Bowtie 2 seems to be working fine (tested command '/home/shared/bowtie2-2.4.4-linux-x86_64/bowtie2 --version' [2.4.4]) Output format is BAM (default) Alignments will be written out in BAM format. Samtools found here: '/usr/bin/samtools' Reference genome folder provided is ../data/genome/ (absolute path is '/home/shared/16TB_HDD_01/sr320/github/Claudia-Halibut/project/data/genome/)' FastQ format assumed (by default) Attention: using more than 4 cores per alignment thread has been reported to have diminishing returns. If possible try to limit -p to a value of 4 Each Bowtie 2 instance is going to be run with 8 threads. Please monitor performance closely and tune down if necessary! Input files to be analysed (in current folder '/home/shared/16TB_HDD_01/sr320/github/Claudia-Halibut/project/code'): /home/shared/16TB_HDD_01/fish546/data/RRBS-run2/raw-data/E1_T1_F2_R1.fq.gz /home/shared/16TB_HDD_01/fish546/data/RRBS-run2/raw-data/E1_T1_F2_R2.fq.gz Library was specified to be not strand-specific (non-directional), therefore alignments to all four possible bisulfite strands (OT, CTOT, OB and CTOB) will be reported Output will be written into the directory: /home/shared/16TB_HDD_01/sr320/github/Claudia-Halibut/project/output/02/ Setting parallelization to single-threaded (default) Summary of all aligner options: -q --score-min L,0,-0.8 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 Current working directory is: /home/shared/16TB_HDD_01/sr320/github/Claudia-Halibut/project/code Now reading in and storing sequence information of the genome specified in: /home/shared/16TB_HDD_01/sr320/github/Claudia-Halibut/project/data/genome/ chr NC_061483.1 (32845235 bp) chr NC_061484.1 (32262650 bp) chr NC_061485.1 (31235357 bp) chr NC_061486.1 (28897756 bp) chr NC_061487.1 (29170857 bp) chr NC_061488.1 (28229454 bp) chr NC_061489.1 (28174910 bp) chr NC_061490.1 (27367920 bp) chr NC_061491.1 (29329910 bp) chr NC_061492.1 (27288303 bp) chr NC_061493.1 (25468064 bp) chr NC_061494.1 (25132731 bp) chr NC_061495.1 (24770608 bp) chr NC_061496.1 (28479552 bp) chr NC_061497.1 (25155020 bp) chr NC_061498.1 (23268637 bp) chr NC_061499.1 (22875723 bp) chr NC_061500.1 (21182577 bp) chr NC_061501.1 (20810600 bp) chr NC_061502.1 (21255706 bp) chr NC_061503.1 (20593300 bp) chr NC_061504.1 (18680547 bp) chr NC_061505.1 (17159251 bp) chr NC_061506.1 (11267222 bp) chr NW_025899746.1 (458395 bp) chr NW_025899747.1 (32022 bp) chr NW_025899748.1 (786 bp) chr NW_025899749.1 (255824 bp) chr NW_025899750.1 (3031 bp) chr NW_025899751.1 (5290 bp) chr NW_025899752.1 (3054 bp) chr NW_025899753.1 (48340 bp) chr NW_025899754.1 (45079 bp) chr NW_025899755.1 (25155 bp) chr NW_025899756.1 (113933 bp) chr NW_025899757.1 (1843 bp) chr NW_025899758.1 (82466 bp) chr NW_025899759.1 (4916 bp) chr NW_025899760.1 (71054 bp) chr NW_025899761.1 (8568 bp) chr NW_025899762.1 (11567 bp) chr NW_025899763.1 (6375 bp) chr NW_025899764.1 (53904 bp) chr NW_025899767.1 (3932 bp) chr NW_025899768.1 (2857 bp) chr NW_025899769.1 (2018 bp) chr NW_025899770.1 (1484 bp) chr NW_025899771.1 (996 bp) chr NW_025899772.1 (680 bp) chr NW_025899773.1 (673 bp) chr NW_025899774.1 (633 bp) chr NW_025899775.1 (582 bp) chr NC_009710.1 (17841 bp) Single-core mode: setting pid to 1 Paired-end alignments will be performed ======================================= The provided filenames for paired-end alignments are /home/shared/16TB_HDD_01/fish546/data/RRBS-run2/raw-data/E1_T1_F2_R1.fq.gz and /home/shared/16TB_HDD_01/fish546/data/RRBS-run2/raw-data/E1_T1_F2_R2.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file E1_T1_F2_R1.fq.gz to E1_T1_F2_R1.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file E1_T1_F2_R1.fq.gz to E1_T1_F2_R1.fq.gz_G_to_A.fastq Created C -> T as well as G -> A converted versions of the FastQ file E1_T1_F2_R1.fq.gz (72879399 sequences in total) Writing a C -> T converted version of the input file E1_T1_F2_R2.fq.gz to E1_T1_F2_R2.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file E1_T1_F2_R2.fq.gz to E1_T1_F2_R2.fq.gz_G_to_A.fastq Created C -> T as well as G -> A converted versions of the FastQ file E1_T1_F2_R2.fq.gz (72879399 sequences in total) Input files are E1_T1_F2_R1.fq.gz_C_to_T.fastq and E1_T1_F2_R1.fq.gz_G_to_A.fastq and E1_T1_F2_R2.fq.gz_C_to_T.fastq and E1_T1_F2_R2.fq.gz_G_to_A.fastq (FastQ) Now running 4 individual instances of Bowtie 2 against the bisulfite genome of /home/shared/16TB_HDD_01/sr320/github/Claudia-Halibut/project/data/genome/ with the specified options: -q --score-min L,0,-0.8 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 Now starting a Bowtie 2 paired-end alignment for CTread1GAread2CTgenome (reading in sequences from E1_T1_F2_R1.fq.gz_C_to_T.fastq and E1_T1_F2_R2.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-0.8 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: lh00134:396:22JL32LT3:2:1101:3238:1064_1:N:0:CCGCGATT+CTAGCGCT:GGTTAGATA/1 77 * 0 0 * * 0 0 TNAAATTATATTAATTTTAATTAAATATTTTTAATAAATAAATAATTATAAAAATATTAAATTAATTTATATTTTAAATATTAAATTAAAATATTATATTATAAAAATTTTAAATTTTTAAAAAAATTAAGATTGGAAGAGTTTATGTTT I#I9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9II9II9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIII-IIIIIIIIIIIIIIIIIIIIIIII-III-II9 YT:Z:UP lh00134:396:22JL32LT3:2:1101:3238:1064_3:N:0:CCGCGATT+CTAGCGCT:GGTTAGATA/2 141 * 0 0 * * 0 0 TAATTTTTTTAAAAATTTAAAATTTTTATAATATAATATTTTAATTTAATATTTAAAATATAAATTAATTTAATATTTTTATAATTATTTATTTATTAAAAATATTTAATTAAAATTAATATAATTTCAAAATCAAAAAAACATCATATA I-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIII-II-III9IIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIII-IIIIIII9IIIIII-III9I YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from E1_T1_F2_R1.fq.gz_G_to_A.fastq and E1_T1_F2_R2.fq.gz_C_to_T.fastq, with the options: -q --score-min L,0,-0.8 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: lh00134:396:22JL32LT3:2:1101:3238:1064_1:N:0:CCGCGATT+CTAGCGCT:GGTTAGATA/1 99 NC_061487.1_GA_converted 13613858 8 131M2I1M7I3M1I5M = 13613845 -153 CNAAACTACATTAATCTTAACTAAATATTCCTAATAAACAAACAACTACAAAAATATCAAATCAACTCATATTTCAAACATTAAACTAAAATACTACATCACAAAAATCCCAAACCCCTAAAAAAACCAAAATCAAAAAAACTCACATCT I#I9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9II9II9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIII-IIIIIIIIIIIIIIIIIIIIIIII-III-II9 AS:i:-64 XN:i:0 XM:i:4 XO:i:3 XG:i:10 NM:i:14 MD:Z:1A123C7A3C2 YS:i:-57 YT:Z:CP lh00134:396:22JL32LT3:2:1101:3238:1064_3:N:0:CCGCGATT+CTAGCGCT:GGTTAGATA/2 147 NC_061487.1_GA_converted 13613845 8 5M6I3M1I5M1I129M = 13613858 153 TACACAACACTCTTCCAATCTCAAAACTACATTAATCTTAACTAAATATTCCTAATAAACAAACAACTACAAAAATATCAAATCAACTCATATTTCAAACATTAAACTAAAATACTACATCACAAAAATCCCAAACCCCTAAAAAAACCA I9III-IIIIII9IIIIIII-IIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIII9III-II-IIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-I AS:i:-57 XN:i:0 XM:i:3 XO:i:3 XG:i:8 NM:i:11 MD:Z:2T7T127C3 YS:i:-64 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from E1_T1_F2_R1.fq.gz_G_to_A.fastq and E1_T1_F2_R2.fq.gz_C_to_T.fastq, with the options: -q --score-min L,0,-0.8 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --nofw)) Found first alignment: lh00134:396:22JL32LT3:2:1101:3238:1064_1:N:0:CCGCGATT+CTAGCGCT:GGTTAGATA/1 77 * 0 0 * * 0 0 CNAAACTACATTAATCTTAACTAAATATTCCTAATAAACAAACAACTACAAAAATATCAAATCAACTCATATTTCAAACATTAAACTAAAATACTACATCACAAAAATCCCAAACCCCTAAAAAAACCAAAATCAAAAAAACTCACATCT I#I9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9II9II9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIII-IIIIIIIIIIIIIIIIIIIIIIII-III-II9 YT:Z:UP lh00134:396:22JL32LT3:2:1101:3238:1064_3:N:0:CCGCGATT+CTAGCGCT:GGTTAGATA/2 141 * 0 0 * * 0 0 TGGTTTTTTTAGGGGTTTGGGATTTTTGTGATGTAGTATTTTAGTTTAATGTTTGAAATATGAGTTGATTTGATATTTTTGTAGTTGTTTGTTTATTAGGAATATTTAGTTAAGATTAATGTAGTTTTGAGATTGGAAGAGTGTTGTGTA I-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIII-II-III9IIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIII-IIIIIII9IIIIII-III9I YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from E1_T1_F2_R1.fq.gz_C_to_T.fastq and E1_T1_F2_R2.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-0.8 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --nofw)) Found first alignment: lh00134:396:22JL32LT3:2:1101:3238:1064_1:N:0:CCGCGATT+CTAGCGCT:GGTTAGATA/1 77 * 0 0 * * 0 0 TNAAATTATATTAATTTTAATTAAATATTTTTAATAAATAAATAATTATAAAAATATTAAATTAATTTATATTTTAAATATTAAATTAAAATATTATATTATAAAAATTTTAAATTTTTAAAAAAATTAAGATTGGAAGAGTTTATGTTT I#I9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9II9II9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIII-IIIIIIIIIIIIIIIIIIIIIIII-III-II9 YT:Z:UP lh00134:396:22JL32LT3:2:1101:3238:1064_3:N:0:CCGCGATT+CTAGCGCT:GGTTAGATA/2 141 * 0 0 * * 0 0 TAATTTTTTTAAAAATTTAAAATTTTTATAATATAATATTTTAATTTAATATTTAAAATATAAATTAATTTAATATTTTTATAATTATTTATTTATTAAAAATATTTAATTAAAATTAATATAATTTCAAAATCAAAAAAACATCATATA I-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIII-II-III9IIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIII-IIIIIII9IIIIII-III9I YT:Z:UP >>> Writing bisulfite mapping results to E1_T1_F2_R1_bismark_bt2_pe.bam <<< Reading in the sequence files /home/shared/16TB_HDD_01/fish546/data/RRBS-run2/raw-data/E1_T1_F2_R1.fq.gz and /home/shared/16TB_HDD_01/fish546/data/RRBS-run2/raw-data/E1_T1_F2_R2.fq.gz Processed 1000000 sequence pairs so far Processed 2000000 sequence pairs so far Processed 3000000 sequence pairs so far Processed 4000000 sequence pairs so far Processed 5000000 sequence pairs so far Processed 6000000 sequence pairs so far Processed 7000000 sequence pairs so far Processed 8000000 sequence pairs so far Processed 9000000 sequence pairs so far Processed 10000000 sequence pairs so far Processed 11000000 sequence pairs so far Processed 12000000 sequence pairs so far Processed 13000000 sequence pairs so far Processed 14000000 sequence pairs so far Processed 15000000 sequence pairs so far Processed 16000000 sequence pairs so far Processed 17000000 sequence pairs so far Processed 18000000 sequence pairs so far Processed 19000000 sequence pairs so far Processed 20000000 sequence pairs so far Processed 21000000 sequence pairs so far Processed 22000000 sequence pairs so far Processed 23000000 sequence pairs so far Processed 24000000 sequence pairs so far Processed 25000000 sequence pairs so far Processed 26000000 sequence pairs so far Processed 27000000 sequence pairs so far Processed 28000000 sequence pairs so far Processed 29000000 sequence pairs so far Chromosomal sequence could not be extracted for lh00134:396:22JL32LT3:2:1254:49625:11430_1:N:0:ACGCGGTT+CTAGCGCT:GCCAGGGAC NW_025899749.1 255676 Processed 30000000 sequence pairs so far Processed 31000000 sequence pairs so far Processed 32000000 sequence pairs so far Processed 33000000 sequence pairs so far Chromosomal sequence could not be extracted for lh00134:396:22JL32LT3:2:1275:23440:18737_1:N:0:CCGCGGTT+CTAGCGCT:GCCAGGGAC NW_025899749.1 255676 Processed 34000000 sequence pairs so far Processed 35000000 sequence pairs so far Processed 36000000 sequence pairs so far Processed 37000000 sequence pairs so far Processed 38000000 sequence pairs so far Processed 39000000 sequence pairs so far Processed 40000000 sequence pairs so far Processed 41000000 sequence pairs so far Processed 42000000 sequence pairs so far Processed 43000000 sequence pairs so far Processed 44000000 sequence pairs so far Processed 45000000 sequence pairs so far Processed 46000000 sequence pairs so far Processed 47000000 sequence pairs so far Processed 48000000 sequence pairs so far Processed 49000000 sequence pairs so far Chromosomal sequence could not be extracted for lh00134:396:22JL32LT3:2:2161:32335:25850_1:N:0:CCGCGGTT+CTAGCGCT:GTTCCAAAT NW_025899749.1 255688 Processed 50000000 sequence pairs so far Processed 51000000 sequence pairs so far Processed 52000000 sequence pairs so far Processed 53000000 sequence pairs so far Processed 54000000 sequence pairs so far Processed 55000000 sequence pairs so far Processed 56000000 sequence pairs so far Processed 57000000 sequence pairs so far Processed 58000000 sequence pairs so far Processed 59000000 sequence pairs so far Processed 60000000 sequence pairs so far Processed 61000000 sequence pairs so far Processed 62000000 sequence pairs so far Processed 63000000 sequence pairs so far Processed 64000000 sequence pairs so far Processed 65000000 sequence pairs so far Processed 66000000 sequence pairs so far Processed 67000000 sequence pairs so far Processed 68000000 sequence pairs so far Processed 69000000 sequence pairs so far Processed 70000000 sequence pairs so far Processed 71000000 sequence pairs so far Processed 72000000 sequence pairs so far 72879399 reads; of these: 72879399 (100.00%) were paired; of these: 72810759 (99.91%) aligned concordantly 0 times 45116 (0.06%) aligned concordantly exactly 1 time 23524 (0.03%) aligned concordantly >1 times 0.09% overall alignment rate 72879399 reads; of these: 72879399 (100.00%) were paired; of these: 72804619 (99.90%) aligned concordantly 0 times 49371 (0.07%) aligned concordantly exactly 1 time 25409 (0.03%) aligned concordantly >1 times 0.10% overall alignment rate 72879399 reads; of these: 72879399 (100.00%) were paired; of these: 48101386 (66.00%) aligned concordantly 0 times 8445104 (11.59%) aligned concordantly exactly 1 time 16332909 (22.41%) aligned concordantly >1 times 34.00% overall alignment rate 72879399 reads; of these: 72879399 (100.00%) were paired; of these: 48142694 (66.06%) aligned concordantly 0 times 8479157 (11.63%) aligned concordantly exactly 1 time 16257548 (22.31%) aligned concordantly >1 times 33.94% overall alignment rate Processed 72879399 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 291517596. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 291517596. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 291517596. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 291517596. Successfully deleted the temporary files E1_T1_F2_R1.fq.gz_C_to_T.fastq, E1_T1_F2_R1.fq.gz_G_to_A.fastq, E1_T1_F2_R2.fq.gz_C_to_T.fastq and E1_T1_F2_R2.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 72879399 Number of paired-end alignments with a unique best hit: 26132779 Mapping efficiency: 35.9% Sequence pairs with no alignments under any condition: 40168425 Sequence pairs did not map uniquely: 6578195 Sequence pairs which were discarded because genomic sequence could not be extracted: 3 Number of sequence pairs with unique best (first) alignment came from the bowtie output: CT/GA/CT: 2877 ((converted) top strand) GA/CT/CT: 12062650 (complementary to (converted) top strand) GA/CT/GA: 14063959 (complementary to (converted) bottom strand) CT/GA/GA: 3290 ((converted) bottom strand) Final Cytosine Methylation Report ================================= Total number of C's analysed: 1909833460 Total methylated C's in CpG context: 156925743 Total methylated C's in CHG context: 13262054 Total methylated C's in CHH context: 35106951 Total methylated C's in Unknown context: 10014781 Total unmethylated C's in CpG context: 151329269 Total unmethylated C's in CHG context: 542360645 Total unmethylated C's in CHH context: 1010848798 Total unmethylated C's in Unknown context: 10484996 C methylated in CpG context: 50.9% C methylated in CHG context: 2.4% C methylated in CHH context: 3.4% C methylated in unknown context (CN or CHN): 48.9% Bismark completed in 0d 6h 55m 7s ==================== Bismark run complete ====================