========================================================================================== Amplicon Sequence Data Preparation Workflow for HPC Euler ------------------------------------------------------------------------------------------ Genetic Diversity Centre (GDC), D-USYS, ETH Zurich https://gdc.ethz.ch ========================================================================================== Project : p700 Run : run200710 Platfom : Illumina MiSeq Provider : GDC Data Typ : PE300 Target : 16S User(s) : Hassan Melhem (hassan.melhem@unibas.ch) ------------------------------------------------------------------------------------------ Project Info ------------------------------------------------------------------------------------------ Organism: Mice WT and Nockout / male and female N(samples) = 46 N(WT) = 20 (10 male / 10 female) N(KO) = 22 (11 male / 11 female) N(NTC) = 1 male / 1 female N(POS) = 1 male / 1 female ------------------------------------------------------------------------------------------ MiSeq Run Information ------------------------------------------------------------------------------------------ Cluster density: 1129 k/mm2 (Optimal 500–850 k/mm2) Reads Total: 27.39 M (goal 25 M) Reads PF: 25.68 M PhiX conc: 5.94 % (loaded 10%) %>=Q30: Total 82.26% (should be at least 70%) ------------------------------------------------------------------------------------------ Primer Info ------------------------------------------------------------------------------------------ 515F: 5'-GTGCCAGCMGCCGCGGTAA-3' 806R: 5-'GGACTACHVGGGTWTCTAAT-3' ========================================================================================== Step [A] - Data Quality Control ------------------------------------------------------------------------------------------ Application(s): usearch v11.0.667_i86linux64 FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) MultiQC (https://multiqc.info) ========================================================================================== Step [B] - Trimming Read End and Merge Read Pairs ------------------------------------------------------------------------------------------ Workflow Overview: ◇ B1 - Check-Point: Verify Sample Names ◇ B2 - Check-Point: Sample File Resolution ◇ B3 - GZ-2-FQ and Renaming Files ◇ B4 - Remove PhiX ................................................................................ Application: usearch v11.0.667_i86linux64 Command: filter_phix ................................................................................ ◇ B5 - Remove Low Complexity Sequences ................................................................................ Application: usearch v11.0.667_i86linux64 Command: filter_lowc Threshold: 25 ................................................................................ ◇ B6a - Trim Read End ................................................................................ Application: usearch v11.0.667_i86linux64 Command: fastx_truncate Trim R1/R2: 40 / 40 ................................................................................ ◇ B6b - Sync Read-Pairs Application: usearch v11.0.667_i86linux64 Command: fastx_syncpairs ................................................................................ ◇ B6c - Merge Read Pairs (Bayesian PE Read Merger) ................................................................................ Application: usearch v11.0.667_i86linux64 Command: fastq_mergepairs Min Overlap: 20 Min %Identity: 60 Min Merged Length: 100 Min Merged Quality: 0 -------------------------------------------------------------------------------- START_Workflow: 14:56:54 16/07/2020 -------------------------------------------------------------------------------- Start_B1_UniqueSampleNames_Check: 14:56:54 16/07/2020 ✔︎ PASS - Unique Sample Names (N=46) End_B1_UniqueSampleNames_Check: 14:56:54 16/07/2020 ................................................................................ Start_B2_SampleResolutionCheck: 14:56:54 16/07/2020 ✔︎ PASS - Sample-Name Resolution Check (n=46) End_B2_SmpleResolutionCheck: 14:57:02 16/07/2020 ................................................................................ Start_B3_GZ-2-FQ: 14:57:02 16/07/2020 ➼ N(FilesR1/FilesR2): 46 / 46 End_B3_GZ-2-FQ: 15:01:40 16/07/2020 ................................................................................ Start_B4_RemovePhiX: 15:01:40 16/07/2020 KO01F: N(PhiX_free)= 594200 (-7) KO01M: N(PhiX_free)= 457294 (-7) KO02F: N(PhiX_free)= 540674 (-16) KO02M: N(PhiX_free)= 478684 (-12) KO03F: N(PhiX_free)= 531430 (-13) KO03M: N(PhiX_free)= 518699 (-10) KO04F: N(PhiX_free)= 561186 (-8) KO04M: N(PhiX_free)= 358733 (-9) KO05F: N(PhiX_free)= 485255 (-6) KO05M: N(PhiX_free)= 473490 (-15) KO06F: N(PhiX_free)= 493458 (-9) KO06M: N(PhiX_free)= 538060 (-12) KO07F: N(PhiX_free)= 498253 (-9) KO07M: N(PhiX_free)= 548023 (-13) KO08F: N(PhiX_free)= 639611 (-11) KO08M: N(PhiX_free)= 519852 (-8) KO09F: N(PhiX_free)= 545607 (-9) KO09M: N(PhiX_free)= 481510 (-4) KO10F: N(PhiX_free)= 515815 (-4) KO10M: N(PhiX_free)= 485012 (-8) KO11F: N(PhiX_free)= 708401 (-12) KO11M: N(PhiX_free)= 542796 (-23) NTCF: N(PhiX_free)= 2186 (-1) NTCM: N(PhiX_free)= 25078 (0) POSF: N(PhiX_free)= 390158 (-6) POSM: N(PhiX_free)= 476725 (-7) WT01F: N(PhiX_free)= 550489 (-8) WT01M: N(PhiX_free)= 498882 (-17) WT02F: N(PhiX_free)= 674145 (-8) WT02M: N(PhiX_free)= 453175 (-27) WT03F: N(PhiX_free)= 523866 (-13) WT03M: N(PhiX_free)= 512725 (-9) WT04F: N(PhiX_free)= 436992 (-7) WT04M: N(PhiX_free)= 485572 (-11) WT05F: N(PhiX_free)= 512593 (-11) WT05M: N(PhiX_free)= 525206 (-14) WT06F: N(PhiX_free)= 545072 (-15) WT06M: N(PhiX_free)= 566992 (-8) WT07F: N(PhiX_free)= 505820 (-6) WT07M: N(PhiX_free)= 501495 (-15) WT08F: N(PhiX_free)= 557735 (-15) WT08M: N(PhiX_free)= 568968 (-14) WT09F: N(PhiX_free)= 545258 (-6) WT09M: N(PhiX_free)= 463074 (-4) WT10F: N(PhiX_free)= 620618 (-15) WT10M: N(PhiX_free)= 526190 (-3) End_B4_RemovePhiX: 15:22:08 16/07/2020 ................................................................................ Start_B5_RemoveLowComplexity: 15:22:08 16/07/2020 KO01F: N(NoLowComplexity)= 589156 (-5044) KO01M: N(NoLowComplexity)= 456228 (-1066) KO02F: N(NoLowComplexity)= 536773 (-3901) KO02M: N(NoLowComplexity)= 476925 (-1759) KO03F: N(NoLowComplexity)= 527484 (-3946) KO03M: N(NoLowComplexity)= 517706 (-993) KO04F: N(NoLowComplexity)= 555438 (-5748) KO04M: N(NoLowComplexity)= 358051 (-682) KO05F: N(NoLowComplexity)= 481837 (-3418) KO05M: N(NoLowComplexity)= 471735 (-1755) KO06F: N(NoLowComplexity)= 490670 (-2788) KO06M: N(NoLowComplexity)= 534230 (-3830) KO07F: N(NoLowComplexity)= 495905 (-2348) KO07M: N(NoLowComplexity)= 546609 (-1414) KO08F: N(NoLowComplexity)= 637041 (-2570) KO08M: N(NoLowComplexity)= 518777 (-1075) KO09F: N(NoLowComplexity)= 542644 (-2963) KO09M: N(NoLowComplexity)= 480444 (-1066) KO10F: N(NoLowComplexity)= 513955 (-1860) KO10M: N(NoLowComplexity)= 483838 (-1174) KO11F: N(NoLowComplexity)= 706341 (-2060) KO11M: N(NoLowComplexity)= 540579 (-2217) NTCF: N(NoLowComplexity)= 663 (-1523) NTCM: N(NoLowComplexity)= 24832 (-246) POSF: N(NoLowComplexity)= 388438 (-1720) POSM: N(NoLowComplexity)= 475884 (-841) WT01F: N(NoLowComplexity)= 547419 (-3070) WT01M: N(NoLowComplexity)= 496853 (-2029) WT02F: N(NoLowComplexity)= 669792 (-4353) WT02M: N(NoLowComplexity)= 451219 (-1956) WT03F: N(NoLowComplexity)= 518034 (-5832) WT03M: N(NoLowComplexity)= 511438 (-1287) WT04F: N(NoLowComplexity)= 433337 (-3655) WT04M: N(NoLowComplexity)= 484242 (-1330) WT05F: N(NoLowComplexity)= 507517 (-5076) WT05M: N(NoLowComplexity)= 523434 (-1772) WT06F: N(NoLowComplexity)= 539287 (-5785) WT06M: N(NoLowComplexity)= 565743 (-1249) WT07F: N(NoLowComplexity)= 501081 (-4739) WT07M: N(NoLowComplexity)= 499629 (-1866) WT08F: N(NoLowComplexity)= 554030 (-3705) WT08M: N(NoLowComplexity)= 567454 (-1514) WT09F: N(NoLowComplexity)= 543835 (-1423) WT09M: N(NoLowComplexity)= 462010 (-1064) WT10F: N(NoLowComplexity)= 618957 (-1661) WT10M: N(NoLowComplexity)= 524899 (-1291) End_B5_RemoveLowComplexity: 15:34:26 16/07/2020 ................................................................................ Start_B6_Trim_and_Merge: 15:34:26 16/07/2020 ‣ KO01F - Merging Summary Merging Rate: 533717 / 589079 (90.6%) Median Merged Length: 298 589079 Pairs (589.1k) 533717 Merged (533.7k, 90.60%) 299916 Alignments with zero diffs (50.91%) 51975 Too many diffs (> 10) (8.82%) 245 Fwd too short (< 64) after tail trimming (0.04%) 3 Rev too short (< 64) after tail trimming (0.00%) 3114 No alignment found (0.53%) 17 Alignment too short (< 20) (0.00%) 8 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 243 Staggered pairs (0.04%) merged & trimmed 218.75 Mean alignment length 298.40 Mean merged length 0.33 Mean fwd expected errors 1.24 Mean rev expected errors 0.09 Mean merged expected errors ----- ‣ KO01M - Merging Summary Merging Rate: 418060 / 456159 (91.6%) Median Merged Length: 299 456159 Pairs (456.2k) 418060 Merged (418.1k, 91.65%) 251707 Alignments with zero diffs (55.18%) 32713 Too many diffs (> 10) (7.17%) 699 Fwd too short (< 64) after tail trimming (0.15%) 17 Rev too short (< 64) after tail trimming (0.00%) 4628 No alignment found (1.01%) 37 Alignment too short (< 20) (0.01%) 5 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 4649 Staggered pairs (1.02%) merged & trimmed 211.56 Mean alignment length 295.84 Mean merged length 0.27 Mean fwd expected errors 1.04 Mean rev expected errors 0.10 Mean merged expected errors ----- ‣ KO02F - Merging Summary Merging Rate: 497527 / 536713 (92.7%) Median Merged Length: 299 536713 Pairs (536.7k) 497527 Merged (497.5k, 92.70%) 312020 Alignments with zero diffs (58.14%) 36132 Too many diffs (> 10) (6.73%) 211 Fwd too short (< 64) after tail trimming (0.04%) 4 Rev too short (< 64) after tail trimming (0.00%) 2766 No alignment found (0.52%) 64 Alignment too short (< 20) (0.01%) 9 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 352 Staggered pairs (0.07%) merged & trimmed 217.99 Mean alignment length 299.36 Mean merged length 0.32 Mean fwd expected errors 0.97 Mean rev expected errors 0.08 Mean merged expected errors ----- ‣ KO02M - Merging Summary Merging Rate: 430541 / 476822 (90.3%) Median Merged Length: 299 476822 Pairs (476.8k) 430541 Merged (430.5k, 90.29%) 267848 Alignments with zero diffs (56.17%) 34452 Too many diffs (> 10) (7.23%) 1185 Fwd too short (< 64) after tail trimming (0.25%) 34 Rev too short (< 64) after tail trimming (0.01%) 10518 No alignment found (2.21%) 82 Alignment too short (< 20) (0.02%) 10 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 8553 Staggered pairs (1.79%) merged & trimmed 203.30 Mean alignment length 292.42 Mean merged length 0.29 Mean fwd expected errors 1.00 Mean rev expected errors 0.11 Mean merged expected errors ----- ‣ KO03F - Merging Summary Merging Rate: 475439 / 527420 (90.1%) Median Merged Length: 299 527420 Pairs (527.4k) 475439 Merged (475.4k, 90.14%) 269812 Alignments with zero diffs (51.16%) 49321 Too many diffs (> 10) (9.35%) 200 Fwd too short (< 64) after tail trimming (0.04%) 3 Rev too short (< 64) after tail trimming (0.00%) 2423 No alignment found (0.46%) 27 Alignment too short (< 20) (0.01%) 7 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 331 Staggered pairs (0.06%) merged & trimmed 216.56 Mean alignment length 301.11 Mean merged length 0.25 Mean fwd expected errors 1.22 Mean rev expected errors 0.09 Mean merged expected errors ----- ‣ KO03M - Merging Summary Merging Rate: 458354 / 517656 (88.5%) Median Merged Length: 299 517656 Pairs (517.7k) 458354 Merged (458.4k, 88.54%) 250874 Alignments with zero diffs (48.46%) 55606 Too many diffs (> 10) (10.74%) 598 Fwd too short (< 64) after tail trimming (0.12%) 12 Rev too short (< 64) after tail trimming (0.00%) 3071 No alignment found (0.59%) 10 Alignment too short (< 20) (0.00%) 5 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 3409 Staggered pairs (0.66%) merged & trimmed 213.36 Mean alignment length 295.96 Mean merged length 0.22 Mean fwd expected errors 1.31 Mean rev expected errors 0.09 Mean merged expected errors ----- ‣ KO04F - Merging Summary Merging Rate: 495762 / 555344 (89.3%) Median Merged Length: 298 555344 Pairs (555.3k) 495762 Merged (495.8k, 89.27%) 269729 Alignments with zero diffs (48.57%) 56574 Too many diffs (> 10) (10.19%) 336 Fwd too short (< 64) after tail trimming (0.06%) 12 Rev too short (< 64) after tail trimming (0.00%) 2640 No alignment found (0.48%) 8 Alignment too short (< 20) (0.00%) 12 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 264 Staggered pairs (0.05%) merged & trimmed 218.59 Mean alignment length 298.46 Mean merged length 0.30 Mean fwd expected errors 1.32 Mean rev expected errors 0.09 Mean merged expected errors ----- ‣ KO04M - Merging Summary Merging Rate: 319371 / 358019 (89.2%) Median Merged Length: 299 358019 Pairs (358.0k) 319371 Merged (319.4k, 89.21%) 163987 Alignments with zero diffs (45.80%) 34080 Too many diffs (> 10) (9.52%) 376 Fwd too short (< 64) after tail trimming (0.11%) 11 Rev too short (< 64) after tail trimming (0.00%) 4157 No alignment found (1.16%) 23 Alignment too short (< 20) (0.01%) 1 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 3881 Staggered pairs (1.08%) merged & trimmed 211.10 Mean alignment length 294.05 Mean merged length 0.33 Mean fwd expected errors 1.26 Mean rev expected errors 0.11 Mean merged expected errors ----- ‣ KO05F - Merging Summary Merging Rate: 444340 / 481769 (92.2%) Median Merged Length: 299 481769 Pairs (481.8k) 444340 Merged (444.3k, 92.23%) 278342 Alignments with zero diffs (57.77%) 34956 Too many diffs (> 10) (7.26%) 158 Fwd too short (< 64) after tail trimming (0.03%) 3 Rev too short (< 64) after tail trimming (0.00%) 2280 No alignment found (0.47%) 25 Alignment too short (< 20) (0.01%) 7 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 230 Staggered pairs (0.05%) merged & trimmed 217.14 Mean alignment length 300.33 Mean merged length 0.26 Mean fwd expected errors 1.04 Mean rev expected errors 0.08 Mean merged expected errors ----- ‣ KO05M - Merging Summary Merging Rate: 418773 / 471648 (88.8%) Median Merged Length: 298 471648 Pairs (471.6k) 418773 Merged (418.8k, 88.79%) 256934 Alignments with zero diffs (54.48%) 35645 Too many diffs (> 10) (7.56%) 891 Fwd too short (< 64) after tail trimming (0.19%) 19 Rev too short (< 64) after tail trimming (0.00%) 16157 No alignment found (3.43%) 158 Alignment too short (< 20) (0.03%) 5 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 9062 Staggered pairs (1.92%) merged & trimmed 203.63 Mean alignment length 291.99 Mean merged length 0.24 Mean fwd expected errors 1.08 Mean rev expected errors 0.11 Mean merged expected errors ----- ‣ KO06F - Merging Summary Merging Rate: 448524 / 490629 (91.4%) Median Merged Length: 298 490629 Pairs (490.6k) 448524 Merged (448.5k, 91.42%) 267055 Alignments with zero diffs (54.43%) 39515 Too many diffs (> 10) (8.05%) 172 Fwd too short (< 64) after tail trimming (0.04%) 4 Rev too short (< 64) after tail trimming (0.00%) 2389 No alignment found (0.49%) 22 Alignment too short (< 20) (0.00%) 3 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 214 Staggered pairs (0.04%) merged & trimmed 218.67 Mean alignment length 298.65 Mean merged length 0.28 Mean fwd expected errors 1.14 Mean rev expected errors 0.08 Mean merged expected errors ----- ‣ KO06M - Merging Summary Merging Rate: 489296 / 533566 (91.7%) Median Merged Length: 299 533566 Pairs (533.6k) 489296 Merged (489.3k, 91.70%) 297848 Alignments with zero diffs (55.82%) 35875 Too many diffs (> 10) (6.72%) 3539 Fwd too short (< 64) after tail trimming (0.66%) 63 Rev too short (< 64) after tail trimming (0.01%) 4745 No alignment found (0.89%) 34 Alignment too short (< 20) (0.01%) 14 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 3584 Staggered pairs (0.67%) merged & trimmed 212.32 Mean alignment length 294.12 Mean merged length 0.23 Mean fwd expected errors 1.02 Mean rev expected errors 0.09 Mean merged expected errors ----- ‣ KO07F - Merging Summary Merging Rate: 452179 / 495853 (91.2%) Median Merged Length: 298 495853 Pairs (495.9k) 452179 Merged (452.2k, 91.19%) 271422 Alignments with zero diffs (54.74%) 40912 Too many diffs (> 10) (8.25%) 276 Fwd too short (< 64) after tail trimming (0.06%) 6 Rev too short (< 64) after tail trimming (0.00%) 2458 No alignment found (0.50%) 19 Alignment too short (< 20) (0.00%) 3 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 253 Staggered pairs (0.05%) merged & trimmed 218.56 Mean alignment length 298.57 Mean merged length 0.29 Mean fwd expected errors 1.13 Mean rev expected errors 0.08 Mean merged expected errors ----- ‣ KO07M - Merging Summary Merging Rate: 497788 / 546509 (91.1%) Median Merged Length: 299 546509 Pairs (546.5k) 497788 Merged (497.8k, 91.09%) 304661 Alignments with zero diffs (55.75%) 39921 Too many diffs (> 10) (7.30%) 837 Fwd too short (< 64) after tail trimming (0.15%) 19 Rev too short (< 64) after tail trimming (0.00%) 7878 No alignment found (1.44%) 62 Alignment too short (< 20) (0.01%) 4 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 5557 Staggered pairs (1.02%) merged & trimmed 209.49 Mean alignment length 293.80 Mean merged length 0.25 Mean fwd expected errors 1.05 Mean rev expected errors 0.10 Mean merged expected errors ----- ‣ KO08F - Merging Summary Merging Rate: 580006 / 636991 (91.1%) Median Merged Length: 298 636991 Pairs (637.0k) 580006 Merged (580.0k, 91.05%) 349734 Alignments with zero diffs (54.90%) 53711 Too many diffs (> 10) (8.43%) 231 Fwd too short (< 64) after tail trimming (0.04%) 5 Rev too short (< 64) after tail trimming (0.00%) 3013 No alignment found (0.47%) 21 Alignment too short (< 20) (0.00%) 4 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 276 Staggered pairs (0.04%) merged & trimmed 218.68 Mean alignment length 298.53 Mean merged length 0.27 Mean fwd expected errors 1.14 Mean rev expected errors 0.08 Mean merged expected errors ----- ‣ KO08M - Merging Summary Merging Rate: 464589 / 518683 (89.6%) Median Merged Length: 299 518683 Pairs (518.7k) 464589 Merged (464.6k, 89.57%) 249774 Alignments with zero diffs (48.16%) 48952 Too many diffs (> 10) (9.44%) 242 Fwd too short (< 64) after tail trimming (0.05%) 8 Rev too short (< 64) after tail trimming (0.00%) 4858 No alignment found (0.94%) 31 Alignment too short (< 20) (0.01%) 3 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 1574 Staggered pairs (0.30%) merged & trimmed 216.02 Mean alignment length 297.34 Mean merged length 0.29 Mean fwd expected errors 1.27 Mean rev expected errors 0.10 Mean merged expected errors ----- ‣ KO09F - Merging Summary Merging Rate: 483315 / 542570 (89.1%) Median Merged Length: 298 542570 Pairs (542.6k) 483315 Merged (483.3k, 89.08%) 266617 Alignments with zero diffs (49.14%) 56101 Too many diffs (> 10) (10.34%) 327 Fwd too short (< 64) after tail trimming (0.06%) 4 Rev too short (< 64) after tail trimming (0.00%) 2793 No alignment found (0.51%) 25 Alignment too short (< 20) (0.00%) 5 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 306 Staggered pairs (0.06%) merged & trimmed 218.60 Mean alignment length 298.61 Mean merged length 0.27 Mean fwd expected errors 1.31 Mean rev expected errors 0.08 Mean merged expected errors ----- ‣ KO09M - Merging Summary Merging Rate: 447678 / 480348 (93.2%) Median Merged Length: 299 480348 Pairs (480.3k) 447678 Merged (447.7k, 93.20%) 296233 Alignments with zero diffs (61.67%) 28430 Too many diffs (> 10) (5.92%) 237 Fwd too short (< 64) after tail trimming (0.05%) 5 Rev too short (< 64) after tail trimming (0.00%) 3966 No alignment found (0.83%) 30 Alignment too short (< 20) (0.01%) 2 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 1504 Staggered pairs (0.31%) merged & trimmed 215.29 Mean alignment length 297.86 Mean merged length 0.25 Mean fwd expected errors 0.87 Mean rev expected errors 0.09 Mean merged expected errors ----- ‣ KO10F - Merging Summary Merging Rate: 461226 / 513922 (89.7%) Median Merged Length: 298 513922 Pairs (513.9k) 461226 Merged (461.2k, 89.75%) 267353 Alignments with zero diffs (52.02%) 50234 Too many diffs (> 10) (9.77%) 142 Fwd too short (< 64) after tail trimming (0.03%) 4 Rev too short (< 64) after tail trimming (0.00%) 2301 No alignment found (0.45%) 14 Alignment too short (< 20) (0.00%) 1 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 185 Staggered pairs (0.04%) merged & trimmed 218.79 Mean alignment length 298.60 Mean merged length 0.27 Mean fwd expected errors 1.23 Mean rev expected errors 0.08 Mean merged expected errors ----- ‣ KO10M - Merging Summary Merging Rate: 451664 / 483759 (93.4%) Median Merged Length: 299 483759 Pairs (483.8k) 451664 Merged (451.7k, 93.37%) 311833 Alignments with zero diffs (64.46%) 26790 Too many diffs (> 10) (5.54%) 715 Fwd too short (< 64) after tail trimming (0.15%) 19 Rev too short (< 64) after tail trimming (0.00%) 4535 No alignment found (0.94%) 31 Alignment too short (< 20) (0.01%) 5 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 5679 Staggered pairs (1.17%) merged & trimmed 206.66 Mean alignment length 292.43 Mean merged length 0.25 Mean fwd expected errors 0.79 Mean rev expected errors 0.09 Mean merged expected errors ----- ‣ KO11F - Merging Summary Merging Rate: 635045 / 706307 (89.9%) Median Merged Length: 299 706307 Pairs (706.3k) 635045 Merged (635.0k, 89.91%) 359607 Alignments with zero diffs (50.91%) 68173 Too many diffs (> 10) (9.65%) 179 Fwd too short (< 64) after tail trimming (0.03%) 4 Rev too short (< 64) after tail trimming (0.00%) 2890 No alignment found (0.41%) 12 Alignment too short (< 20) (0.00%) 4 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 221 Staggered pairs (0.03%) merged & trimmed 219.01 Mean alignment length 298.66 Mean merged length 0.24 Mean fwd expected errors 1.26 Mean rev expected errors 0.08 Mean merged expected errors ----- ‣ KO11M - Merging Summary Merging Rate: 479679 / 540356 (88.8%) Median Merged Length: 298 540356 Pairs (540.4k) 479679 Merged (479.7k, 88.77%) 289555 Alignments with zero diffs (53.59%) 49284 Too many diffs (> 10) (9.12%) 1497 Fwd too short (< 64) after tail trimming (0.28%) 25 Rev too short (< 64) after tail trimming (0.00%) 9776 No alignment found (1.81%) 88 Alignment too short (< 20) (0.02%) 7 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 6584 Staggered pairs (1.22%) merged & trimmed 204.65 Mean alignment length 289.03 Mean merged length 0.23 Mean fwd expected errors 1.16 Mean rev expected errors 0.09 Mean merged expected errors ----- ‣ NTCF - Merging Summary Merging Rate: 264 / 636 (41.5%) Median Merged Length: 299 636 Pairs (636) 264 Merged (264, 41.51%) 97 Alignments with zero diffs (15.25%) 253 Too many diffs (> 10) (39.78%) 42 Fwd too short (< 64) after tail trimming (6.60%) 4 Rev too short (< 64) after tail trimming (0.63%) 70 No alignment found (11.01%) 0 Alignment too short (< 20) (0.00%) 3 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 7 Staggered pairs (1.10%) merged & trimmed 216.34 Mean alignment length 296.14 Mean merged length 0.40 Mean fwd expected errors 2.00 Mean rev expected errors 0.15 Mean merged expected errors ----- ‣ NTCM - Merging Summary Merging Rate: 22238 / 24791 (89.7%) Median Merged Length: 299 24791 Pairs (24.8k) 22238 Merged (22.2k, 89.70%) 12441 Alignments with zero diffs (50.18%) 2150 Too many diffs (> 10) (8.67%) 280 Fwd too short (< 64) after tail trimming (1.13%) 3 Rev too short (< 64) after tail trimming (0.01%) 119 No alignment found (0.48%) 1 Alignment too short (< 20) (0.00%) 0 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 19 Staggered pairs (0.08%) merged & trimmed 218.78 Mean alignment length 298.72 Mean merged length 0.23 Mean fwd expected errors 1.24 Mean rev expected errors 0.08 Mean merged expected errors ----- ‣ POSF - Merging Summary Merging Rate: 348424 / 388401 (89.7%) Median Merged Length: 298 388401 Pairs (388.4k) 348424 Merged (348.4k, 89.71%) 187653 Alignments with zero diffs (48.31%) 38775 Too many diffs (> 10) (9.98%) 72 Fwd too short (< 64) after tail trimming (0.02%) 0 Rev too short (< 64) after tail trimming (0.00%) 1120 No alignment found (0.29%) 3 Alignment too short (< 20) (0.00%) 7 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 90 Staggered pairs (0.02%) merged & trimmed 219.06 Mean alignment length 297.92 Mean merged length 0.27 Mean fwd expected errors 1.31 Mean rev expected errors 0.08 Mean merged expected errors ----- ‣ POSM - Merging Summary Merging Rate: 431993 / 475811 (90.8%) Median Merged Length: 298 475811 Pairs (475.8k) 431993 Merged (432.0k, 90.79%) 247288 Alignments with zero diffs (51.97%) 42537 Too many diffs (> 10) (8.94%) 137 Fwd too short (< 64) after tail trimming (0.03%) 3 Rev too short (< 64) after tail trimming (0.00%) 1140 No alignment found (0.24%) 1 Alignment too short (< 20) (0.00%) 0 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 192 Staggered pairs (0.04%) merged & trimmed 218.88 Mean alignment length 297.83 Mean merged length 0.27 Mean fwd expected errors 1.20 Mean rev expected errors 0.08 Mean merged expected errors ----- ‣ WT01F - Merging Summary Merging Rate: 496635 / 547365 (90.7%) Median Merged Length: 299 547365 Pairs (547.4k) 496635 Merged (496.6k, 90.73%) 290313 Alignments with zero diffs (53.04%) 47926 Too many diffs (> 10) (8.76%) 130 Fwd too short (< 64) after tail trimming (0.02%) 1 Rev too short (< 64) after tail trimming (0.00%) 2657 No alignment found (0.49%) 8 Alignment too short (< 20) (0.00%) 8 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 268 Staggered pairs (0.05%) merged & trimmed 217.61 Mean alignment length 300.12 Mean merged length 0.31 Mean fwd expected errors 1.15 Mean rev expected errors 0.09 Mean merged expected errors ----- ‣ WT01M - Merging Summary Merging Rate: 443368 / 496740 (89.3%) Median Merged Length: 298 496740 Pairs (496.7k) 443368 Merged (443.4k, 89.26%) 298099 Alignments with zero diffs (60.01%) 31440 Too many diffs (> 10) (6.33%) 2385 Fwd too short (< 64) after tail trimming (0.48%) 54 Rev too short (< 64) after tail trimming (0.01%) 19321 No alignment found (3.89%) 159 Alignment too short (< 20) (0.03%) 13 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 12593 Staggered pairs (2.54%) merged & trimmed 191.47 Mean alignment length 283.33 Mean merged length 0.26 Mean fwd expected errors 0.87 Mean rev expected errors 0.12 Mean merged expected errors ----- ‣ WT02F - Merging Summary Merging Rate: 607106 / 669726 (90.6%) Median Merged Length: 298 669726 Pairs (669.7k) 607106 Merged (607.1k, 90.65%) 359717 Alignments with zero diffs (53.71%) 58972 Too many diffs (> 10) (8.81%) 193 Fwd too short (< 64) after tail trimming (0.03%) 7 Rev too short (< 64) after tail trimming (0.00%) 3422 No alignment found (0.51%) 17 Alignment too short (< 20) (0.00%) 9 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 243 Staggered pairs (0.04%) merged & trimmed 217.31 Mean alignment length 300.31 Mean merged length 0.31 Mean fwd expected errors 1.15 Mean rev expected errors 0.08 Mean merged expected errors ----- ‣ WT02M - Merging Summary Merging Rate: 398919 / 451148 (88.4%) Median Merged Length: 298 451148 Pairs (451.1k) 398919 Merged (398.9k, 88.42%) 271942 Alignments with zero diffs (60.28%) 28659 Too many diffs (> 10) (6.35%) 1025 Fwd too short (< 64) after tail trimming (0.23%) 24 Rev too short (< 64) after tail trimming (0.01%) 22310 No alignment found (4.95%) 204 Alignment too short (< 20) (0.05%) 7 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 10808 Staggered pairs (2.40%) merged & trimmed 192.60 Mean alignment length 287.24 Mean merged length 0.26 Mean fwd expected errors 0.86 Mean rev expected errors 0.12 Mean merged expected errors ----- ‣ WT03F - Merging Summary Merging Rate: 462158 / 517930 (89.2%) Median Merged Length: 299 517930 Pairs (517.9k) 462158 Merged (462.2k, 89.23%) 254778 Alignments with zero diffs (49.19%) 52898 Too many diffs (> 10) (10.21%) 301 Fwd too short (< 64) after tail trimming (0.06%) 6 Rev too short (< 64) after tail trimming (0.00%) 2545 No alignment found (0.49%) 15 Alignment too short (< 20) (0.00%) 7 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 276 Staggered pairs (0.05%) merged & trimmed 218.28 Mean alignment length 299.04 Mean merged length 0.27 Mean fwd expected errors 1.31 Mean rev expected errors 0.08 Mean merged expected errors ----- ‣ WT03M - Merging Summary Merging Rate: 459056 / 511389 (89.8%) Median Merged Length: 299 511389 Pairs (511.4k) 459056 Merged (459.1k, 89.77%) 271537 Alignments with zero diffs (53.10%) 40291 Too many diffs (> 10) (7.88%) 587 Fwd too short (< 64) after tail trimming (0.11%) 12 Rev too short (< 64) after tail trimming (0.00%) 11364 No alignment found (2.22%) 73 Alignment too short (< 20) (0.01%) 6 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 5503 Staggered pairs (1.08%) merged & trimmed 210.01 Mean alignment length 296.08 Mean merged length 0.24 Mean fwd expected errors 1.12 Mean rev expected errors 0.10 Mean merged expected errors ----- ‣ WT04F - Merging Summary Merging Rate: 378423 / 433281 (87.3%) Median Merged Length: 298 433281 Pairs (433.3k) 378423 Merged (378.4k, 87.34%) 186546 Alignments with zero diffs (43.05%) 52490 Too many diffs (> 10) (12.11%) 196 Fwd too short (< 64) after tail trimming (0.05%) 1 Rev too short (< 64) after tail trimming (0.00%) 2152 No alignment found (0.50%) 10 Alignment too short (< 20) (0.00%) 9 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 209 Staggered pairs (0.05%) merged & trimmed 218.65 Mean alignment length 298.84 Mean merged length 0.34 Mean fwd expected errors 1.48 Mean rev expected errors 0.09 Mean merged expected errors ----- ‣ WT04M - Merging Summary Merging Rate: 429109 / 484192 (88.6%) Median Merged Length: 299 484192 Pairs (484.2k) 429109 Merged (429.1k, 88.62%) 244105 Alignments with zero diffs (50.41%) 44670 Too many diffs (> 10) (9.23%) 847 Fwd too short (< 64) after tail trimming (0.17%) 25 Rev too short (< 64) after tail trimming (0.01%) 9461 No alignment found (1.95%) 75 Alignment too short (< 20) (0.02%) 5 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 6921 Staggered pairs (1.43%) merged & trimmed 209.24 Mean alignment length 294.24 Mean merged length 0.26 Mean fwd expected errors 1.20 Mean rev expected errors 0.10 Mean merged expected errors ----- ‣ WT05F - Merging Summary Merging Rate: 464226 / 507417 (91.5%) Median Merged Length: 298 507417 Pairs (507.4k) 464226 Merged (464.2k, 91.49%) 288244 Alignments with zero diffs (56.81%) 40202 Too many diffs (> 10) (7.92%) 243 Fwd too short (< 64) after tail trimming (0.05%) 11 Rev too short (< 64) after tail trimming (0.00%) 2710 No alignment found (0.53%) 12 Alignment too short (< 20) (0.00%) 13 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 206 Staggered pairs (0.04%) merged & trimmed 218.64 Mean alignment length 298.46 Mean merged length 0.26 Mean fwd expected errors 1.09 Mean rev expected errors 0.08 Mean merged expected errors ----- ‣ WT05M - Merging Summary Merging Rate: 478354 / 523313 (91.4%) Median Merged Length: 299 523313 Pairs (523.3k) 478354 Merged (478.4k, 91.41%) 322707 Alignments with zero diffs (61.67%) 31525 Too many diffs (> 10) (6.02%) 933 Fwd too short (< 64) after tail trimming (0.18%) 20 Rev too short (< 64) after tail trimming (0.00%) 12378 No alignment found (2.37%) 100 Alignment too short (< 20) (0.02%) 3 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 6100 Staggered pairs (1.17%) merged & trimmed 204.30 Mean alignment length 292.38 Mean merged length 0.23 Mean fwd expected errors 0.87 Mean rev expected errors 0.10 Mean merged expected errors ----- ‣ WT06F - Merging Summary Merging Rate: 494796 / 539187 (91.8%) Median Merged Length: 299 539187 Pairs (539.2k) 494796 Merged (494.8k, 91.77%) 304638 Alignments with zero diffs (56.50%) 41583 Too many diffs (> 10) (7.71%) 252 Fwd too short (< 64) after tail trimming (0.05%) 3 Rev too short (< 64) after tail trimming (0.00%) 2530 No alignment found (0.47%) 10 Alignment too short (< 20) (0.00%) 13 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 211 Staggered pairs (0.04%) merged & trimmed 218.80 Mean alignment length 298.46 Mean merged length 0.28 Mean fwd expected errors 1.08 Mean rev expected errors 0.08 Mean merged expected errors ----- ‣ WT06M - Merging Summary Merging Rate: 520383 / 565635 (92.0%) Median Merged Length: 299 565635 Pairs (565.6k) 520383 Merged (520.4k, 92.00%) 321705 Alignments with zero diffs (56.88%) 41775 Too many diffs (> 10) (7.39%) 254 Fwd too short (< 64) after tail trimming (0.04%) 2 Rev too short (< 64) after tail trimming (0.00%) 3198 No alignment found (0.57%) 20 Alignment too short (< 20) (0.00%) 3 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 1131 Staggered pairs (0.20%) merged & trimmed 216.54 Mean alignment length 297.35 Mean merged length 0.26 Mean fwd expected errors 1.05 Mean rev expected errors 0.08 Mean merged expected errors ----- ‣ WT07F - Merging Summary Merging Rate: 462780 / 500999 (92.4%) Median Merged Length: 298 500999 Pairs (501.0k) 462780 Merged (462.8k, 92.37%) 288523 Alignments with zero diffs (57.59%) 35804 Too many diffs (> 10) (7.15%) 155 Fwd too short (< 64) after tail trimming (0.03%) 4 Rev too short (< 64) after tail trimming (0.00%) 2233 No alignment found (0.45%) 13 Alignment too short (< 20) (0.00%) 10 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 215 Staggered pairs (0.04%) merged & trimmed 218.82 Mean alignment length 298.52 Mean merged length 0.31 Mean fwd expected errors 1.05 Mean rev expected errors 0.08 Mean merged expected errors ----- ‣ WT07M - Merging Summary Merging Rate: 453378 / 499532 (90.8%) Median Merged Length: 298 499532 Pairs (499.5k) 453378 Merged (453.4k, 90.76%) 311305 Alignments with zero diffs (62.32%) 29681 Too many diffs (> 10) (5.94%) 506 Fwd too short (< 64) after tail trimming (0.10%) 14 Rev too short (< 64) after tail trimming (0.00%) 15795 No alignment found (3.16%) 155 Alignment too short (< 20) (0.03%) 3 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 12379 Staggered pairs (2.48%) merged & trimmed 190.29 Mean alignment length 278.33 Mean merged length 0.26 Mean fwd expected errors 0.82 Mean rev expected errors 0.10 Mean merged expected errors ----- ‣ WT08F - Merging Summary Merging Rate: 502146 / 553937 (90.7%) Median Merged Length: 298 553937 Pairs (553.9k) 502146 Merged (502.1k, 90.65%) 301987 Alignments with zero diffs (54.52%) 48852 Too many diffs (> 10) (8.82%) 281 Fwd too short (< 64) after tail trimming (0.05%) 5 Rev too short (< 64) after tail trimming (0.00%) 2615 No alignment found (0.47%) 28 Alignment too short (< 20) (0.01%) 10 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 237 Staggered pairs (0.04%) merged & trimmed 218.69 Mean alignment length 298.43 Mean merged length 0.28 Mean fwd expected errors 1.15 Mean rev expected errors 0.08 Mean merged expected errors ----- ‣ WT08M - Merging Summary Merging Rate: 515211 / 567361 (90.8%) Median Merged Length: 298 567361 Pairs (567.4k) 515211 Merged (515.2k, 90.81%) 326992 Alignments with zero diffs (57.63%) 41738 Too many diffs (> 10) (7.36%) 1158 Fwd too short (< 64) after tail trimming (0.20%) 25 Rev too short (< 64) after tail trimming (0.00%) 9138 No alignment found (1.61%) 88 Alignment too short (< 20) (0.02%) 3 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 5890 Staggered pairs (1.04%) merged & trimmed 206.16 Mean alignment length 289.14 Mean merged length 0.27 Mean fwd expected errors 1.00 Mean rev expected errors 0.09 Mean merged expected errors ----- ‣ WT09F - Merging Summary Merging Rate: 487917 / 543820 (89.7%) Median Merged Length: 298 543820 Pairs (543.8k) 487917 Merged (487.9k, 89.72%) 273281 Alignments with zero diffs (50.25%) 53440 Too many diffs (> 10) (9.83%) 124 Fwd too short (< 64) after tail trimming (0.02%) 3 Rev too short (< 64) after tail trimming (0.00%) 2302 No alignment found (0.42%) 30 Alignment too short (< 20) (0.01%) 4 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 201 Staggered pairs (0.04%) merged & trimmed 218.87 Mean alignment length 298.47 Mean merged length 0.27 Mean fwd expected errors 1.29 Mean rev expected errors 0.09 Mean merged expected errors ----- ‣ WT09M - Merging Summary Merging Rate: 425447 / 461924 (92.1%) Median Merged Length: 299 461924 Pairs (461.9k) 425447 Merged (425.4k, 92.10%) 259021 Alignments with zero diffs (56.07%) 32516 Too many diffs (> 10) (7.04%) 433 Fwd too short (< 64) after tail trimming (0.09%) 8 Rev too short (< 64) after tail trimming (0.00%) 3471 No alignment found (0.75%) 45 Alignment too short (< 20) (0.01%) 4 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 2283 Staggered pairs (0.49%) merged & trimmed 213.35 Mean alignment length 294.55 Mean merged length 0.29 Mean fwd expected errors 1.06 Mean rev expected errors 0.09 Mean merged expected errors ----- ‣ WT10F - Merging Summary Merging Rate: 554420 / 618924 (89.6%) Median Merged Length: 298 618924 Pairs (618.9k) 554420 Merged (554.4k, 89.58%) 314105 Alignments with zero diffs (50.75%) 61651 Too many diffs (> 10) (9.96%) 194 Fwd too short (< 64) after tail trimming (0.03%) 6 Rev too short (< 64) after tail trimming (0.00%) 2629 No alignment found (0.42%) 22 Alignment too short (< 20) (0.00%) 2 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 268 Staggered pairs (0.04%) merged & trimmed 218.83 Mean alignment length 298.49 Mean merged length 0.27 Mean fwd expected errors 1.26 Mean rev expected errors 0.08 Mean merged expected errors ----- ‣ WT10M - Merging Summary Merging Rate: 485948 / 524755 (92.6%) Median Merged Length: 299 524755 Pairs (524.8k) 485948 Merged (485.9k, 92.60%) 295813 Alignments with zero diffs (56.37%) 34234 Too many diffs (> 10) (6.52%) 373 Fwd too short (< 64) after tail trimming (0.07%) 10 Rev too short (< 64) after tail trimming (0.00%) 4158 No alignment found (0.79%) 31 Alignment too short (< 20) (0.01%) 1 Merged too short (< 100) 0 Min Q too low (<0) (0.00%) 2691 Staggered pairs (0.51%) merged & trimmed 214.86 Mean alignment length 296.46 Mean merged length 0.23 Mean fwd expected errors 1.01 Mean rev expected errors 0.09 Mean merged expected errors ----- End_B6_Trim_and_Merge: 15:54:24 16/07/2020 ................................................................................ END_Workflow: 15:54:24 16/07/2020 ========================================================================================== Step [C] - Remove/Trim Primer Sites ------------------------------------------------------------------------------------------ Application: usearch v11.0.667_i86linux64 Command: search_pcr Amplicon Size Range: 100-600 Number of Mis-Matches: 2 Coverage: full-length (no end gaps) Wildcards enabled: IUPAC codes Additional Filtering: -Exclude PCR-hits with mismatches at the primer end. -Exclude amplicons in the wrong (reverse) orientation. -Remove sequences with more than 1 amplicon. -------------------------------------------------------------------------------- START_In-Silico_PCR: 15:54:34 16/07/2020 ................................................................................ KO01F: N(allPCRHits)= 512053 (-21664) KO01F: N(SingleHits)= 512047 (-21670) KO01F: N(CleanHits) = 496514 (-37203) ◇ Amplicon Orientation: 512045 KO01F-PF KO01F-PR 8 KO01F-PR KO01F-PF ◇ Mis-Matches (top3): PrimerF 692 ...C..........G.... PrimerF 70025 ................... PrimerF 435921 ...C............... PrimerR 11804 ...................T PrimerR 104251 .............W...... PrimerR 385564 .................... - - - KO01M: N(allPCRHits)= 380951 (-37109) KO01M: N(SingleHits)= 380941 (-37119) KO01M: N(CleanHits) = 378829 (-39231) ◇ Amplicon Orientation: 380944 KO01M-PF KO01M-PR 7 KO01M-PR KO01M-PF ◇ Mis-Matches (top3): PrimerF 9268 ...C.............A. PrimerF 50468 ................... PrimerF 315437 ...C............... PrimerR 6083 .................A.. PrimerR 95241 .............W...... PrimerR 270312 .................... - - - KO02F: N(allPCRHits)= 475607 (-21920) KO02F: N(SingleHits)= 475603 (-21924) KO02F: N(CleanHits) = 468915 (-28612) ◇ Amplicon Orientation: 475602 KO02F-PF KO02F-PR 5 KO02F-PR KO02F-PF ◇ Mis-Matches (top3): PrimerF 629 G..C............... PrimerF 64591 ................... PrimerF 405589 ...C............... PrimerR 4931 ...................T PrimerR 98620 .............W...... PrimerR 360627 .................... - - - KO02M: N(allPCRHits)= 356950 (-73591) KO02M: N(SingleHits)= 356942 (-73599) KO02M: N(CleanHits) = 351968 (-78573) ◇ Amplicon Orientation: 356947 KO02M-PF KO02M-PR 3 KO02M-PR KO02M-PF ◇ Mis-Matches (top3): PrimerF 25187 ...C.............A. PrimerF 43835 ................... PrimerF 279097 ...C............... PrimerR 2802 .................A.. PrimerR 86942 .............W...... PrimerR 256900 .................... - - - KO03F: N(allPCRHits)= 455840 (-19599) KO03F: N(SingleHits)= 455832 (-19607) KO03F: N(CleanHits) = 451632 (-23807) ◇ Amplicon Orientation: 455839 KO03F-PF KO03F-PR 1 KO03F-PR KO03F-PF ◇ Mis-Matches (top3): PrimerF 609 ...C..........G.... PrimerF 62295 ................... PrimerF 388167 ...C............... PrimerR 14493 .................A.. PrimerR 91337 .............W...... PrimerR 335481 .................... - - - KO03M: N(allPCRHits)= 430370 (-27984) KO03M: N(SingleHits)= 430332 (-28022) KO03M: N(CleanHits) = 422861 (-35493) ◇ Amplicon Orientation: 430366 KO03M-PF KO03M-PR 4 KO03M-PR KO03M-PF ◇ Mis-Matches (top3): PrimerF 13823 ...C.............A. PrimerF 57190 ................... PrimerF 352585 ...C............... PrimerR 6078 .................A.. PrimerR 105958 .............W...... PrimerR 303043 .................... - - - KO04F: N(allPCRHits)= 475054 (-20708) KO04F: N(SingleHits)= 475046 (-20716) KO04F: N(CleanHits) = 463606 (-32156) ◇ Amplicon Orientation: 475050 KO04F-PF KO04F-PR 4 KO04F-PR KO04F-PF ◇ Mis-Matches (top3): PrimerF 653 ...C..........G.... PrimerF 65341 ................... PrimerF 404284 ...C............... PrimerR 8644 ...................T PrimerR 97984 .............W...... PrimerR 358968 .................... - - - KO04M: N(allPCRHits)= 293617 (-25754) KO04M: N(SingleHits)= 293601 (-25770) KO04M: N(CleanHits) = 290805 (-28566) ◇ Amplicon Orientation: 293610 KO04M-PF KO04M-PR 7 KO04M-PR KO04M-PF ◇ Mis-Matches (top3): PrimerF 13806 ...C.............A. PrimerF 37600 ................... PrimerF 236739 ...C............... PrimerR 2337 .................A.. PrimerR 72228 .............W...... PrimerR 211193 .................... - - - KO05F: N(allPCRHits)= 425351 (-18989) KO05F: N(SingleHits)= 425341 (-18999) KO05F: N(CleanHits) = 419869 (-24471) ◇ Amplicon Orientation: 425349 KO05F-PF KO05F-PR 2 KO05F-PR KO05F-PF ◇ Mis-Matches (top3): PrimerF 580 ...C..........G.... PrimerF 58843 ................... PrimerF 361818 ...C............... PrimerR 9104 .................A.. PrimerR 86487 .............W...... PrimerR 316531 .................... - - - KO05M: N(allPCRHits)= 352621 (-66152) KO05M: N(SingleHits)= 352617 (-66156) KO05M: N(CleanHits) = 347662 (-71111) ◇ Amplicon Orientation: 352619 KO05M-PF KO05M-PR 2 KO05M-PR KO05M-PF ◇ Mis-Matches (top3): PrimerF 26361 ...C.............A. PrimerF 43034 ................... PrimerF 274690 ...C............... PrimerR 2325 .................A.. PrimerR 84815 .............W...... PrimerR 255124 .................... - - - KO06F: N(allPCRHits)= 429833 (-18691) KO06F: N(SingleHits)= 429827 (-18697) KO06F: N(CleanHits) = 410539 (-37985) ◇ Amplicon Orientation: 429832 KO06F-PF KO06F-PR 1 KO06F-PR KO06F-PF ◇ Mis-Matches (top3): PrimerF 2207 ...C.............A. PrimerF 59069 ................... PrimerF 363869 ...C............... PrimerR 14753 ...................T PrimerR 86999 .............W...... PrimerR 318146 .................... - - - KO06M: N(allPCRHits)= 457767 (-31529) KO06M: N(SingleHits)= 457765 (-31531) KO06M: N(CleanHits) = 454709 (-34587) ◇ Amplicon Orientation: 457764 KO06M-PF KO06M-PR 3 KO06M-PR KO06M-PF ◇ Mis-Matches (top3): PrimerF 17856 ...C.............A. PrimerF 57416 ................... PrimerF 374631 ...C............... PrimerR 3263 .................A.. PrimerR 106974 .............W...... PrimerR 337074 .................... - - - KO07F: N(allPCRHits)= 432859 (-19320) KO07F: N(SingleHits)= 432855 (-19324) KO07F: N(CleanHits) = 422409 (-29770) ◇ Amplicon Orientation: 432852 KO07F-PF KO07F-PR 7 KO07F-PR KO07F-PF ◇ Mis-Matches (top3): PrimerF 1252 ...C.............A. PrimerF 59913 ................... PrimerF 367272 ...C............... PrimerR 7901 ...................T PrimerR 89803 .............W...... PrimerR 327333 .................... - - - KO07M: N(allPCRHits)= 454251 (-43537) KO07M: N(SingleHits)= 454247 (-43541) KO07M: N(CleanHits) = 449341 (-48447) ◇ Amplicon Orientation: 454246 KO07M-PF KO07M-PR 5 KO07M-PR KO07M-PF ◇ Mis-Matches (top3): PrimerF 23258 ...C.............A. PrimerF 57682 ................... PrimerF 364728 ...C............... PrimerR 6389 .................A.. PrimerR 109528 .............W...... PrimerR 325344 .................... - - - KO08F: N(allPCRHits)= 555463 (-24543) KO08F: N(SingleHits)= 555451 (-24555) KO08F: N(CleanHits) = 530680 (-49326) ◇ Amplicon Orientation: 555456 KO08F-PF KO08F-PR 7 KO08F-PR KO08F-PF ◇ Mis-Matches (top3): PrimerF 3776 ...C.............A. PrimerF 76053 ................... PrimerF 469653 ...C............... PrimerR 18914 ...................T PrimerR 112545 .............W...... PrimerR 411429 .................... - - - KO08M: N(allPCRHits)= 441264 (-23325) KO08M: N(SingleHits)= 441254 (-23335) KO08M: N(CleanHits) = 429965 (-34624) ◇ Amplicon Orientation: 441261 KO08M-PF KO08M-PR 3 KO08M-PR KO08M-PF ◇ Mis-Matches (top3): PrimerF 8359 ...C.............A. PrimerF 57347 ................... PrimerF 369185 ...C............... PrimerR 8118 ...................T PrimerR 101885 .............W...... PrimerR 320225 .................... - - - KO09F: N(allPCRHits)= 463271 (-20044) KO09F: N(SingleHits)= 463257 (-20058) KO09F: N(CleanHits) = 449028 (-34287) ◇ Amplicon Orientation: 463268 KO09F-PF KO09F-PR 3 KO09F-PR KO09F-PF ◇ Mis-Matches (top3): PrimerF 2808 ...C.............A. PrimerF 62735 ................... PrimerF 392582 ...C............... PrimerR 10730 ...................T PrimerR 95537 .............W...... PrimerR 347366 .................... - - - KO09M: N(allPCRHits)= 424551 (-23127) KO09M: N(SingleHits)= 424547 (-23131) KO09M: N(CleanHits) = 421833 (-25845) ◇ Amplicon Orientation: 424549 KO09M-PF KO09M-PR 2 KO09M-PR KO09M-PF ◇ Mis-Matches (top3): PrimerF 7908 ...C.............A. PrimerF 54875 ................... PrimerF 355695 ...C............... PrimerR 4884 .................A.. PrimerR 99806 .............W...... PrimerR 310366 .................... - - - KO10F: N(allPCRHits)= 443730 (-17496) KO10F: N(SingleHits)= 443720 (-17506) KO10F: N(CleanHits) = 422195 (-39031) ◇ Amplicon Orientation: 443727 KO10F-PF KO10F-PR 3 KO10F-PR KO10F-PF ◇ Mis-Matches (top3): PrimerF 1022 ...C.............A. PrimerF 60404 ................... PrimerF 377843 ...C............... PrimerR 16598 ...................T PrimerR 89407 .............W...... PrimerR 326756 .................... - - - KO10M: N(allPCRHits)= 401521 (-50143) KO10M: N(SingleHits)= 401517 (-50147) KO10M: N(CleanHits) = 398631 (-53033) ◇ Amplicon Orientation: 401517 KO10M-PF KO10M-PR 4 KO10M-PR KO10M-PF ◇ Mis-Matches (top3): PrimerF 26624 ...C.............A. PrimerF 50009 ................... PrimerF 316328 ...C............... PrimerR 8121 .................A.. PrimerR 97098 .............W...... PrimerR 286135 .................... - - - KO11F: N(allPCRHits)= 611079 (-23966) KO11F: N(SingleHits)= 611075 (-23970) KO11F: N(CleanHits) = 587008 (-48037) ◇ Amplicon Orientation: 611079 KO11F-PF KO11F-PR ◇ Mis-Matches (top3): PrimerF 1808 ...C.............A. PrimerF 83969 ................... PrimerF 518831 ...C............... PrimerR 18396 ...................T PrimerR 124625 .............W...... PrimerR 453818 .................... - - - KO11M: N(allPCRHits)= 425621 (-54058) KO11M: N(SingleHits)= 425615 (-54064) KO11M: N(CleanHits) = 416516 (-63163) ◇ Amplicon Orientation: 425616 KO11M-PF KO11M-PR 5 KO11M-PR KO11M-PF ◇ Mis-Matches (top3): PrimerF 42774 ...C.............A. PrimerF 49230 ................... PrimerF 322464 ...C............... PrimerR 5599 ...................T PrimerR 99581 .............W...... PrimerR 310289 .................... - - - NTCF: N(allPCRHits)= 247 (-17) NTCF: N(SingleHits)= 247 (-17) NTCF: N(CleanHits) = 243 (-21) ◇ Amplicon Orientation: 247 NTCF-PF NTCF-PR ◇ Mis-Matches (top3): PrimerF 3 ...C.............A. PrimerF 34 ................... PrimerF 206 ...C............... PrimerR 3 ...................T PrimerR 54 .............W...... PrimerR 182 .................... - - - NTCM: N(allPCRHits)= 21539 (-699) NTCM: N(SingleHits)= 21539 (-699) NTCM: N(CleanHits) = 20898 (-1340) ◇ Amplicon Orientation: 21539 NTCM-PF NTCM-PR ◇ Mis-Matches (top3): PrimerF 551 ...C.............A. PrimerF 2737 ................... PrimerF 17944 ...C............... PrimerR 469 ...................T PrimerR 4891 .............W...... PrimerR 15742 .................... - - - POSF: N(allPCRHits)= 336425 (-11999) POSF: N(SingleHits)= 336399 (-12025) POSF: N(CleanHits) = 336107 (-12317) ◇ Amplicon Orientation: 336425 POSF-PF POSF-PR ◇ Mis-Matches (top3): PrimerF 580 ...C..........G.... PrimerF 45085 ................... PrimerF 287608 ...C............... PrimerR 553 ............T....... PrimerR 71316 .............W...... PrimerR 260185 .................... - - - POSM: N(allPCRHits)= 419681 (-12312) POSM: N(SingleHits)= 419675 (-12318) POSM: N(CleanHits) = 419244 (-12749) ◇ Amplicon Orientation: 419680 POSM-PF POSM-PR 1 POSM-PR POSM-PF ◇ Mis-Matches (top3): PrimerF 665 ...C..........G.... PrimerF 55245 ................... PrimerF 359923 ...C............... PrimerR 585 ............T....... PrimerR 103421 .............W...... PrimerR 310850 .................... - - - WT01F: N(allPCRHits)= 476783 (-19852) WT01F: N(SingleHits)= 476777 (-19858) WT01F: N(CleanHits) = 476298 (-20337) ◇ Amplicon Orientation: 476781 WT01F-PF WT01F-PR 2 WT01F-PR WT01F-PF ◇ Mis-Matches (top3): PrimerF 663 ...C..........G.... PrimerF 64938 ................... PrimerF 406645 ...C............... PrimerR 8427 .................A.. PrimerR 99766 .............W...... PrimerR 360015 .................... - - - WT01M: N(allPCRHits)= 343707 (-99661) WT01M: N(SingleHits)= 343691 (-99677) WT01M: N(CleanHits) = 338460 (-104908) ◇ Amplicon Orientation: 343707 WT01M-PF WT01M-PR ◇ Mis-Matches (top3): PrimerF 38242 ................... PrimerF 50671 ...C.............A. PrimerF 242002 ...C............... PrimerR 6506 .................A.. PrimerR 83252 .............W...... PrimerR 242418 .................... - - - WT02F: N(allPCRHits)= 583826 (-23280) WT02F: N(SingleHits)= 583820 (-23286) WT02F: N(CleanHits) = 583280 (-23826) ◇ Amplicon Orientation: 583820 WT02F-PF WT02F-PR 6 WT02F-PR WT02F-PF ◇ Mis-Matches (top3): PrimerF 806 ...C..........G.... PrimerF 79473 ................... PrimerF 498005 ...C............... PrimerR 12601 .................A.. PrimerR 121097 .............W...... PrimerR 439285 .................... - - - WT02M: N(allPCRHits)= 303430 (-95489) WT02M: N(SingleHits)= 303424 (-95495) WT02M: N(CleanHits) = 298144 (-100775) ◇ Amplicon Orientation: 303428 WT02M-PF WT02M-PR 2 WT02M-PR WT02M-PF ◇ Mis-Matches (top3): PrimerF 33969 ................... PrimerF 44100 ...C.............A. PrimerF 214101 ...C............... PrimerR 4150 .................A.. PrimerR 73856 .............W...... PrimerR 215313 .................... - - - WT03F: N(allPCRHits)= 442968 (-19190) WT03F: N(SingleHits)= 442960 (-19198) WT03F: N(CleanHits) = 442516 (-19642) ◇ Amplicon Orientation: 442967 WT03F-PF WT03F-PR 1 WT03F-PR WT03F-PF ◇ Mis-Matches (top3): PrimerF 638 ...C..........G.... PrimerF 60526 ................... PrimerF 377771 ...C............... PrimerR 3001 .................A.. PrimerR 93663 .............W...... PrimerR 339596 .................... - - - WT03M: N(allPCRHits)= 412581 (-46475) WT03M: N(SingleHits)= 412577 (-46479) WT03M: N(CleanHits) = 408768 (-50288) ◇ Amplicon Orientation: 412569 WT03M-PF WT03M-PR 12 WT03M-PR WT03M-PF ◇ Mis-Matches (top3): PrimerF 13876 ...C.............A. PrimerF 53815 ................... PrimerF 337856 ...C............... PrimerR 5227 .................A.. PrimerR 103044 .............W...... PrimerR 293601 .................... - - - WT04F: N(allPCRHits)= 363158 (-15265) WT04F: N(SingleHits)= 363152 (-15271) WT04F: N(CleanHits) = 362761 (-15662) ◇ Amplicon Orientation: 363157 WT04F-PF WT04F-PR 1 WT04F-PR WT04F-PF ◇ Mis-Matches (top3): PrimerF 492 ...C..........G.... PrimerF 49617 ................... PrimerF 309699 ...C............... PrimerR 1652 .................A.. PrimerR 75921 .............W...... PrimerR 279891 .................... - - - WT04M: N(allPCRHits)= 378275 (-50834) WT04M: N(SingleHits)= 378271 (-50838) WT04M: N(CleanHits) = 371824 (-57285) ◇ Amplicon Orientation: 378270 WT04M-PF WT04M-PR 5 WT04M-PR WT04M-PF ◇ Mis-Matches (top3): PrimerF 14134 ...C.............A. PrimerF 49431 ................... PrimerF 307868 ...C............... PrimerR 3613 ...................T PrimerR 94608 .............W...... PrimerR 269833 .................... - - - WT05F: N(allPCRHits)= 444784 (-19442) WT05F: N(SingleHits)= 444784 (-19442) WT05F: N(CleanHits) = 425484 (-38742) ◇ Amplicon Orientation: 444783 WT05F-PF WT05F-PR 1 WT05F-PR WT05F-PF ◇ Mis-Matches (top3): PrimerF 622 ...C.............A. PrimerF 60932 ................... PrimerF 378715 ...C............... PrimerR 14717 ...................T PrimerR 90322 .............W...... PrimerR 329446 .................... - - - WT05M: N(allPCRHits)= 423256 (-55098) WT05M: N(SingleHits)= 423246 (-55108) WT05M: N(CleanHits) = 419914 (-58440) ◇ Amplicon Orientation: 423251 WT05M-PF WT05M-PR 5 WT05M-PR WT05M-PF ◇ Mis-Matches (top3): PrimerF 35354 ...C.............A. PrimerF 51080 ................... PrimerF 326469 ...C............... PrimerR 9678 .................A.. PrimerR 99866 .............W...... PrimerR 302225 .................... - - - WT06F: N(allPCRHits)= 474258 (-20538) WT06F: N(SingleHits)= 474252 (-20544) WT06F: N(CleanHits) = 456985 (-37811) ◇ Amplicon Orientation: 474253 WT06F-PF WT06F-PR 5 WT06F-PR WT06F-PF ◇ Mis-Matches (top3): PrimerF 655 ...C..........G.... PrimerF 65202 ................... PrimerF 403979 ...C............... PrimerR 13295 ...................T PrimerR 96757 .............W...... PrimerR 354744 .................... - - - WT06M: N(allPCRHits)= 498913 (-21470) WT06M: N(SingleHits)= 498911 (-21472) WT06M: N(CleanHits) = 487912 (-32471) ◇ Amplicon Orientation: 498901 WT06M-PF WT06M-PR 12 WT06M-PR WT06M-PF ◇ Mis-Matches (top3): PrimerF 8342 ...C.............A. PrimerF 65930 ................... PrimerF 417956 ...C............... PrimerR 7996 ...................T PrimerR 118879 .............W...... PrimerR 360460 .................... - - - WT07F: N(allPCRHits)= 443702 (-19078) WT07F: N(SingleHits)= 443700 (-19080) WT07F: N(CleanHits) = 421828 (-40952) ◇ Amplicon Orientation: 443698 WT07F-PF WT07F-PR 4 WT07F-PR WT07F-PF ◇ Mis-Matches (top3): PrimerF 1920 ...C.............A. PrimerF 60409 ................... PrimerF 376723 ...C............... PrimerR 16842 ...................T PrimerR 89811 .............W...... PrimerR 327288 .................... - - - WT07M: N(allPCRHits)= 366528 (-86850) WT07M: N(SingleHits)= 366526 (-86852) WT07M: N(CleanHits) = 360079 (-93299) ◇ Amplicon Orientation: 366526 WT07M-PF WT07M-PR 2 WT07M-PR WT07M-PF ◇ Mis-Matches (top3): PrimerF 35372 ................... PrimerF 85811 ...C.............A. PrimerF 227986 ...C............... PrimerR 2695 ...................T PrimerR 86529 .............W...... PrimerR 268934 .................... - - - WT08F: N(allPCRHits)= 481491 (-20655) WT08F: N(SingleHits)= 481479 (-20667) WT08F: N(CleanHits) = 465844 (-36302) ◇ Amplicon Orientation: 481487 WT08F-PF WT08F-PR 4 WT08F-PR WT08F-PF ◇ Mis-Matches (top3): PrimerF 655 ...C..........G.... PrimerF 65957 ................... PrimerF 409984 ...C............... PrimerR 11851 ...................T PrimerR 98318 .............W...... PrimerR 361820 .................... - - - WT08M: N(allPCRHits)= 464210 (-51001) WT08M: N(SingleHits)= 464206 (-51005) WT08M: N(CleanHits) = 453557 (-61654) ◇ Amplicon Orientation: 464203 WT08M-PF WT08M-PR 7 WT08M-PR WT08M-PF ◇ Mis-Matches (top3): PrimerF 37531 ...C.............A. PrimerF 57015 ................... PrimerF 358431 ...C............... PrimerR 6842 ...................T PrimerR 112123 .............W...... PrimerR 333854 .................... - - - WT09F: N(allPCRHits)= 468537 (-19380) WT09F: N(SingleHits)= 468527 (-19390) WT09F: N(CleanHits) = 443985 (-43932) ◇ Amplicon Orientation: 468536 WT09F-PF WT09F-PR 1 WT09F-PR WT09F-PF ◇ Mis-Matches (top3): PrimerF 636 ...C..........G.... PrimerF 64595 ................... PrimerF 399051 ...C............... PrimerR 18823 ...................T PrimerR 93243 .............W...... PrimerR 344203 .................... - - - WT09M: N(allPCRHits)= 400994 (-24453) WT09M: N(SingleHits)= 400982 (-24465) WT09M: N(CleanHits) = 393821 (-31626) ◇ Amplicon Orientation: 400989 WT09M-PF WT09M-PR 5 WT09M-PR WT09M-PF ◇ Mis-Matches (top3): PrimerF 15258 ...C.............A. PrimerF 51718 ................... PrimerF 327241 ...C............... PrimerR 4876 ...................T PrimerR 96560 .............W...... PrimerR 291491 .................... - - - WT10F: N(allPCRHits)= 532174 (-22246) WT10F: N(SingleHits)= 532170 (-22250) WT10F: N(CleanHits) = 509334 (-45086) ◇ Amplicon Orientation: 532170 WT10F-PF WT10F-PR 4 WT10F-PR WT10F-PF ◇ Mis-Matches (top3): PrimerF 1151 ...C.............A. PrimerF 72098 ................... PrimerF 453469 ...C............... PrimerR 17499 ...................T PrimerR 107549 .............W...... PrimerR 395084 .................... - - - WT10M: N(allPCRHits)= 458411 (-27537) WT10M: N(SingleHits)= 458399 (-27549) WT10M: N(CleanHits) = 457043 (-28905) ◇ Amplicon Orientation: 458409 WT10M-PF WT10M-PR 2 WT10M-PR WT10M-PF ◇ Mis-Matches (top3): PrimerF 10311 ...C.............A. PrimerF 58548 ................... PrimerF 382975 ...C............... PrimerR 2246 .................A.. PrimerR 108961 .............W...... PrimerR 338186 .................... - - - ................................................................................ END_In-Silico_PCR: 16:41:24 16/07/2020 ========================================================================================== Step [D] - Size Selection and Quality Filtering ------------------------------------------------------------------------------------------ Application : PRINSEQ-lite 0.20.4 Size Range : 150-350 GC Range : 30-70 Min Q Mean : 20 Number of Ns : 0 Low Complexity: dust / 30 -------------------------------------------------------------------------------- START_QualityFiltering: 15:09:43 20/07/2020 ................................................................................ Start_QF: 15:09:43 20/07/2020 ➼ KO01F: N(filtered)= 496185 (-329) ➼ KO01M: N(filtered)= 378162 (-667) ➼ KO02F: N(filtered)= 468522 (-393) ➼ KO02M: N(filtered)= 350902 (-1066) ➼ KO03F: N(filtered)= 451283 (-349) ➼ KO03M: N(filtered)= 422157 (-704) ➼ KO04F: N(filtered)= 463312 (-294) ➼ KO04M: N(filtered)= 290174 (-631) ➼ KO05F: N(filtered)= 419530 (-339) ➼ KO05M: N(filtered)= 346584 (-1078) ➼ KO06F: N(filtered)= 410236 (-303) ➼ KO06M: N(filtered)= 453815 (-894) ➼ KO07F: N(filtered)= 422070 (-339) ➼ KO07M: N(filtered)= 448270 (-1071) ➼ KO08F: N(filtered)= 530315 (-365) ➼ KO08M: N(filtered)= 429466 (-499) ➼ KO09F: N(filtered)= 448689 (-339) ➼ KO09M: N(filtered)= 421278 (-555) ➼ KO10F: N(filtered)= 421874 (-321) ➼ KO10M: N(filtered)= 397426 (-1205) ➼ KO11F: N(filtered)= 586565 (-443) ➼ KO11M: N(filtered)= 415060 (-1456) ➼ NTCF: N(filtered)= 243 (0) ➼ NTCM: N(filtered)= 20879 (-19) ➼ POSF: N(filtered)= 335892 (-215) ➼ POSM: N(filtered)= 418954 (-290) ➼ WT01F: N(filtered)= 475933 (-365) ➼ WT01M: N(filtered)= 336616 (-1844) ➼ WT02F: N(filtered)= 582850 (-430) ➼ WT02M: N(filtered)= 296501 (-1643) ➼ WT03F: N(filtered)= 442201 (-315) ➼ WT03M: N(filtered)= 407923 (-845) ➼ WT04F: N(filtered)= 362549 (-212) ➼ WT04M: N(filtered)= 370984 (-840) ➼ WT05F: N(filtered)= 425170 (-314) ➼ WT05M: N(filtered)= 418497 (-1417) ➼ WT06F: N(filtered)= 456627 (-358) ➼ WT06M: N(filtered)= 487301 (-611) ➼ WT07F: N(filtered)= 421525 (-303) ➼ WT07M: N(filtered)= 357430 (-2649) ➼ WT08F: N(filtered)= 465445 (-399) ➼ WT08M: N(filtered)= 452144 (-1413) ➼ WT09F: N(filtered)= 443657 (-328) ➼ WT09M: N(filtered)= 393117 (-704) ➼ WT10F: N(filtered)= 508947 (-387) ➼ WT10M: N(filtered)= 456354 (-689) End_QF: 16:12:24 20/07/2020 ................................................................................ Start_CombineSequences: 16:12:24 20/07/2020 ➼ N(all) : 18809614 (== 18809614) ➼ N(clean): 18229722 (d=579892) End_CombineSequences: 16:14:00 20/07/2020 ................................................................................ Start_ReadReport: 16:14:00 20/07/2020 End_ReadReport: 16:22:33 20/07/2020 ................................................................................ END_QualityFiltering: 16:22:33 20/07/2020 ========================================================================================== Step [D1] - Read-Stats ------------------------------------------------------------------------------------------ Sample : Sample ID Raw : Number of raw reads NoPhiX : Number of reads after step [B] Merged : Number of reads after step [B] Primer : Number of reads after step [C] Clean : Number of reads after step [D] MeanLength: Average trimmed amplicon length ------------------------------------------------------------------------------------------ Sample;Raw;NoPhiX;Merged;Primer;Clean;MeanLength KO01F;594207;594200;533717;496514;496185;252.519 KO01M;457301;457294;418060;378829;378162;251.857 KO02F;540690;540674;497527;468915;468522;253.366 KO02M;478696;478684;430541;351968;350902;246.918 KO03F;531443;531430;475439;451632;451283;255.214 KO03M;518709;518699;458354;422861;422157;251.394 KO04F;561194;561186;495762;463606;463312;252.663 KO04M;358742;358733;319371;290805;290174;249.174 KO05F;485261;485255;444340;419869;419530;254.331 KO05M;473505;473490;418773;347662;346584;246.231 KO06F;493467;493458;448524;410539;410236;252.714 KO06M;538072;538060;489296;454709;453815;249.79 KO07F;498262;498253;452179;422409;422070;252.678 KO07M;548036;548023;497788;449341;448270;249.077 KO08F;639622;639611;580006;530680;530315;252.674 KO08M;519860;519852;464589;429965;429466;251.198 KO09F;545616;545607;483315;449028;448689;252.712 KO09M;481514;481510;447678;421833;421278;252.12 KO10F;515819;515815;461226;422195;421874;252.753 KO10M;485020;485012;451664;398631;397426;248.144 KO11F;708413;708401;635045;587008;586565;252.799 KO11M;542819;542796;479679;416516;415060;243.261 NTCF;2187;2186;264;243;243;251.775 NTCM;25078;25078;22238;20898;20879;252.796 POSF;390164;390158;348424;336107;335892;251.997 POSM;476732;476725;431993;419244;418954;252.014 WT01F;550497;550489;496635;476298;475933;253.986 WT01M;498899;498882;443368;338460;336616;240.178 WT02F;674153;674145;607106;583280;582850;254.163 WT02M;453202;453175;398919;298144;296501;239.944 WT03F;523879;523866;462158;442516;442201;253.099 WT03M;512734;512725;459056;408768;407923;250.7 WT04F;436999;436992;378423;362761;362549;252.895 WT04M;485583;485572;429109;371824;370984;249.684 WT05F;512604;512593;464226;425484;425170;252.633 WT05M;525220;525206;478354;419914;418497;246.818 WT06F;545087;545072;494796;456985;456627;252.68 WT06M;567000;566992;520383;487912;487301;251.513 WT07F;505826;505820;462780;421828;421525;252.686 WT07M;501510;501495;453378;360079;357430;230.289 WT08F;557750;557735;502146;465844;465445;252.606 WT08M;568982;568968;515211;453557;452144;245.161 WT09F;545264;545258;487917;443985;443657;252.661 WT09M;463078;463074;425447;393821;393117;249.339 WT10F;620633;620618;554420;509334;508947;252.663 WT10M;526193;526190;485948;457043;456354;251.232 ========================================================================================== Step [E] - Clustering - UPARSE for OTUs & UNOISE for ZOTUs ------------------------------------------------------------------------------------------ N(sequences): 18229722 -------------------------------------------------------------------------------- UPARSE : usearch v11.0.667_i86linux64 Min Abundance Size: 2 ................................................................................ UNOISE3: usearch v11.0.667_i86linux64 Min Abundance Size: 8 -------------------------------------------------------------------------------- START_(Z)OTU: 17:12:57 30/07/2020 -------------------------------------------------------------------------------- ▶ Deduplicate Amplicons ................................................................................ De-replicate amplicons to obtain unique amplicons. Determine error rates of amplicon reads > *error.report ⇨ Number of Unique Amplicons: 532047 -------------------------------------------------------------------------------- ▶ UPARSE - Cluster OTU (97%) ................................................................................ Clusters OTU at 97% using the UPARSE-OTU algorithm. ⇨ Number of OTUs: 676 -------------------------------------------------------------------------------- ▶ UNOISE3 - Amplicon Sequence Variants ................................................................................ Uses the UNOISE algorithm to perform denoising (error-correction) of amplicon sequence variants (zero OTUs). ⇨ Number of ZOTUs: 692 -------------------------------------------------------------------------------- ▶ Additional Clustering ................................................................................ Clusters ZOTUs at different identity levels (i.e. 97%,98% and 99%). • Sort ZOTUs ⇨ Number of ZOTUs 99%: 565 ⇨ Number of ZOTUs 98%: 503 ⇨ Number of ZOTUs 97%: 463 -------------------------------------------------------------------------------- ▶ Count Table ................................................................................ Generate count tables by mapping reads to (Z)OTUs. -------------------------------------------------------------------------------- ▶ ZOTU Table Report ................................................................................ Create summary-reports for the (Z)OTU tables > *_Count.summary -------------------------------------------------------------------------------- ▶ Distance matrices (for Octave plots) ................................................................................ Pairwise distances between (Z)OTU sequences. -------------------------------------------------------------------------------- ▶ Octave plots ................................................................................ Octave plots with low-abundance (Z)OTUs and cross-talk information. -------------------------------------------------------------------------------- ▶ Approximate Phylogenetic Trees ................................................................................ Create tree files. The trees will be very approximate in both cases. ◦ Multiple Sequence Alignments (MSA) MUSCLE v3.8.1551 by Robert C. Edgar ◦ Cluster Trees (CLU) usearch v11.0.667_i86linux64 -------------------------------------------------------------------------------- ▶ Uncross ................................................................................ Detects and filters cross-talk (sample mis-assignment) in a OTU table using the UNCROSS algorithm. -------------------------------------------------------------------------------- ▶ Back-Mapping Efficiency ................................................................................ N(Amplicons) = 18229722 N(Map-2-OTU) = 17968021 (.985) N(Map-2-ZOTU) = 18002811 (.987) N(Map-2-ZOTU_c99) = 17995375 (.987) N(Map-2-ZOTU_c98) = 17981570 (.986) N(Map-2-ZOTU_c97) = 17898490 (.981) -------------------------------------------------------------------------------- END_(Z)OTU: 03:02:18 31/07/2020 ========================================================================================== Step [F] - Taxonomic Assignment-Predictions with SINTAX ------------------------------------------------------------------------------------------ Application : SINTAX (v11.0.667_i86linux64) Reference : SILVA_128_16S_utax_work.fa Tax Filter : 0.85 ========================================================================================== Tax-Prediction Workflow Summary: (F1) Prepare Reference (F2) Assign Taxa with SINTAX (F3) Reformat Tax-Information (F4) Combine Count-Table and Taxa for Phyloseq Import (F5) Tax-Assignment Report for Phylum Level (F6) Additional Chimera Check (Not Needed) -------------------------------------------------- START_Tax-Prediction: 17:21:15 31/07/2020 Start_F1_Restrict_Reference: 17:21:15 31/07/2020 ‣Number of Reference Sequences: 341146 ‣OTU related References: 965744 ‣ZOTU related References: 964326 End_F1_Restrict_Reference: 17:44:45 31/07/2020 -------------------------------------------------- Start_F1_Unique_Records: 17:44:45 31/07/2020 ‣OTU: N(seq)= 965744 / N(uniques)= 229679 / N(singletons)= 2033 (0.9%) ‣OTU: Min size 1 / median 3 / max 2154 / avg 4.20 ‣ZOTU: N(seq)= 964326 / N(uniques)= 229147 / N(singletons)= 1862 (0.8%) ‣ZOTU: Min size 1 / median 3 / max 2154 / avg 4.21 End_F1_Unique_Records: 17:45:01 31/07/2020 -------------------------------------------------- Start_F1_Build_UPD_Reference: 17:45:01 31/07/2020 End_F1_Build_UPD_Reference: 17:45:12 31/07/2020 -------------------------------------------------- Start_F2_Tax-Assignment: 17:45:12 31/07/2020 End_F2_Tax-Assignment: 17:52:00 31/07/2020 -------------------------------------------------- Start_F3_Re-Format: 17:52:00 31/07/2020 End_F3_Re-Format: 17:52:00 31/07/2020 -------------------------------------------------- Start_F4_Combine_CountTable_TaxLabel: 17:52:00 31/07/2020 End_F4_Combine_CountTable_TaxLabel: 17:52:00 31/07/2020 -------------------------------------------------- Start_F5_Tax-Summary: 17:52:00 31/07/2020 Tax Summary for OTU: Firmicutes 306 45.3 45.3 Proteobacteria 97 14.3 59.6 Bacteroidetes 95 14.1 73.7 (Unassigned) 70 10.4 84.0 Actinobacteria 51 7.5 91.6 Cyanobacteria 14 2.1 93.6 Planctomycetes 11 1.6 95.3 Tenericutes 5 0.7 96.0 Saccharibacteria 4 0.6 96.6 Deferribacteres 4 0.6 97.2 Acidobacteria 4 0.6 97.8 Chloroflexi 3 0.4 98.2 Armatimonadetes 3 0.4 98.7 Metamonada 3 0.4 99.1 Deinococcus-Thermus 2 0.3 99.4 Chlamydiae 2 0.3 99.7 Verrucomicrobia 1 0.1 99.9 Fusobacteria 1 0.1 100.0 Tax Summary for ZOTU: Firmicutes 373 53.9 53.9 Bacteroidetes 155 22.4 76.3 Proteobacteria 73 10.5 86.8 Actinobacteria 34 4.9 91.8 (Unassigned) 20 2.9 94.7 Cyanobacteria 16 2.3 97.0 Saccharibacteria 4 0.6 97.5 Planctomycetes 4 0.6 98.1 Metamonada 3 0.4 98.6 Tenericutes 2 0.3 98.8 Acidobacteria 2 0.3 99.1 Chlamydiae 2 0.3 99.4 Deferribacteres 2 0.3 99.7 Verrucomicrobia 1 0.1 99.9 Armatimonadetes 1 0.1 100.0 End_F5_Tax-Summary: 17:52:00 31/07/2020 -------------------------------------------------- Start_F6_Chimera-Check: 17:52:00 31/07/2020 Additional Chimera-Check Note: Although the high_confidence mode is used for chimera detection, this is an extra step with a high risk of false positives. There is already a sufficient built-in de novo chimera filtering as part of the UPARSE or UNOISE workflow. This extra step might be useful if applied with caution. For details see -> e_OTU/p700_run200710_16S_(Z)OTU_chimera.txt .................................................. Chimera-Check for OTU: 21 Chimera-Check for ZOTU: 28 Chimera-Check for ZOTU_c99: 26 Chimera-Check for ZOTU_c98: 23 Chimera-Check for ZOTU_c97: 21 End_F6_Chimera-Check: 17:52:32 31/07/2020 -------------------------------------------------- END_Tax-Prediction: 17:52:32 31/07/2020 ==========================================================================================