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) Processing sequences up to read no. 10000 from the input file 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 is assumed to be strand-specific (directional), alignments to strands complementary to the original top or bottom strands will be ignored (i.e. not performed!) Output will be written into the directory: /home/shared/16TB_HDD_01/sr320/github/Claudia-Halibut/project/output/03/ 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 Processing reads up to sequence no. 10000 from /home/shared/16TB_HDD_01/fish546/data/RRBS-run2/raw-data/E1_T1_F2_R1.fq.gz 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 Created C -> T converted version of the FastQ file E1_T1_F2_R1.fq.gz (10001 sequences in total) gzip: stdout: Broken pipe Processing reads up to sequence no. 10000 from /home/shared/16TB_HDD_01/fish546/data/RRBS-run2/raw-data/E1_T1_F2_R2.fq.gz 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 G -> A converted version of the FastQ file E1_T1_F2_R2.fq.gz (10001 sequences in total) Input files are E1_T1_F2_R1.fq.gz_C_to_T.fastq and E1_T1_F2_R2.fq.gz_G_to_A.fastq (FastQ) Now running 2 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 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 10000 reads; of these: 10000 (100.00%) were paired; of these:10000 9992 (99.92% reads; of these:) aligned concordantly 0 times 100008 ( (0.08%100.00) aligned concordantly exactly 1 time% ) were paired; of these: 0 (99950.00 (%99.95) aligned concordantly >1 times% ) aligned concordantly 0 times0.08 % overall alignment rate4 (0.04%) aligned concordantly exactly 1 time 1 (0.01%) aligned concordantly >1 times 0.05% overall alignment rate Processed 10000 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 40004. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 40004. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 40004. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 40004. Successfully deleted the temporary files E1_T1_F2_R1.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: 10000 Number of paired-end alignments with a unique best hit: 13 Mapping efficiency: 0.1% Sequence pairs with no alignments under any condition: 9987 Sequence pairs did not map uniquely: 0 Sequence pairs which were discarded because genomic sequence could not be extracted: 0 Number of sequence pairs with unique best (first) alignment came from the bowtie output: CT/GA/CT: 5 ((converted) top strand) GA/CT/CT: 0 (complementary to (converted) top strand) GA/CT/GA: 0 (complementary to (converted) bottom strand) CT/GA/GA: 8 ((converted) bottom strand) Number of alignments to (merely theoretical) complementary strands being rejected in total: 0 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1223 Total methylated C's in CpG context: 174 Total methylated C's in CHG context: 162 Total methylated C's in CHH context: 862 Total methylated C's in Unknown context: 3 Total unmethylated C's in CpG context: 2 Total unmethylated C's in CHG context: 4 Total unmethylated C's in CHH context: 19 Total unmethylated C's in Unknown context: 2 C methylated in CpG context: 98.9% C methylated in CHG context: 97.6% C methylated in CHH context: 97.8% C methylated in unknown context (CN or CHN): 60.0% Bismark completed in 0d 0h 0m 15s ==================== Bismark run complete ==================== gzip: stdout: Broken pipe gzip: stdout: Broken pipe gzip: stdout: Broken pipe