed off to remove potential methylation-biased bases from the end-repair reaction Running FastQC on the data once trimming has completed Running FastQC with the following extra arguments: '--outdir /gscratch/scrubbed/strigg/analyses/20200306/FASTQC' Output file(s) will be GZIP compressed >>> Now performing adaptive quality trimming with a Phred-score cutoff of: 20 <<< This is cutadapt 2.4 with Python 3.7.3 Command line parameters: -f fastq -e 0.1 -q 20 -a X /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth15_R1_001.fastq.gz WARNING: Option --format is deprecated and ignored because the input file format is always auto-detected Processing reads on 1 core in single-end mode ... 10000000 sequences processed 20000000 sequences processed 30000000 sequences processed 40000000 sequences processed 50000000 sequences processed 60000000 sequences processed 70000000 sequences processed 80000000 sequences processed 90000000 sequences processed 100000000 sequences processed Finished in 1018.98 s (9 us/read; 6.45 M reads/minute). === Summary === Total reads processed: 109,570,006 Reads with adapters: 0 (0.0%) Reads written (passing filters): 109,570,006 (100.0%) Total basepairs processed: 16,435,500,900 bp Quality-trimmed: 17,058,606 bp (0.1%) Total written (filtered): 16,418,442,294 bp (99.9%) === Adapter 1 === Sequence: X; Type: regular 3'; Length: 1; Trimmed: 0 times. >>> Quality trimming completed <<< 109570006 sequences processed in total Writing final adapter and quality trimmed output to Meth15_R1_001_trimmed.fq.gz >>> Now performing adapter trimming for the adapter sequence: 'AGATCGGAAGAGC' from file Meth15_R1_001.fastq.gz_qual_trimmed.fastq <<< 10000000 sequences processed 20000000 sequences processed 30000000 sequences processed 40000000 sequences processed 50000000 sequences processed 60000000 sequences processed 70000000 sequences processed 80000000 sequences processed 90000000 sequences processed 100000000 sequences processed This is cutadapt 2.4 with Python 3.7.3 Command line parameters: -f fastq -e 0.1 -O 1 -a AGATCGGAAGAGC /gscratch/scrubbed/strigg/analyses/20200306/Meth15_R1_001.fastq.gz_qual_trimmed.fastq WARNING: Option --format is deprecated and ignored because the input file format is always auto-detected Processing reads on 1 core in single-end mode ... Finished in 2634.44 s (24 us/read; 2.50 M reads/minute). === Summary === Total reads processed: 109,570,006 Reads with adapters: 85,450,494 (78.0%) Reads written (passing filters): 109,570,006 (100.0%) Total basepairs processed: 16,418,442,294 bp Total written (filtered): 11,708,570,940 bp (71.3%) === Adapter 1 === Sequence: AGATCGGAAGAGC; Type: regular 3'; Length: 13; Trimmed: 85450494 times. No. of allowed errors: 0-9 bp: 0; 10-13 bp: 1 Bases preceding removed adapters: A: 47.5% C: 3.1% G: 43.1% T: 6.4% none/other: 0.0% Overview of removed sequences length count expect max.err error counts 1 15881685 27392501.5 0 15881685 2 1326314 6848125.4 0 1326314 3 987908 1712031.3 0 987908 4 479892 428007.8 0 479892 5 315963 107002.0 0 315963 6 284231 26750.5 0 284231 7 332502 6687.6 0 332502 8 336661 1671.9 0 336661 9 319375 418.0 0 315014 4361 10 376078 104.5 1 343524 32554 11 652211 26.1 1 584608 67603 12 385526 6.5 1 346589 38937 13 367125 1.6 1 327068 40057 14 345421 1.6 1 306617 38804 15 308922 1.6 1 274813 34109 16 325782 1.6 1 288166 37616 17 343774 1.6 1 304581 39193 18 351421 1.6 1 313771 37650 19 366477 1.6 1 326737 39740 20 357719 1.6 1 318838 38881 21 355737 1.6 1 316455 39282 22 396415 1.6 1 353903 42512 23 534508 1.6 1 476805 57703 24 438039 1.6 1 388134 49905 25 392153 1.6 1 349639 42514 26 326147 1.6 1 290198 35949 27 367964 1.6 1 326870 41094 28 589600 1.6 1 526234 63366 29 392793 1.6 1 347702 45091 30 371448 1.6 1 331148 40300 31 385679 1.6 1 343409 42270 32 413960 1.6 1 368784 45176 33 380206 1.6 1 337064 43142 34 433647 1.6 1 385023 48624 35 384518 1.6 1 341965 42553 36 393763 1.6 1 350251 43512 37 524656 1.6 1 465666 58990 38 548952 1.6 1 487732 61220 39 538971 1.6 1 481224 57747 40 575079 1.6 1 510147 64932 41 629906 1.6 1 558113 71793 42 406006 1.6 1 361276 44730 43 509761 1.6 1 450931 58830 44 418557 1.6 1 367831 50726 45 657598 1.6 1 588206 69392 46 301467 1.6 1 266028 35439 47 384563 1.6 1 341752 42811 48 479560 1.6 1 424891 54669 49 919153 1.6 1 819152 100001 50 482536 1.6 1 425678 56858 51 565122 1.6 1 496604 68518 52 646514 1.6 1 574128 72386 53 520868 1.6 1 461228 59640 54 954083 1.6 1 846167 107916 55 621495 1.6 1 550973 70522 56 475172 1.6 1 421258 53914 57 568249 1.6 1 502917 65332 58 866998 1.6 1 768288 98710 59 472499 1.6 1 416890 55609 60 1000128 1.6 1 890090 110038 61 558077 1.6 1 493433 64644 62 505737 1.6 1 447064 58673 63 455947 1.6 1 403334 52613 64 520537 1.6 1 461227 59310 65 422608 1.6 1 372821 49787 66 506601 1.6 1 446321 60280 67 681931 1.6 1 600525 81406 68 844668 1.6 1 744047 100621 69 797831 1.6 1 685016 112815 70 2455220 1.6 1 2209337 245883 71 165028 1.6 1 143638 21390 72 75859 1.6 1 63042 12817 73 627173 1.6 1 547548 79625 74 2053322 1.6 1 1816689 236633 75 504092 1.6 1 440997 63095 76 600709 1.6 1 529073 71636 77 637126 1.6 1 560130 76996 78 494842 1.6 1 435516 59326 79 522272 1.6 1 459493 62779 80 543212 1.6 1 477983 65229 81 465519 1.6 1 409098 56421 82 504755 1.6 1 443375 61380 83 475264 1.6 1 417280 57984 84 699035 1.6 1 614950 84085 85 611586 1.6 1 536803 74783 86 490097 1.6 1 429187 60910 87 577172 1.6 1 506192 70980 88 521333 1.6 1 456853 64480 89 634181 1.6 1 555529 78652 90 593188 1.6 1 519930 73258 91 679111 1.6 1 594973 84138 92 471265 1.6 1 412028 59237 93 583880 1.6 1 510091 73789 94 660442 1.6 1 577533 82909 95 454088 1.6 1 395746 58342 96 489013 1.6 1 425639 63374 97 636451 1.6 1 554620 81831 98 619482 1.6 1 539222 80260 99 550951 1.6 1 478829 72122 100 611930 1.6 1 531343 80587 101 477720 1.6 1 414868 62852 102 469210 1.6 1 406380 62830 103 559469 1.6 1 485715 73754 104 496435 1.6 1 429327 67108 105 478535 1.6 1 413559 64976 106 774275 1.6 1 669839 104436 107 617133 1.6 1 532627 84506 108 613075 1.6 1 527738 85337 109 696205 1.6 1 600909 95296 110 467767 1.6 1 403306 64461 111 444303 1.6 1 381719 62584 112 518609 1.6 1 446790 71819 113 446848 1.6 1 383017 63831 114 756680 1.6 1 650302 106378 115 1428651 1.6 1 1230013 198638 116 451175 1.6 1 384952 66223 117 589655 1.6 1 504024 85631 118 583890 1.6 1 498851 85039 119 633978 1.6 1 541847 92131 120 357006 1.6 1 304026 52980 121 371769 1.6 1 316182 55587 122 339599 1.6 1 289245 50354 123 378713 1.6 1 322408 56305 124 381818 1.6 1 324294 57524 125 491769 1.6 1 417384 74385 126 304155 1.6 1 257227 46928 127 265411 1.6 1 224767 40644 128 173625 1.6 1 147409 26216 129 144139 1.6 1 121958 22181 130 106601 1.6 1 90012 16589 131 141712 1.6 1 119681 22031 132 71531 1.6 1 60372 11159 133 26436 1.6 1 22268 4168 134 14419 1.6 1 12179 2240 135 4404 1.6 1 3659 745 136 1873 1.6 1 1614 259 137 111 1.6 1 91 20 138 30 1.6 1 24 6 139 10 1.6 1 9 1 140 4 1.6 1 4 141 3 1.6 1 2 1 142 1 1.6 1 1 144 6 1.6 1 2 4 147 8 1.6 1 4 4 148 147 1.6 1 126 21 149 29 1.6 1 26 3 150 560 1.6 1 491 69 Successfully deleted temporary file Meth15_R1_001.fastq.gz_qual_trimmed.fastq RUN STATISTICS FOR INPUT FILE: /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth15_R1_001.fastq.gz ============================================= 109570006 sequences processed in total Sequences were truncated to a varying degree because of deteriorating qualities (Phred score quality cutoff: 20): 5866846 (5.4%) The length threshold of paired-end sequences gets evaluated later on (in the validation step) RRBS reads trimmed by additional 2 bp when adapter contamination was detected: 41006195 (37.4%) RRBS reads trimmed by 2 bp at the start when read started with CAA (1231660) or CGA (54436579) in total: 55668239 (50.8%) Writing report to '/gscratch/scrubbed/strigg/analyses/20200306/Meth15_R2_001.fastq.gz_trimming_report.txt' SUMMARISING RUN PARAMETERS ========================== Input filename: /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth15_R2_001.fastq.gz Trimming mode: paired-end Trim Galore version: 0.4.4_dev Cutadapt version: 2.4 Quality Phred score cutoff: 20 Quality encoding type selected: ASCII+33 Adapter sequence: 'AGATCGGAAGAGC' (Illumina TruSeq, Sanger iPCR; auto-detected) Maximum trimming error rate: 0.1 (default) Minimum required adapter overlap (stringency): 1 bp Minimum required sequence length for both reads before a sequence pair gets removed: 20 bp File was specified to be an MspI-digested RRBS sample. Read 1 sequences with adapter contamination will be trimmed a further 2 bp from their 3' end, and Read 2 sequences will be trimmed by 2 bp from their 5' end to remove potential methylation-biased bases from the end-repair reaction File was specified to be a non-directional MspI-digested RRBS sample. Sequences starting with either 'CAA' or 'CGA' will have the first 2 bp trimmed off to remove potential methylation-biased bases from the end-repair reaction Running FastQC on the data once trimming has completed Running FastQC with the following extra arguments: '--outdir /gscratch/scrubbed/strigg/analyses/20200306/FASTQC' Output file(s) will be GZIP compressed >>> Now performing adaptive quality trimming with a Phred-score cutoff of: 20 <<< This is cutadapt 2.4 with Python 3.7.3 Command line parameters: -f fastq -e 0.1 -q 20 -a X /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth15_R2_001.fastq.gz WARNING: Option --format is deprecated and ignored because the input file format is always auto-detected Processing reads on 1 core in single-end mode ... 10000000 sequences processed 20000000 sequences processed 30000000 sequences processed 40000000 sequences processed 50000000 sequences processed 60000000 sequences processed 70000000 sequences processed 80000000 sequences processed 90000000 sequences processed 100000000 sequences processed Finished in 1010.75 s (9 us/read; 6.50 M reads/minute). === Summary === Total reads processed: 109,570,006 Reads with adapters: 0 (0.0%) Reads written (passing filters): 109,570,006 (100.0%) Total basepairs processed: 16,435,500,900 bp Quality-trimmed: 157,545,324 bp (1.0%) Total written (filtered): 16,277,955,576 bp (99.0%) === Adapter 1 === Sequence: X; Type: regular 3'; Length: 1; Trimmed: 0 times. >>> Quality trimming completed <<< 109570006 sequences processed in total Writing final adapter and quality trimmed output to Meth15_R2_001_trimmed.fq.gz >>> Now performing adapter trimming for the adapter sequence: 'AGATCGGAAGAGC' from file Meth15_R2_001.fastq.gz_qual_trimmed.fastq <<< 10000000 sequences processed 20000000 sequences processed 30000000 sequences processed 40000000 sequences processed 50000000 sequences processed 60000000 sequences processed 70000000 sequences processed 80000000 sequences processed 90000000 sequences processed 100000000 sequences processed This is cutadapt 2.4 with Python 3.7.3 Command line parameters: -f fastq -e 0.1 -O 1 -a AGATCGGAAGAGC /gscratch/scrubbed/strigg/analyses/20200306/Meth15_R2_001.fastq.gz_qual_trimmed.fastq WARNING: Option --format is deprecated and ignored because the input file format is always auto-detected Processing reads on 1 core in single-end mode ... Finished in 2714.96 s (25 us/read; 2.42 M reads/minute). === Summary === Total reads processed: 109,570,006 Reads with adapters: 83,811,500 (76.5%) Reads written (passing filters): 109,570,006 (100.0%) Total basepairs processed: 16,277,955,576 bp Total written (filtered): 11,729,149,239 bp (72.1%) === Adapter 1 === Sequence: AGATCGGAAGAGC; Type: regular 3'; Length: 13; Trimmed: 83811500 times. No. of allowed errors: 0-9 bp: 0; 10-13 bp: 1 Bases preceding removed adapters: A: 46.2% C: 2.6% G: 44.4% T: 6.7% none/other: 0.0% Overview of removed sequences length count expect max.err error counts 1 15643604 27392501.5 0 15643604 2 1404461 6848125.4 0 1404461 3 1032068 1712031.3 0 1032068 4 495162 428007.8 0 495162 5 315265 107002.0 0 315265 6 281806 26750.5 0 281806 7 315056 6687.6 0 315056 8 343362 1671.9 0 343362 9 303966 418.0 0 301786 2180 10 364458 104.5 1 337350 27108 11 636950 26.1 1 577475 59475 12 415500 6.5 1 378195 37305 13 327101 1.6 1 291246 35855 14 334728 1.6 1 300065 34663 15 293162 1.6 1 262741 30421 16 333254 1.6 1 297273 35981 17 328131 1.6 1 295009 33122 18 321080 1.6 1 289708 31372 19 382369 1.6 1 344782 37587 20 357602 1.6 1 321903 35699 21 333890 1.6 1 299589 34301 22 403737 1.6 1 364438 39299 23 495561 1.6 1 447621 47940 24 430568 1.6 1 388613 41955 25 435718 1.6 1 393815 41903 26 279865 1.6 1 251513 28352 27 353301 1.6 1 315930 37371 28 569412 1.6 1 518365 51047 29 386976 1.6 1 347737 39239 30 361508 1.6 1 327534 33974 31 384181 1.6 1 345906 38275 32 398982 1.6 1 361474 37508 33 378366 1.6 1 340059 38307 34 422614 1.6 1 382314 40300 35 367792 1.6 1 332596 35196 36 403291 1.6 1 361515 41776 37 529705 1.6 1 478848 50857 38 528089 1.6 1 472928 55161 39 511744 1.6 1 457170 54574 40 641148 1.6 1 582863 58285 41 597201 1.6 1 539426 57775 42 443261 1.6 1 402607 40654 43 432281 1.6 1 390543 41738 44 452376 1.6 1 409941 42435 45 537153 1.6 1 484438 52715 46 510405 1.6 1 458984 51421 47 700998 1.6 1 639352 61646 48 312850 1.6 1 281030 31820 49 724516 1.6 1 656801 67715 50 504634 1.6 1 457939 46695 51 433338 1.6 1 390768 42570 52 619613 1.6 1 560502 59111 53 524596 1.6 1 472405 52191 54 1164400 1.6 1 1061414 102986 55 336683 1.6 1 302509 34174 56 506802 1.6 1 452593 54209 57 1713467 1.6 1 1562674 150793 58 380124 1.6 1 339541 40583 59 350769 1.6 1 312681 38088 60 1486641 1.6 1 1353823 132818 61 474221 1.6 1 428726 45495 62 389291 1.6 1 339128 50163 63 2403019 1.6 1 2192515 210504 64 664617 1.6 1 603401 61216 65 209309 1.6 1 187984 21325 66 259805 1.6 1 230002 29803 67 1021196 1.6 1 929011 92185 68 556813 1.6 1 502779 54034 69 498388 1.6 1 449011 49377 70 799861 1.6 1 725414 74447 71 549560 1.6 1 496408 53152 72 431189 1.6 1 387803 43386 73 912672 1.6 1 825986 86686 74 1215964 1.6 1 1101520 114444 75 387223 1.6 1 348654 38569 76 439150 1.6 1 397016 42134 77 308592 1.6 1 275729 32863 78 390003 1.6 1 351604 38399 79 465817 1.6 1 419820 45997 80 500520 1.6 1 450662 49858 81 439923 1.6 1 395631 44292 82 486249 1.6 1 437371 48878 83 499883 1.6 1 449436 50447 84 685820 1.6 1 616711 69109 85 633416 1.6 1 568645 64771 86 491581 1.6 1 440530 51051 87 556922 1.6 1 499982 56940 88 500754 1.6 1 450100 50654 89 600638 1.6 1 539777 60861 90 567327 1.6 1 509781 57546 91 639615 1.6 1 574511 65104 92 449676 1.6 1 403204 46472 93 555974 1.6 1 499491 56483 94 618669 1.6 1 555245 63424 95 428774 1.6 1 383862 44912 96 464401 1.6 1 416024 48377 97 609349 1.6 1 545451 63898 98 599249 1.6 1 536342 62907 99 546292 1.6 1 488390 57902 100 600200 1.6 1 535861 64339 101 463544 1.6 1 413256 50288 102 447382 1.6 1 398994 48388 103 527840 1.6 1 471266 56574 104 466116 1.6 1 415619 50497 105 457490 1.6 1 407507 49983 106 729732 1.6 1 649934 79798 107 586569 1.6 1 521881 64688 108 585355 1.6 1 520142 65213 109 655586 1.6 1 582992 72594 110 445079 1.6 1 395391 49688 111 423064 1.6 1 375407 47657 112 491877 1.6 1 436210 55667 113 426838 1.6 1 377878 48960 114 718856 1.6 1 636056 82800 115 1343866 1.6 1 1190996 152870 116 433588 1.6 1 383499 50089 117 562583 1.6 1 496034 66549 118 564042 1.6 1 497114 66928 119 608898 1.6 1 537036 71862 120 345174 1.6 1 303547 41627 121 358548 1.6 1 315632 42916 122 324348 1.6 1 284899 39449 123 363526 1.6 1 319323 44203 124 366908 1.6 1 322053 44855 125 475676 1.6 1 416659 59017 126 294723 1.6 1 258353 36370 127 256017 1.6 1 224163 31854 128 168906 1.6 1 147377 21529 129 138126 1.6 1 120620 17506 130 102655 1.6 1 89603 13052 131 134692 1.6 1 117223 17469 132 68185 1.6 1 59411 8774 133 25423 1.6 1 21912 3511 134 13817 1.6 1 11938 1879 135 4221 1.6 1 3660 561 136 1815 1.6 1 1547 268 137 97 1.6 1 82 15 138 26 1.6 1 25 1 139 9 1.6 1 8 1 140 6 1.6 1 6 141 3 1.6 1 3 142 3 1.6 1 0 3 144 6 1.6 1 2 4 145 1 1.6 1 0 1 146 1 1.6 1 0 1 147 3 1.6 1 3 148 145 1.6 1 129 16 149 30 1.6 1 21 9 150 542 1.6 1 498 44 Successfully deleted temporary file Meth15_R2_001.fastq.gz_qual_trimmed.fastq RUN STATISTICS FOR INPUT FILE: /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth15_R2_001.fastq.gz ============================================= 109570006 sequences processed in total Sequences were truncated to a varying degree because of deteriorating qualities (Phred score quality cutoff: 20): 14718642 (13.4%) The length threshold of paired-end sequences gets evaluated later on (in the validation step) RRBS reads trimmed by additional 2 bp when adapter contamination was detected: 0 (0.0%) RRBS reads trimmed by 2 bp at the start when read started with CAA (1128168) or CGA (49375162) in total: 50503330 (46.1%) Validate paired-end files Meth15_R1_001_trimmed.fq.gz and Meth15_R2_001_trimmed.fq.gz file_1: Meth15_R1_001_trimmed.fq.gz, file_2: Meth15_R2_001_trimmed.fq.gz >>>>> Now validing the length of the 2 paired-end infiles: Meth15_R1_001_trimmed.fq.gz and Meth15_R2_001_trimmed.fq.gz <<<<< Writing validated paired-end read 1 reads to Meth15_R1_001_val_1.fq.gz Writing validated paired-end read 2 reads to Meth15_R2_001_val_2.fq.gz Total number of sequences analysed: 109570006 Number of sequence pairs removed because at least one read was shorter than the length cutoff (20 bp): 658759 (0.60%) >>> Now running FastQC on the validated data Meth15_R1_001_val_1.fq.gz<<< Can't exec "fastqc": No such file or directory at /gscratch/srlab/programs/TrimGalore-0.4.5/trim_galore line 821, line 1120149308. >>> Now running FastQC on the validated data Meth15_R2_001_val_2.fq.gz<<< Can't exec "fastqc": No such file or directory at /gscratch/srlab/programs/TrimGalore-0.4.5/trim_galore line 831, line 1120149308. Deleting both intermediate output files Meth15_R1_001_trimmed.fq.gz and Meth15_R2_001_trimmed.fq.gz ==================================================================================================== Writing report to '/gscratch/scrubbed/strigg/analyses/20200306/Meth4_R1_001.fastq.gz_trimming_report.txt' SUMMARISING RUN PARAMETERS ========================== Input filename: /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth4_R1_001.fastq.gz Trimming mode: paired-end Trim Galore version: 0.4.4_dev Cutadapt version: 2.4 Quality Phred score cutoff: 20 Quality encoding type selected: ASCII+33 Adapter sequence: 'AGATCGGAAGAGC' (Illumina TruSeq, Sanger iPCR; auto-detected) Maximum trimming error rate: 0.1 (default) Minimum required adapter overlap (stringency): 1 bp Minimum required sequence length for both reads before a sequence pair gets removed: 20 bp File was specified to be an MspI-digested RRBS sample. Read 1 sequences with adapter contamination will be trimmed a further 2 bp from their 3' end, and Read 2 sequences will be trimmed by 2 bp from their 5' end to remove potential methylation-biased bases from the end-repair reaction File was specified to be a non-directional MspI-digested RRBS sample. Sequences starting with either 'CAA' or 'CGA' will have the first 2 bp trimmed off to remove potential methylation-biased bases from the end-repair reaction Running FastQC on the data once trimming has completed Running FastQC with the following extra arguments: '--outdir /gscratch/scrubbed/strigg/analyses/20200306/FASTQC' Output file(s) will be GZIP compressed >>> Now performing adaptive quality trimming with a Phred-score cutoff of: 20 <<< This is cutadapt 2.4 with Python 3.7.3 Command line parameters: -f fastq -e 0.1 -q 20 -a X /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth4_R1_001.fastq.gz WARNING: Option --format is deprecated and ignored because the input file format is always auto-detected Processing reads on 1 core in single-end mode ... 10000000 sequences processed 20000000 sequences processed 30000000 sequences processed 40000000 sequences processed 50000000 sequences processed 60000000 sequences processed 70000000 sequences processed 80000000 sequences processed 90000000 sequences processed 100000000 sequences processed 110000000 sequences processed 120000000 sequences processed 130000000 sequences processed 140000000 sequences processed 150000000 sequences processed Finished in 1398.08 s (9 us/read; 6.46 M reads/minute). === Summary === Total reads processed: 150,624,528 Reads with adapters: 0 (0.0%) Reads written (passing filters): 150,624,528 (100.0%) Total basepairs processed: 22,593,679,200 bp Quality-trimmed: 28,858,391 bp (0.1%) Total written (filtered): 22,564,820,809 bp (99.9%) === Adapter 1 === Sequence: X; Type: regular 3'; Length: 1; Trimmed: 0 times. >>> Quality trimming completed <<< 150624528 sequences processed in total Writing final adapter and quality trimmed output to Meth4_R1_001_trimmed.fq.gz >>> Now performing adapter trimming for the adapter sequence: 'AGATCGGAAGAGC' from file Meth4_R1_001.fastq.gz_qual_trimmed.fastq <<< 10000000 sequences processed 20000000 sequences processed 30000000 sequences processed 40000000 sequences processed 50000000 sequences processed 60000000 sequences processed 70000000 sequences processed 80000000 sequences processed 90000000 sequences processed 100000000 sequences processed 110000000 sequences processed 120000000 sequences processed 130000000 sequences processed 140000000 sequences processed 150000000 sequences processed This is cutadapt 2.4 with Python 3.7.3 Command line parameters: -f fastq -e 0.1 -O 1 -a AGATCGGAAGAGC /gscratch/scrubbed/strigg/analyses/20200306/Meth4_R1_001.fastq.gz_qual_trimmed.fastq WARNING: Option --format is deprecated and ignored because the input file format is always auto-detected Processing reads on 1 core in single-end mode ... Finished in 3585.43 s (24 us/read; 2.52 M reads/minute). === Summary === Total reads processed: 150,624,528 Reads with adapters: 123,278,388 (81.8%) Reads written (passing filters): 150,624,528 (100.0%) Total basepairs processed: 22,564,820,809 bp Total written (filtered): 15,728,080,367 bp (69.7%) === Adapter 1 === Sequence: AGATCGGAAGAGC; Type: regular 3'; Length: 13; Trimmed: 123278388 times. No. of allowed errors: 0-9 bp: 0; 10-13 bp: 1 Bases preceding removed adapters: A: 47.3% C: 2.9% G: 44.7% T: 5.2% none/other: 0.0% Overview of removed sequences length count expect max.err error counts 1 17926941 37656132.0 0 17926941 2 1629060 9414033.0 0 1629060 3 1127081 2353508.2 0 1127081 4 4408237 588377.1 0 4408237 5 507349 147094.3 0 507349 6 409510 36773.6 0 409510 7 436136 9193.4 0 436136 8 402372 2298.3 0 402372 9 371021 574.6 0 365110 5911 10 559431 143.6 1 513570 45861 11 488616 35.9 1 442071 46545 12 503385 9.0 1 455371 48014 13 526794 2.2 1 470871 55923 14 552304 2.2 1 491787 60517 15 674521 2.2 1 603250 71271 16 763387 2.2 1 676764 86623 17 524698 2.2 1 465066 59632 18 549959 2.2 1 492758 57201 19 534333 2.2 1 477885 56448 20 930236 2.2 1 832975 97261 21 563541 2.2 1 501928 61613 22 747074 2.2 1 669031 78043 23 550154 2.2 1 490338 59816 24 573693 2.2 1 507658 66035 25 1011625 2.2 1 904878 106747 26 551110 2.2 1 490832 60278 27 761201 2.2 1 677236 83965 28 2074838 2.2 1 1858685 216153 29 698868 2.2 1 619185 79683 30 634708 2.2 1 567752 66956 31 697946 2.2 1 623660 74286 32 911986 2.2 1 815557 96429 33 620881 2.2 1 551932 68949 34 710617 2.2 1 633940 76677 35 656720 2.2 1 584849 71871 36 604486 2.2 1 534170 70316 37 649973 2.2 1 581767 68206 38 970085 2.2 1 867378 102707 39 737457 2.2 1 657190 80267 40 901784 2.2 1 805308 96476 41 1224543 2.2 1 1092982 131561 42 704106 2.2 1 623714 80392 43 994656 2.2 1 871526 123130 44 952359 2.2 1 841944 110415 45 1141074 2.2 1 1021530 119544 46 543061 2.2 1 480898 62163 47 595940 2.2 1 527957 67983 48 744798 2.2 1 661862 82936 49 805957 2.2 1 718270 87687 50 581810 2.2 1 510966 70844 51 955412 2.2 1 855088 100324 52 643803 2.2 1 568882 74921 53 561556 2.2 1 496265 65291 54 844459 2.2 1 749828 94631 55 985435 2.2 1 875588 109847 56 729733 2.2 1 647451 82282 57 940576 2.2 1 837633 102943 58 1047851 2.2 1 926197 121654 59 1255922 2.2 1 1123839 132083 60 925301 2.2 1 823105 102196 61 835208 2.2 1 740018 95190 62 787102 2.2 1 695244 91858 63 925875 2.2 1 827116 98759 64 750734 2.2 1 665032 85702 65 752704 2.2 1 667155 85549 66 947743 2.2 1 836316 111427 67 776177 2.2 1 686068 90109 68 732667 2.2 1 646315 86352 69 834630 2.2 1 720687 113943 70 2073402 2.2 1 1865772 207630 71 346381 2.2 1 309848 36533 72 88612 2.2 1 76012 12600 73 189138 2.2 1 160288 28850 74 527145 2.2 1 462450 64695 75 706430 2.2 1 624059 82371 76 1099520 2.2 1 971235 128285 77 744676 2.2 1 655670 89006 78 592279 2.2 1 521339 70940 79 808120 2.2 1 712479 95641 80 730827 2.2 1 642344 88483 81 720896 2.2 1 634443 86453 82 951089 2.2 1 836563 114526 83 762900 2.2 1 670404 92496 84 905291 2.2 1 795140 110151 85 933124 2.2 1 816240 116884 86 788744 2.2 1 687756 100988 87 835429 2.2 1 731643 103786 88 1058598 2.2 1 927737 130861 89 961249 2.2 1 841738 119511 90 1105458 2.2 1 968895 136563 91 1119746 2.2 1 980263 139483 92 1016580 2.2 1 888416 128164 93 1014188 2.2 1 885659 128529 94 1175409 2.2 1 1024452 150957 95 826076 2.2 1 718798 107278 96 1292670 2.2 1 1128212 164458 97 1142779 2.2 1 995220 147559 98 1104950 2.2 1 961366 143584 99 955719 2.2 1 830972 124747 100 1014295 2.2 1 881529 132766 101 787403 2.2 1 682689 104714 102 722257 2.2 1 626532 95725 103 951202 2.2 1 825115 126087 104 814717 2.2 1 704603 110114 105 769224 2.2 1 665301 103923 106 948544 2.2 1 820515 128029 107 686527 2.2 1 591875 94652 108 798243 2.2 1 687617 110626 109 870079 2.2 1 747453 122626 110 765858 2.2 1 658273 107585 111 743948 2.2 1 638808 105140 112 1170274 2.2 1 1005227 165047 113 712644 2.2 1 609774 102870 114 913331 2.2 1 781221 132110 115 1064364 2.2 1 911825 152539 116 685707 2.2 1 585330 100377 117 706162 2.2 1 602201 103961 118 1015924 2.2 1 866717 149207 119 1238461 2.2 1 1055942 182519 120 615046 2.2 1 522969 92077 121 672403 2.2 1 571120 101283 122 593766 2.2 1 504215 89551 123 627170 2.2 1 532776 94394 124 711539 2.2 1 604566 106973 125 365643 2.2 1 309999 55644 126 399539 2.2 1 338517 61022 127 548562 2.2 1 465380 83182 128 223311 2.2 1 188582 34729 129 168704 2.2 1 142915 25789 130 149921 2.2 1 126976 22945 131 151895 2.2 1 128658 23237 132 76747 2.2 1 64877 11870 133 29756 2.2 1 25187 4569 134 5398 2.2 1 4590 808 135 1162 2.2 1 939 223 136 1058 2.2 1 881 177 137 83 2.2 1 68 15 138 38 2.2 1 30 8 139 24 2.2 1 23 1 140 16 2.2 1 15 1 141 12 2.2 1 10 2 142 5 2.2 1 3 2 143 3 2.2 1 1 2 144 2 2.2 1 1 1 145 3 2.2 1 3 146 5 2.2 1 5 147 14 2.2 1 9 5 148 211 2.2 1 171 40 149 306 2.2 1 260 46 150 4147 2.2 1 3579 568 Successfully deleted temporary file Meth4_R1_001.fastq.gz_qual_trimmed.fastq RUN STATISTICS FOR INPUT FILE: /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth4_R1_001.fastq.gz ============================================= 150624528 sequences processed in total Sequences were truncated to a varying degree because of deteriorating qualities (Phred score quality cutoff: 20): 9496390 (6.3%) The length threshold of paired-end sequences gets evaluated later on (in the validation step) RRBS reads trimmed by additional 2 bp when adapter contamination was detected: 61328973 (40.7%) RRBS reads trimmed by 2 bp at the start when read started with CAA (1764891) or CGA (72831693) in total: 74596584 (49.5%) Writing report to '/gscratch/scrubbed/strigg/analyses/20200306/Meth4_R2_001.fastq.gz_trimming_report.txt' SUMMARISING RUN PARAMETERS ========================== Input filename: /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth4_R2_001.fastq.gz Trimming mode: paired-end Trim Galore version: 0.4.4_dev Cutadapt version: 2.4 Quality Phred score cutoff: 20 Quality encoding type selected: ASCII+33 Adapter sequence: 'AGATCGGAAGAGC' (Illumina TruSeq, Sanger iPCR; auto-detected) Maximum trimming error rate: 0.1 (default) Minimum required adapter overlap (stringency): 1 bp Minimum required sequence length for both reads before a sequence pair gets removed: 20 bp File was specified to be an MspI-digested RRBS sample. Read 1 sequences with adapter contamination will be trimmed a further 2 bp from their 3' end, and Read 2 sequences will be trimmed by 2 bp from their 5' end to remove potential methylation-biased bases from the end-repair reaction File was specified to be a non-directional MspI-digested RRBS sample. Sequences starting with either 'CAA' or 'CGA' will have the first 2 bp trimmed off to remove potential methylation-biased bases from the end-repair reaction Running FastQC on the data once trimming has completed Running FastQC with the following extra arguments: '--outdir /gscratch/scrubbed/strigg/analyses/20200306/FASTQC' Output file(s) will be GZIP compressed >>> Now performing adaptive quality trimming with a Phred-score cutoff of: 20 <<< This is cutadapt 2.4 with Python 3.7.3 Command line parameters: -f fastq -e 0.1 -q 20 -a X /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth4_R2_001.fastq.gz WARNING: Option --format is deprecated and ignored because the input file format is always auto-detected Processing reads on 1 core in single-end mode ... 10000000 sequences processed 20000000 sequences processed 30000000 sequences processed 40000000 sequences processed 50000000 sequences processed 60000000 sequences processed 70000000 sequences processed 80000000 sequences processed 90000000 sequences processed 100000000 sequences processed 110000000 sequences processed 120000000 sequences processed 130000000 sequences processed 140000000 sequences processed 150000000 sequences processed Finished in 1410.41 s (9 us/read; 6.41 M reads/minute). === Summary === Total reads processed: 150,624,528 Reads with adapters: 0 (0.0%) Reads written (passing filters): 150,624,528 (100.0%) Total basepairs processed: 22,593,679,200 bp Quality-trimmed: 196,540,406 bp (0.9%) Total written (filtered): 22,397,138,794 bp (99.1%) === Adapter 1 === Sequence: X; Type: regular 3'; Length: 1; Trimmed: 0 times. >>> Quality trimming completed <<< 150624528 sequences processed in total Writing final adapter and quality trimmed output to Meth4_R2_001_trimmed.fq.gz >>> Now performing adapter trimming for the adapter sequence: 'AGATCGGAAGAGC' from file Meth4_R2_001.fastq.gz_qual_trimmed.fastq <<< 10000000 sequences processed 20000000 sequences processed 30000000 sequences processed 40000000 sequences processed 50000000 sequences processed 60000000 sequences processed 70000000 sequences processed 80000000 sequences processed 90000000 sequences processed 100000000 sequences processed 110000000 sequences processed 120000000 sequences processed 130000000 sequences processed 140000000 sequences processed 150000000 sequences processed This is cutadapt 2.4 with Python 3.7.3 Command line parameters: -f fastq -e 0.1 -O 1 -a AGATCGGAAGAGC /gscratch/scrubbed/strigg/analyses/20200306/Meth4_R2_001.fastq.gz_qual_trimmed.fastq WARNING: Option --format is deprecated and ignored because the input file format is always auto-detected Processing reads on 1 core in single-end mode ... Finished in 3650.38 s (24 us/read; 2.48 M reads/minute). === Summary === Total reads processed: 150,624,528 Reads with adapters: 120,971,047 (80.3%) Reads written (passing filters): 150,624,528 (100.0%) Total basepairs processed: 22,397,138,794 bp Total written (filtered): 15,763,645,799 bp (70.4%) === Adapter 1 === Sequence: AGATCGGAAGAGC; Type: regular 3'; Length: 13; Trimmed: 120971047 times. No. of allowed errors: 0-9 bp: 0; 10-13 bp: 1 Bases preceding removed adapters: A: 46.9% C: 2.4% G: 45.1% T: 5.6% none/other: 0.0% Overview of removed sequences length count expect max.err error counts 1 17704778 37656132.0 0 17704778 2 1751103 9414033.0 0 1751103 3 1263294 2353508.2 0 1263294 4 4365421 588377.1 0 4365421 5 508068 147094.3 0 508068 6 402402 36773.6 0 402402 7 410156 9193.4 0 410156 8 410374 2298.3 0 410374 9 353798 574.6 0 350726 3072 10 539760 143.6 1 503994 35766 11 477668 35.9 1 436982 40686 12 532131 9.0 1 487671 44460 13 486822 2.2 1 436506 50316 14 539545 2.2 1 484419 55126 15 639200 2.2 1 577264 61936 16 761846 2.2 1 679999 81847 17 495288 2.2 1 446356 48932 18 507725 2.2 1 461048 46677 19 565907 2.2 1 512127 53780 20 916493 2.2 1 830102 86391 21 526657 2.2 1 474040 52617 22 738304 2.2 1 672767 65537 23 504904 2.2 1 458802 46102 24 563879 2.2 1 510652 53227 25 1056223 2.2 1 961389 94834 26 497243 2.2 1 448937 48306 27 788720 2.2 1 700456 88264 28 1960766 2.2 1 1792799 167967 29 686529 2.2 1 621548 64981 30 617053 2.2 1 563016 54037 31 693488 2.2 1 629748 63740 32 875248 2.2 1 798864 76384 33 612393 2.2 1 554151 58242 34 716742 2.2 1 648612 68130 35 627810 2.2 1 566068 61742 36 583703 2.2 1 530958 52745 37 648382 2.2 1 589829 58553 38 1010711 2.2 1 920975 89736 39 752123 2.2 1 683221 68902 40 845533 2.2 1 772514 73019 41 1180412 2.2 1 1073473 106939 42 682186 2.2 1 622808 59378 43 912325 2.2 1 831696 80629 44 924149 2.2 1 844398 79751 45 912963 2.2 1 829264 83699 46 823104 2.2 1 747224 75880 47 847707 2.2 1 777784 69923 48 475945 2.2 1 432201 43744 49 675107 2.2 1 613943 61164 50 553917 2.2 1 505794 48123 51 507775 2.2 1 460521 47254 52 892849 2.2 1 814507 78342 53 649067 2.2 1 591115 57952 54 935579 2.2 1 856816 78763 55 601790 2.2 1 545633 56157 56 820426 2.2 1 743091 77335 57 1857360 2.2 1 1700456 156904 58 621978 2.2 1 560827 61151 59 708920 2.2 1 641179 67741 60 1741444 2.2 1 1594528 146916 61 967071 2.2 1 881412 85659 62 563156 2.2 1 497672 65484 63 3045739 2.2 1 2793845 251894 64 811007 2.2 1 736692 74315 65 342532 2.2 1 310142 32390 66 439834 2.2 1 392775 47059 67 1275204 2.2 1 1165564 109640 68 579467 2.2 1 526231 53236 69 548629 2.2 1 497301 51328 70 787077 2.2 1 717268 69809 71 582894 2.2 1 530001 52893 72 397882 2.2 1 360012 37870 73 556894 2.2 1 506122 50772 74 464822 2.2 1 420788 44034 75 551862 2.2 1 499878 51984 76 625742 2.2 1 566750 58992 77 442317 2.2 1 400503 41814 78 500512 2.2 1 453768 46744 79 728733 2.2 1 661483 67250 80 679047 2.2 1 614607 64440 81 687573 2.2 1 623341 64232 82 908445 2.2 1 821639 86806 83 788613 2.2 1 711731 76882 84 893243 2.2 1 806888 86355 85 964196 2.2 1 867348 96848 86 784286 2.2 1 702026 82260 87 809792 2.2 1 730495 79297 88 1004633 2.2 1 907807 96826 89 912131 2.2 1 823742 88389 90 1051789 2.2 1 951255 100534 91 1056348 2.2 1 953368 102980 92 961691 2.2 1 867792 93899 93 963799 2.2 1 870026 93773 94 1101267 2.2 1 990532 110735 95 785924 2.2 1 707576 78348 96 1220856 2.2 1 1100359 120497 97 1090480 2.2 1 981335 109145 98 1066093 2.2 1 960124 105969 99 942920 2.2 1 848644 94276 100 990754 2.2 1 890669 100085 101 756926 2.2 1 679074 77852 102 686249 2.2 1 615696 70553 103 893629 2.2 1 802176 91453 104 761610 2.2 1 682225 79385 105 729367 2.2 1 652853 76514 106 892685 2.2 1 799804 92881 107 653036 2.2 1 584237 68799 108 761204 2.2 1 680292 80912 109 819521 2.2 1 730935 88586 110 726323 2.2 1 647765 78558 111 723001 2.2 1 644213 78788 112 1128978 2.2 1 1005814 123164 113 707572 2.2 1 629023 78549 114 895709 2.2 1 795691 100018 115 1014650 2.2 1 901547 113103 116 660820 2.2 1 586918 73902 117 679832 2.2 1 602661 77171 118 986966 2.2 1 874932 112034 119 1188741 2.2 1 1053713 135028 120 596497 2.2 1 527773 68724 121 649928 2.2 1 575276 74652 122 567927 2.2 1 501179 66748 123 604361 2.2 1 534320 70041 124 683263 2.2 1 603279 79984 125 358064 2.2 1 316245 41819 126 391821 2.2 1 345278 46543 127 528991 2.2 1 466161 62830 128 217432 2.2 1 191228 26204 129 162497 2.2 1 142759 19738 130 145048 2.2 1 127349 17699 131 145578 2.2 1 127871 17707 132 73729 2.2 1 64689 9040 133 28631 2.2 1 25101 3530 134 5200 2.2 1 4565 635 135 1120 2.2 1 966 154 136 1060 2.2 1 904 156 137 74 2.2 1 69 5 138 34 2.2 1 33 1 139 24 2.2 1 17 7 140 19 2.2 1 18 1 141 13 2.2 1 7 6 142 9 2.2 1 5 4 143 1 2.2 1 1 144 2 2.2 1 0 2 145 3 2.2 1 3 146 7 2.2 1 4 3 147 13 2.2 1 9 4 148 193 2.2 1 164 29 149 311 2.2 1 266 45 150 3931 2.2 1 3534 397 Successfully deleted temporary file Meth4_R2_001.fastq.gz_qual_trimmed.fastq RUN STATISTICS FOR INPUT FILE: /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth4_R2_001.fastq.gz ============================================= 150624528 sequences processed in total Sequences were truncated to a varying degree because of deteriorating qualities (Phred score quality cutoff: 20): 17381248 (11.5%) The length threshold of paired-end sequences gets evaluated later on (in the validation step) RRBS reads trimmed by additional 2 bp when adapter contamination was detected: 0 (0.0%) RRBS reads trimmed by 2 bp at the start when read started with CAA (1609237) or CGA (67354262) in total: 68963499 (45.8%) Validate paired-end files Meth4_R1_001_trimmed.fq.gz and Meth4_R2_001_trimmed.fq.gz file_1: Meth4_R1_001_trimmed.fq.gz, file_2: Meth4_R2_001_trimmed.fq.gz >>>>> Now validing the length of the 2 paired-end infiles: Meth4_R1_001_trimmed.fq.gz and Meth4_R2_001_trimmed.fq.gz <<<<< Writing validated paired-end read 1 reads to Meth4_R1_001_val_1.fq.gz Writing validated paired-end read 2 reads to Meth4_R2_001_val_2.fq.gz Total number of sequences analysed: 150624528 Number of sequence pairs removed because at least one read was shorter than the length cutoff (20 bp): 795739 (0.53%) >>> Now running FastQC on the validated data Meth4_R1_001_val_1.fq.gz<<< Can't exec "fastqc": No such file or directory at /gscratch/srlab/programs/TrimGalore-0.4.5/trim_galore line 821, line 1722647420. >>> Now running FastQC on the validated data Meth4_R2_001_val_2.fq.gz<<< Can't exec "fastqc": No such file or directory at /gscratch/srlab/programs/TrimGalore-0.4.5/trim_galore line 831, line 1722647420. Deleting both intermediate output files Meth4_R1_001_trimmed.fq.gz and Meth4_R2_001_trimmed.fq.gz ==================================================================================================== Writing report to '/gscratch/scrubbed/strigg/analyses/20200306/Meth5_R1_001.fastq.gz_trimming_report.txt' SUMMARISING RUN PARAMETERS ========================== Input filename: /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth5_R1_001.fastq.gz Trimming mode: paired-end Trim Galore version: 0.4.4_dev Cutadapt version: 2.4 Quality Phred score cutoff: 20 Quality encoding type selected: ASCII+33 Adapter sequence: 'AGATCGGAAGAGC' (Illumina TruSeq, Sanger iPCR; auto-detected) Maximum trimming error rate: 0.1 (default) Minimum required adapter overlap (stringency): 1 bp Minimum required sequence length for both reads before a sequence pair gets removed: 20 bp File was specified to be an MspI-digested RRBS sample. Read 1 sequences with adapter contamination will be trimmed a further 2 bp from their 3' end, and Read 2 sequences will be trimmed by 2 bp from their 5' end to remove potential methylation-biased bases from the end-repair reaction File was specified to be a non-directional MspI-digested RRBS sample. Sequences starting with either 'CAA' or 'CGA' will have the first 2 bp trimmed off to remove potential methylation-biased bases from the end-repair reaction Running FastQC on the data once trimming has completed Running FastQC with the following extra arguments: '--outdir /gscratch/scrubbed/strigg/analyses/20200306/FASTQC' Output file(s) will be GZIP compressed >>> Now performing adaptive quality trimming with a Phred-score cutoff of: 20 <<< This is cutadapt 2.4 with Python 3.7.3 Command line parameters: -f fastq -e 0.1 -q 20 -a X /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth5_R1_001.fastq.gz WARNING: Option --format is deprecated and ignored because the input file format is always auto-detected Processing reads on 1 core in single-end mode ... 10000000 sequences processed 20000000 sequences processed 30000000 sequences processed 40000000 sequences processed 50000000 sequences processed 60000000 sequences processed 70000000 sequences processed 80000000 sequences processed 90000000 sequences processed 100000000 sequences processed 110000000 sequences processed 120000000 sequences processed 130000000 sequences processed 140000000 sequences processed 150000000 sequences processed 160000000 sequences processed 170000000 sequences processed 180000000 sequences processed 190000000 sequences processed 200000000 sequences processed 210000000 sequences processed 220000000 sequences processed 230000000 sequences processed 240000000 sequences processed 250000000 sequences processed 260000000 sequences processed 270000000 sequences processed 280000000 sequences processed Finished in 2589.98 s (9 us/read; 6.62 M reads/minute). === Summary === Total reads processed: 285,607,307 Reads with adapters: 0 (0.0%) Reads written (passing filters): 285,607,307 (100.0%) Total basepairs processed: 42,841,096,050 bp Quality-trimmed: 42,177,750 bp (0.1%) Total written (filtered): 42,798,918,300 bp (99.9%) === Adapter 1 === Sequence: X; Type: regular 3'; Length: 1; Trimmed: 0 times. >>> Quality trimming completed <<< 285607307 sequences processed in total Writing final adapter and quality trimmed output to Meth5_R1_001_trimmed.fq.gz >>> Now performing adapter trimming for the adapter sequence: 'AGATCGGAAGAGC' from file Meth5_R1_001.fastq.gz_qual_trimmed.fastq <<< 10000000 sequences processed 20000000 sequences processed 30000000 sequences processed 40000000 sequences processed 50000000 sequences processed 60000000 sequences processed 70000000 sequences processed 80000000 sequences processed 90000000 sequences processed 100000000 sequences processed 110000000 sequences processed 120000000 sequences processed 130000000 sequences processed 140000000 sequences processed 150000000 sequences processed 160000000 sequences processed 170000000 sequences processed 180000000 sequences processed 190000000 sequences processed 200000000 sequences processed 210000000 sequences processed 220000000 sequences processed 230000000 sequences processed 240000000 sequences processed 250000000 sequences processed 260000000 sequences processed 270000000 sequences processed 280000000 sequences processed This is cutadapt 2.4 with Python 3.7.3 Command line parameters: -f fastq -e 0.1 -O 1 -a AGATCGGAAGAGC /gscratch/scrubbed/strigg/analyses/20200306/Meth5_R1_001.fastq.gz_qual_trimmed.fastq WARNING: Option --format is deprecated and ignored because the input file format is always auto-detected Processing reads on 1 core in single-end mode ... Finished in 6820.87 s (24 us/read; 2.51 M reads/minute). === Summary === Total reads processed: 285,607,307 Reads with adapters: 229,365,416 (80.3%) Reads written (passing filters): 285,607,307 (100.0%) Total basepairs processed: 42,798,918,300 bp Total written (filtered): 30,082,664,292 bp (70.3%) === Adapter 1 === Sequence: AGATCGGAAGAGC; Type: regular 3'; Length: 13; Trimmed: 229365416 times. No. of allowed errors: 0-9 bp: 0; 10-13 bp: 1 Bases preceding removed adapters: A: 48.5% C: 2.8% G: 43.1% T: 5.7% none/other: 0.0% Overview of removed sequences length count expect max.err error counts 1 37615404 71401826.8 0 37615404 2 3823272 17850456.7 0 3823272 3 1929705 4462614.2 0 1929705 4 8216086 1115653.5 0 8216086 5 874551 278913.4 0 874551 6 729732 69728.3 0 729732 7 719271 17432.1 0 719271 8 636616 4358.0 0 636616 9 610015 1089.5 0 601088 8927 10 897943 272.4 1 826202 71741 11 835081 68.1 1 758841 76240 12 732250 17.0 1 664625 67625 13 855682 4.3 1 768432 87250 14 934838 4.3 1 833256 101582 15 1237634 4.3 1 1110781 126853 16 1385243 4.3 1 1230937 154306 17 877314 4.3 1 779091 98223 18 908164 4.3 1 816927 91237 19 831362 4.3 1 745063 86299 20 1555687 4.3 1 1398148 157539 21 2741198 4.3 1 2465529 275669 22 1820876 4.3 1 1637482 183394 23 899390 4.3 1 803614 95776 24 918471 4.3 1 814304 104167 25 1671292 4.3 1 1502852 168440 26 867775 4.3 1 775720 92055 27 1324017 4.3 1 1180731 143286 28 4561981 4.3 1 4104006 457975 29 1146671 4.3 1 1017804 128867 30 1027972 4.3 1 924415 103557 31 1085728 4.3 1 972859 112869 32 1611448 4.3 1 1448791 162657 33 886562 4.3 1 789986 96576 34 1119780 4.3 1 1003394 116386 35 1031703 4.3 1 919280 112423 36 943899 4.3 1 846420 97479 37 1021879 4.3 1 914638 107241 38 1703806 4.3 1 1531207 172599 39 1123930 4.3 1 1003160 120770 40 1482557 4.3 1 1327082 155475 41 1692413 4.3 1 1520244 172169 42 1153325 4.3 1 1033386 119939 43 1672983 4.3 1 1496297 176686 44 1714346 4.3 1 1528267 186079 45 1935829 4.3 1 1747015 188814 46 768909 4.3 1 685312 83597 47 896871 4.3 1 802485 94386 48 1053398 4.3 1 944221 109177 49 1302932 4.3 1 1168808 134124 50 885825 4.3 1 788797 97028 51 1010152 4.3 1 899387 110765 52 1404553 4.3 1 1260893 143660 53 930746 4.3 1 831140 99606 54 1129444 4.3 1 1007089 122355 55 1433735 4.3 1 1282800 150935 56 1194201 4.3 1 1067503 126698 57 1324099 4.3 1 1183222 140877 58 2002838 4.3 1 1793889 208949 59 1371764 4.3 1 1224247 147517 60 2124403 4.3 1 1907764 216639 61 2020633 4.3 1 1811014 209619 62 1081769 4.3 1 965189 116580 63 1231632 4.3 1 1101342 130290 64 1763384 4.3 1 1582772 180612 65 1092017 4.3 1 973871 118146 66 1470910 4.3 1 1313554 157356 67 1593438 4.3 1 1422138 171300 68 1348060 4.3 1 1201756 146304 69 1376068 4.3 1 1200593 175475 70 3341550 4.3 1 3030992 310558 71 249878 4.3 1 218574 31304 72 104243 4.3 1 85095 19148 73 534650 4.3 1 469177 65473 74 1018477 4.3 1 907342 111135 75 1252802 4.3 1 1117960 134842 76 2034078 4.3 1 1819042 215036 77 1298399 4.3 1 1156782 141617 78 1009250 4.3 1 898957 110293 79 1326076 4.3 1 1182564 143512 80 1241862 4.3 1 1105651 136211 81 1246431 4.3 1 1107915 138516 82 1860293 4.3 1 1632119 228174 83 1544206 4.3 1 1361468 182738 84 1473965 4.3 1 1309253 164712 85 1514414 4.3 1 1347322 167092 86 1131141 4.3 1 1004718 126423 87 1457872 4.3 1 1296908 160964 88 1729833 4.3 1 1539759 190074 89 1706854 4.3 1 1517143 189711 90 2084838 4.3 1 1854550 230288 91 1887471 4.3 1 1675495 211976 92 2101576 4.3 1 1863429 238147 93 2084969 4.3 1 1849308 235661 94 2336410 4.3 1 2066675 269735 95 1590695 4.3 1 1407361 183334 96 2684007 4.3 1 2384074 299933 97 2256733 4.3 1 1998241 258492 98 2219956 4.3 1 1964200 255756 99 1737602 4.3 1 1536908 200694 100 1881866 4.3 1 1664340 217526 101 1686004 4.3 1 1489669 196335 102 1364518 4.3 1 1204809 159709 103 2414501 4.3 1 2136096 278405 104 1521021 4.3 1 1339449 181572 105 1471000 4.3 1 1296875 174125 106 1849669 4.3 1 1632079 217590 107 1356605 4.3 1 1193999 162606 108 2158876 4.3 1 1902708 256168 109 1684151 4.3 1 1477712 206439 110 1457010 4.3 1 1280244 176766 111 1398943 4.3 1 1228355 170588 112 2115042 4.3 1 1858382 256660 113 1446018 4.3 1 1267467 178551 114 2191531 4.3 1 1920127 271404 115 2230740 4.3 1 1957130 273610 116 1344965 4.3 1 1176649 168316 117 1386741 4.3 1 1214186 172555 118 1947265 4.3 1 1704881 242384 119 2807501 4.3 1 2458055 349446 120 1145668 4.3 1 999495 146173 121 1344777 4.3 1 1174737 170040 122 1151281 4.3 1 1004528 146753 123 1360292 4.3 1 1188619 171673 124 1273651 4.3 1 1112417 161234 125 736748 4.3 1 642212 94536 126 739344 4.3 1 644508 94836 127 1094512 4.3 1 954291 140221 128 424513 4.3 1 369000 55513 129 295771 4.3 1 257526 38245 130 264580 4.3 1 230384 34196 131 367593 4.3 1 319865 47728 132 135721 4.3 1 117681 18040 133 54134 4.3 1 47126 7008 134 10091 4.3 1 8754 1337 135 2902 4.3 1 2533 369 136 7458 4.3 1 6469 989 137 264 4.3 1 206 58 138 56 4.3 1 39 17 139 40 4.3 1 36 4 140 24 4.3 1 16 8 141 18 4.3 1 16 2 142 15 4.3 1 7 8 143 2 4.3 1 2 144 10 4.3 1 3 7 145 3 4.3 1 2 1 146 1 4.3 1 1 147 8 4.3 1 5 3 148 193 4.3 1 164 29 149 207 4.3 1 190 17 150 3579 4.3 1 3140 439 Successfully deleted temporary file Meth5_R1_001.fastq.gz_qual_trimmed.fastq RUN STATISTICS FOR INPUT FILE: /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth5_R1_001.fastq.gz ============================================= 285607307 sequences processed in total Sequences were truncated to a varying degree because of deteriorating qualities (Phred score quality cutoff: 20): 12455645 (4.4%) The length threshold of paired-end sequences gets evaluated later on (in the validation step) RRBS reads trimmed by additional 2 bp when adapter contamination was detected: 108911143 (38.1%) RRBS reads trimmed by 2 bp at the start when read started with CAA (3181836) or CGA (143429518) in total: 146611354 (51.3%) Writing report to '/gscratch/scrubbed/strigg/analyses/20200306/Meth5_R2_001.fastq.gz_trimming_report.txt' SUMMARISING RUN PARAMETERS ========================== Input filename: /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth5_R2_001.fastq.gz Trimming mode: paired-end Trim Galore version: 0.4.4_dev Cutadapt version: 2.4 Quality Phred score cutoff: 20 Quality encoding type selected: ASCII+33 Adapter sequence: 'AGATCGGAAGAGC' (Illumina TruSeq, Sanger iPCR; auto-detected) Maximum trimming error rate: 0.1 (default) Minimum required adapter overlap (stringency): 1 bp Minimum required sequence length for both reads before a sequence pair gets removed: 20 bp File was specified to be an MspI-digested RRBS sample. Read 1 sequences with adapter contamination will be trimmed a further 2 bp from their 3' end, and Read 2 sequences will be trimmed by 2 bp from their 5' end to remove potential methylation-biased bases from the end-repair reaction File was specified to be a non-directional MspI-digested RRBS sample. Sequences starting with either 'CAA' or 'CGA' will have the first 2 bp trimmed off to remove potential methylation-biased bases from the end-repair reaction Running FastQC on the data once trimming has completed Running FastQC with the following extra arguments: '--outdir /gscratch/scrubbed/strigg/analyses/20200306/FASTQC' Output file(s) will be GZIP compressed >>> Now performing adaptive quality trimming with a Phred-score cutoff of: 20 <<< This is cutadapt 2.4 with Python 3.7.3 Command line parameters: -f fastq -e 0.1 -q 20 -a X /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth5_R2_001.fastq.gz WARNING: Option --format is deprecated and ignored because the input file format is always auto-detected Processing reads on 1 core in single-end mode ... 10000000 sequences processed 20000000 sequences processed 30000000 sequences processed 40000000 sequences processed 50000000 sequences processed 60000000 sequences processed 70000000 sequences processed 80000000 sequences processed 90000000 sequences processed 100000000 sequences processed 110000000 sequences processed 120000000 sequences processed 130000000 sequences processed 140000000 sequences processed 150000000 sequences processed 160000000 sequences processed 170000000 sequences processed 180000000 sequences processed 190000000 sequences processed 200000000 sequences processed 210000000 sequences processed 220000000 sequences processed 230000000 sequences processed 240000000 sequences processed 250000000 sequences processed 260000000 sequences processed 270000000 sequences processed 280000000 sequences processed Finished in 2596.47 s (9 us/read; 6.60 M reads/minute). === Summary === Total reads processed: 285,607,307 Reads with adapters: 0 (0.0%) Reads written (passing filters): 285,607,307 (100.0%) Total basepairs processed: 42,841,096,050 bp Quality-trimmed: 324,449,136 bp (0.8%) Total written (filtered): 42,516,646,914 bp (99.2%) === Adapter 1 === Sequence: X; Type: regular 3'; Length: 1; Trimmed: 0 times. >>> Quality trimming completed <<< 285607307 sequences processed in total Writing final adapter and quality trimmed output to Meth5_R2_001_trimmed.fq.gz >>> Now performing adapter trimming for the adapter sequence: 'AGATCGGAAGAGC' from file Meth5_R2_001.fastq.gz_qual_trimmed.fastq <<< 10000000 sequences processed 20000000 sequences processed 30000000 sequences processed 40000000 sequences processed 50000000 sequences processed 60000000 sequences processed 70000000 sequences processed 80000000 sequences processed 90000000 sequences processed 100000000 sequences processed 110000000 sequences processed 120000000 sequences processed 130000000 sequences processed 140000000 sequences processed 150000000 sequences processed 160000000 sequences processed 170000000 sequences processed 180000000 sequences processed 190000000 sequences processed 200000000 sequences processed 210000000 sequences processed 220000000 sequences processed 230000000 sequences processed 240000000 sequences processed 250000000 sequences processed 260000000 sequences processed 270000000 sequences processed 280000000 sequences processed This is cutadapt 2.4 with Python 3.7.3 Command line parameters: -f fastq -e 0.1 -O 1 -a AGATCGGAAGAGC /gscratch/scrubbed/strigg/analyses/20200306/Meth5_R2_001.fastq.gz_qual_trimmed.fastq WARNING: Option --format is deprecated and ignored because the input file format is always auto-detected Processing reads on 1 core in single-end mode ... Finished in 7056.11 s (25 us/read; 2.43 M reads/minute). === Summary === Total reads processed: 285,607,307 Reads with adapters: 225,585,200 (79.0%) Reads written (passing filters): 285,607,307 (100.0%) Total basepairs processed: 42,516,646,914 bp Total written (filtered): 30,155,326,446 bp (70.9%) === Adapter 1 === Sequence: AGATCGGAAGAGC; Type: regular 3'; Length: 13; Trimmed: 225585200 times. No. of allowed errors: 0-9 bp: 0; 10-13 bp: 1 Bases preceding removed adapters: A: 45.7% C: 2.4% G: 45.8% T: 6.1% none/other: 0.0% Overview of removed sequences length count expect max.err error counts 1 37550958 71401826.8 0 37550958 2 3999282 17850456.7 0 3999282 3 2189402 4462614.2 0 2189402 4 8119262 1115653.5 0 8119262 5 873671 278913.4 0 873671 6 709476 69728.3 0 709476 7 656407 17432.1 0 656407 8 637060 4358.0 0 637060 9 590881 1089.5 0 586446 4435 10 862637 272.4 1 804753 57884 11 824942 68.1 1 751663 73279 12 800616 17.0 1 733556 67060 13 776504 4.3 1 694301 82203 14 914479 4.3 1 818113 96366 15 1168832 4.3 1 1050977 117855 16 1375438 4.3 1 1219584 155854 17 821395 4.3 1 736617 84778 18 836446 4.3 1 756621 79825 19 883523 4.3 1 796330 87193 20 1661465 4.3 1 1500619 160846 21 2530193 4.3 1 2289437 240756 22 1790993 4.3 1 1630887 160106 23 816304 4.3 1 739867 76437 24 910457 4.3 1 821999 88458 25 1742369 4.3 1 1582488 159881 26 775650 4.3 1 695991 79659 27 1392418 4.3 1 1223561 168857 28 4306874 4.3 1 3932359 374515 29 1126540 4.3 1 1016211 110329 30 998742 4.3 1 909760 88982 31 1089490 4.3 1 985790 103700 32 1541836 4.3 1 1407132 134704 33 886377 4.3 1 802233 84144 34 1142805 4.3 1 1028498 114307 35 1013539 4.3 1 920862 92677 36 873015 4.3 1 797615 75400 37 1020651 4.3 1 927408 93243 38 1610806 4.3 1 1464903 145903 39 1178072 4.3 1 1067170 110902 40 1452147 4.3 1 1315194 136953 41 1658733 4.3 1 1514018 144715 42 1128479 4.3 1 1028781 99698 43 1542277 4.3 1 1405387 136890 44 1725407 4.3 1 1575400 150007 45 1474146 4.3 1 1339373 134773 46 1343257 4.3 1 1217202 126055 47 1088504 4.3 1 998241 90263 48 902566 4.3 1 820574 81992 49 1156175 4.3 1 1052588 103587 50 904523 4.3 1 824983 79540 51 844725 4.3 1 767457 77268 52 1414166 4.3 1 1290004 124162 53 932731 4.3 1 850233 82498 54 1439177 4.3 1 1317857 121320 55 962029 4.3 1 873407 88622 56 1249213 4.3 1 1129428 119785 57 2495058 4.3 1 2280619 214439 58 1082649 4.3 1 978738 103911 59 1196960 4.3 1 1086582 110378 60 2655580 4.3 1 2434981 220599 61 1878629 4.3 1 1715325 163304 62 955590 4.3 1 850632 104958 63 4125480 4.3 1 3793647 331833 64 1156038 4.3 1 1052382 103656 65 633976 4.3 1 576567 57409 66 827307 4.3 1 747215 80092 67 2040729 4.3 1 1870475 170254 68 1138467 4.3 1 1037794 100673 69 953600 4.3 1 868744 84856 70 1330080 4.3 1 1216756 113324 71 1065370 4.3 1 973142 92228 72 910235 4.3 1 828615 81620 73 1301346 4.3 1 1187735 113611 74 1212881 4.3 1 1103622 109259 75 1426499 4.3 1 1299462 127037 76 1442267 4.3 1 1318503 123764 77 616653 4.3 1 556610 60043 78 796348 4.3 1 724140 72208 79 1173497 4.3 1 1069659 103838 80 1147825 4.3 1 1043683 104142 81 1190242 4.3 1 1078511 111731 82 1766597 4.3 1 1579901 186696 83 1596348 4.3 1 1436533 159815 84 1469557 4.3 1 1333989 135568 85 1611221 4.3 1 1464960 146261 86 1178902 4.3 1 1070210 108692 87 1436923 4.3 1 1305971 130952 88 1674824 4.3 1 1523974 150850 89 1637483 4.3 1 1488217 149266 90 1988478 4.3 1 1809837 178641 91 1795727 4.3 1 1630997 164730 92 1986686 4.3 1 1803966 182720 93 1981772 4.3 1 1800991 180781 94 2188078 4.3 1 1982590 205488 95 1515832 4.3 1 1374456 141376 96 2527317 4.3 1 2299131 228186 97 2154423 4.3 1 1958347 196076 98 2140234 4.3 1 1945322 194912 99 1721542 4.3 1 1563192 158350 100 1854808 4.3 1 1684175 170633 101 1626385 4.3 1 1475357 151028 102 1311137 4.3 1 1188590 122547 103 2263843 4.3 1 2054809 209034 104 1424019 4.3 1 1289898 134121 105 1393582 4.3 1 1262542 131040 106 1734960 4.3 1 1571195 163765 107 1291706 4.3 1 1169023 122683 108 2035934 4.3 1 1842451 193483 109 1576095 4.3 1 1422638 153457 110 1373934 4.3 1 1241688 132246 111 1325579 4.3 1 1196575 129004 112 2004815 4.3 1 1811963 192852 113 1416896 4.3 1 1279101 137795 114 2124004 4.3 1 1916759 207245 115 2170659 4.3 1 1958563 212096 116 1324252 4.3 1 1193624 130628 117 1343993 4.3 1 1211034 132959 118 1904040 4.3 1 1716333 187707 119 2690112 4.3 1 2423928 266184 120 1115731 4.3 1 1003786 111945 121 1300134 4.3 1 1169857 130277 122 1101233 4.3 1 990008 111225 123 1307813 4.3 1 1176141 131672 124 1219643 4.3 1 1097092 122551 125 720809 4.3 1 648163 72646 126 725237 4.3 1 651779 73458 127 1052435 4.3 1 944640 107795 128 411841 4.3 1 369359 42482 129 284093 4.3 1 255062 29031 130 256111 4.3 1 229972 26139 131 350724 4.3 1 314098 36626 132 129417 4.3 1 116452 12965 133 51882 4.3 1 46490 5392 134 9690 4.3 1 8650 1040 135 2831 4.3 1 2512 319 136 7300 4.3 1 6487 813 137 246 4.3 1 222 24 138 52 4.3 1 47 5 139 39 4.3 1 34 5 140 20 4.3 1 19 1 141 19 4.3 1 14 5 142 13 4.3 1 9 4 143 9 4.3 1 3 6 144 13 4.3 1 5 8 145 3 4.3 1 2 1 146 2 4.3 1 0 2 147 12 4.3 1 9 3 148 189 4.3 1 163 26 149 221 4.3 1 197 24 150 3373 4.3 1 3088 285 Successfully deleted temporary file Meth5_R2_001.fastq.gz_qual_trimmed.fastq RUN STATISTICS FOR INPUT FILE: /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth5_R2_001.fastq.gz ============================================= 285607307 sequences processed in total Sequences were truncated to a varying degree because of deteriorating qualities (Phred score quality cutoff: 20): 29966360 (10.5%) The length threshold of paired-end sequences gets evaluated later on (in the validation step) RRBS reads trimmed by additional 2 bp when adapter contamination was detected: 0 (0.0%) RRBS reads trimmed by 2 bp at the start when read started with CAA (2851016) or CGA (127749902) in total: 130600918 (45.7%) Validate paired-end files Meth5_R1_001_trimmed.fq.gz and Meth5_R2_001_trimmed.fq.gz file_1: Meth5_R1_001_trimmed.fq.gz, file_2: Meth5_R2_001_trimmed.fq.gz >>>>> Now validing the length of the 2 paired-end infiles: Meth5_R1_001_trimmed.fq.gz and Meth5_R2_001_trimmed.fq.gz <<<<< Writing validated paired-end read 1 reads to Meth5_R1_001_val_1.fq.gz Writing validated paired-end read 2 reads to Meth5_R2_001_val_2.fq.gz Total number of sequences analysed: 285607307 Number of sequence pairs removed because at least one read was shorter than the length cutoff (20 bp): 1490937 (0.52%) >>> Now running FastQC on the validated data Meth5_R1_001_val_1.fq.gz<<< Can't exec "fastqc": No such file or directory at /gscratch/srlab/programs/TrimGalore-0.4.5/trim_galore line 821, line 2865076648. >>> Now running FastQC on the validated data Meth5_R2_001_val_2.fq.gz<<< Can't exec "fastqc": No such file or directory at /gscratch/srlab/programs/TrimGalore-0.4.5/trim_galore line 831, line 2865076648. Deleting both intermediate output files Meth5_R1_001_trimmed.fq.gz and Meth5_R2_001_trimmed.fq.gz ==================================================================================================== Writing report to '/gscratch/scrubbed/strigg/analyses/20200306/Meth6_R1_001.fastq.gz_trimming_report.txt' SUMMARISING RUN PARAMETERS ========================== Input filename: /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth6_R1_001.fastq.gz Trimming mode: paired-end Trim Galore version: 0.4.4_dev Cutadapt version: 2.4 Quality Phred score cutoff: 20 Quality encoding type selected: ASCII+33 Adapter sequence: 'AGATCGGAAGAGC' (Illumina TruSeq, Sanger iPCR; auto-detected) Maximum trimming error rate: 0.1 (default) Minimum required adapter overlap (stringency): 1 bp Minimum required sequence length for both reads before a sequence pair gets removed: 20 bp File was specified to be an MspI-digested RRBS sample. Read 1 sequences with adapter contamination will be trimmed a further 2 bp from their 3' end, and Read 2 sequences will be trimmed by 2 bp from their 5' end to remove potential methylation-biased bases from the end-repair reaction File was specified to be a non-directional MspI-digested RRBS sample. Sequences starting with either 'CAA' or 'CGA' will have the first 2 bp trimmed off to remove potential methylation-biased bases from the end-repair reaction Running FastQC on the data once trimming has completed Running FastQC with the following extra arguments: '--outdir /gscratch/scrubbed/strigg/analyses/20200306/FASTQC' Output file(s) will be GZIP compressed >>> Now performing adaptive quality trimming with a Phred-score cutoff of: 20 <<< This is cutadapt 2.4 with Python 3.7.3 Command line parameters: -f fastq -e 0.1 -q 20 -a X /gscratch/scrubbed/sr320/froger-raw/00_fastq/Meth6_R1_001.fastq.gz WARNING: Option --format is deprecated and ignored because the input file format is always auto-detected Processing reads on 1 core in single-end mode ... 10000000 sequences processed 20000000 sequences processed 30000000 sequences processed 40000000 sequences processed 50000000 sequences processed 60000000 sequences processed 70000000 sequences processed 80000000 sequences processed 90000000 sequences processed 100000000 sequences processed 110000000 sequences processed 120000000 sequences processed 130000000 sequences processed 140000000 sequences processed Finished in 1344.99 s (10 us/read; 6.30 M reads/minute). === Summary === Total reads processed: 141,156,454 Reads with adapters: 0 (0.0%) Reads written (passing filters): 141,156,454 (100.0%) Total basepairs processed: 21,173,468,100 bp Quality-trimmed: 20,236,937 bp (0.1%) Total written (filtered): 21,153,231,163 bp (99.9%) === Adapter 1 === Sequence: X; Type: regular 3'; Length: 1; Trimmed: 0 times. >>> Quality trimming completed <<< 141156454 sequences processed in total Writing final adapter and quality trimmed output to Meth6_R1_001_trimmed.fq.gz >>> Now performing adapter trimming for the adapter sequence: 'AGATCGGAAGAGC' from file Meth6_R1_001.fastq.gz_qual_trimmed.fastq <<< 10000000 sequences processed 20000000 sequences processed 30000000 sequences processed slurmstepd: error: *** JOB 2025665 ON n2193 CANCELLED AT 2020-03-07T09:30:10 ***