CRISPResso version 2.2.12 [Command used]: /Users/idala384/anaconda3/bin/CRISPResso --fastq_r1 CS5KO_HT_FKDN230242857-1A_HC23NDRX3_L2_1_SOX6_WT.fq --fastq_r2 CS5KO_HT_FKDN230242857-1A_HC23NDRX3_L2_2_SOX6_WT.fq --amplicon_seq ACCTGGTAGTTGTTTCATGATCTACACAAAAATAACTCTAAGGTCATCTATTTCCCTAAAATATCTATTTATAATGAGGACATTCTAAGGAATGCATTTAGTGGCAGTGGTCAGATTTTAAAGGCTCACCATTCTTTGCTGAGATGACAGAACGCTGTCCCAGTCAGCATCTTGTTGAATGGTACTGACAAGTGTTGGTAGCTCCTCAGAGTGAGGTTTGTTGTGCATTATGGGGTGCAGAGGCAGATGGGAGGCCACATGTTGATCACTGCCCTCTTCCTTTTCCCTTGAGGTTAAATCCTGGGTCATTGCATCCTCTCCATCAGCTGCACAGGCAAATGGAGAGGTGGCTTGCTTGGAAGACATTCTTCTGCAGAAAAAAAACAGAACGGATTAAAAATGGCTGTCAATAACAAACCATAAACCAATCCTTGCATTTGGCTAGTTCAGGAAAGAAGGAAAGTTATTTAACTTTAGAGATATTTGTGGTCCACTCTAAACCCCTG --guide_seq TATGGGGTGCAGAGGCAGAT,TTCCCTTGAGGTTAAATCCT,AAATGGAGAGGTGGCTTGCT --fastq_output [Execution log]: Writing fastq output file: CRISPResso_on_CS5KO_HT_FKDN230242857-1A_HC23NDRX3_L2_1_SOX6_WT_CS5KO_HT_FKDN230242857-1A_HC23NDRX3_L2_2_SOX6_WT/CRISPResso_output.fastq.gz Computing quantification windows Estimating average read length... Merging paired sequences with Flash... Running FLASH command: flash "CS5KO_HT_FKDN230242857-1A_HC23NDRX3_L2_1_SOX6_WT.fq" "CS5KO_HT_FKDN230242857-1A_HC23NDRX3_L2_2_SOX6_WT.fq" --min-overlap 10 --max-overlap 100 --allow-outies -z -d CRISPResso_on_CS5KO_HT_FKDN230242857-1A_HC23NDRX3_L2_1_SOX6_WT_CS5KO_HT_FKDN230242857-1A_HC23NDRX3_L2_2_SOX6_WT -o out >>CRISPResso_on_CS5KO_HT_FKDN230242857-1A_HC23NDRX3_L2_1_SOX6_WT_CS5KO_HT_FKDN230242857-1A_HC23NDRX3_L2_2_SOX6_WT/CRISPResso_RUNNING_LOG.txt 2>&1 [FLASH] Starting FLASH v1.2.11 [FLASH] Fast Length Adjustment of SHort reads [FLASH] [FLASH] Input files: [FLASH] CS5KO_HT_FKDN230242857-1A_HC23NDRX3_L2_1_SOX6_WT.fq [FLASH] CS5KO_HT_FKDN230242857-1A_HC23NDRX3_L2_2_SOX6_WT.fq [FLASH] [FLASH] Output files: [FLASH] CRISPResso_on_CS5KO_HT_FKDN230242857-1A_HC23NDRX3_L2_1_SOX6_WT_CS5KO_HT_FKDN230242857-1A_HC23NDRX3_L2_2_SOX6_WT/out.extendedFrags.fastq.gz [FLASH] CRISPResso_on_CS5KO_HT_FKDN230242857-1A_HC23NDRX3_L2_1_SOX6_WT_CS5KO_HT_FKDN230242857-1A_HC23NDRX3_L2_2_SOX6_WT/out.notCombined_1.fastq.gz [FLASH] CRISPResso_on_CS5KO_HT_FKDN230242857-1A_HC23NDRX3_L2_1_SOX6_WT_CS5KO_HT_FKDN230242857-1A_HC23NDRX3_L2_2_SOX6_WT/out.notCombined_2.fastq.gz [FLASH] CRISPResso_on_CS5KO_HT_FKDN230242857-1A_HC23NDRX3_L2_1_SOX6_WT_CS5KO_HT_FKDN230242857-1A_HC23NDRX3_L2_2_SOX6_WT/out.hist [FLASH] CRISPResso_on_CS5KO_HT_FKDN230242857-1A_HC23NDRX3_L2_1_SOX6_WT_CS5KO_HT_FKDN230242857-1A_HC23NDRX3_L2_2_SOX6_WT/out.histogram [FLASH] [FLASH] Parameters: [FLASH] Min overlap: 10 [FLASH] Max overlap: 100 [FLASH] Max mismatch density: 0.250000 [FLASH] Allow "outie" pairs: true [FLASH] Cap mismatch quals: false [FLASH] Combiner threads: 16 [FLASH] Input format: FASTQ, phred_offset=33 [FLASH] Output format: FASTQ, phred_offset=33, gzip [FLASH] [FLASH] Starting reader and writer threads [FLASH] Starting 16 combiner threads [FLASH] Processed 25000 read pairs [FLASH] Processed 50000 read pairs [FLASH] Processed 75000 read pairs [FLASH] Processed 100000 read pairs [FLASH] Processed 125000 read pairs [FLASH] Processed 150000 read pairs [FLASH] Processed 175000 read pairs [FLASH] Processed 200000 read pairs [FLASH] Processed 225000 read pairs [FLASH] Processed 250000 read pairs [FLASH] Processed 275000 read pairs [FLASH] Processed 300000 read pairs [FLASH] Processed 325000 read pairs [FLASH] Processed 350000 read pairs [FLASH] Processed 375000 read pairs [FLASH] Processed 400000 read pairs [FLASH] Processed 425000 read pairs [FLASH] Processed 450000 read pairs [FLASH] Processed 475000 read pairs [FLASH] Processed 500000 read pairs [FLASH] Processed 525000 read pairs [FLASH] Processed 550000 read pairs [FLASH] Processed 575000 read pairs [FLASH] Processed 600000 read pairs [FLASH] Processed 625000 read pairs [FLASH] Processed 650000 read pairs [FLASH] Processed 675000 read pairs [FLASH] Processed 700000 read pairs [FLASH] Processed 725000 read pairs [FLASH] Processed 750000 read pairs [FLASH] Processed 775000 read pairs [FLASH] Processed 800000 read pairs [FLASH] Processed 825000 read pairs [FLASH] Processed 850000 read pairs [FLASH] Processed 875000 read pairs [FLASH] Processed 900000 read pairs [FLASH] Processed 922081 read pairs [FLASH] [FLASH] Read combination statistics: [FLASH] Total pairs: 922081 [FLASH] Combined pairs: 5020 [FLASH] Innie pairs: 3359 (66.91% of combined) [FLASH] Outie pairs: 1661 (33.09% of combined) [FLASH] Uncombined pairs: 917061 [FLASH] Percent combined: 0.54% [FLASH] [FLASH] Writing histogram files. [FLASH] WARNING: An unexpectedly high proportion of combined pairs (63.61%) overlapped by more than 100 bp, the --max-overlap (-M) parameter. Consider increasing this parameter. (As-is, FLASH is penalizing overlaps longer than 100 bp when considering them for possible combining!) [FLASH] [FLASH] FLASH v1.2.11 complete! [FLASH] 27.767 seconds elapsed [FLASH] Finished with 1 warning (see above) Done! Aligning sequences... Processing reads; N_TOT_READS: 0 N_COMPUTED_ALN: 0 N_CACHED_ALN: 0 N_COMPUTED_NOTALN: 0 N_CACHED_NOTALN: 0 Finished reads; N_TOT_READS: 5020 N_COMPUTED_ALN: 1102 N_CACHED_ALN: 1404 N_COMPUTED_NOTALN: 1574 N_CACHED_NOTALN: 940 Done! Quantifying indels/substitutions... Done! Calculating allele frequencies... Done! Saving processed data... Making Plots... Plotting read bar plot Plotting read class pie chart and bar plot Begin processing plots for amplicon Reference Plotting nucleotide quilt across amplicon Plotting nucleotide distribuition around sgRNA TATGGGGTGCAGAGGCAGAT for Reference Plotting nucleotide distribuition around sgRNA TTCCCTTGAGGTTAAATCCT for Reference Plotting nucleotide distribuition around sgRNA AAATGGAGAGGTGGCTTGCT for Reference Plotting indel size distribution for Reference Plotting frequency deletions/insertions for Reference Plotting amplication modifications for Reference Plotting modification frequency for Reference Plotting quantification window locations for Reference Plotting position dependent indel for Reference Plotting allele distribution around cut for Reference Plotting allele distribution around cut for Reference Plotting allele distribution around cut for Reference Done! Done! Removing Intermediate files... Analysis Complete!