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/ (absolute path is '/home/shared/16TB_HDD_01/sr320/github/project-mytilus-methylation/data/)' 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/project-mytilus-methylation/code'): ../data/79M_R1.fastp-trim.fq.gz ../data/79M_R2.fastp-trim.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/project-mytilus-methylation/output/01.1-bismark-min_score/79M_L0-1.0/ Setting parallelization to single-threaded (default) Summary of all aligner options: -q --score-min L,0,-1.0 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 Current working directory is: /home/shared/16TB_HDD_01/sr320/github/project-mytilus-methylation/code Now reading in and storing sequence information of the genome specified in: /home/shared/16TB_HDD_01/sr320/github/project-mytilus-methylation/data/ Single-core mode: setting pid to 1 Paired-end alignments will be performed ======================================= The provided filenames for paired-end alignments are ../data/79M_R1.fastp-trim.fq.gz and ../data/79M_R2.fastp-trim.fq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from ../data/79M_R1.fastp-trim.fq.gz Writing a C -> T converted version of the input file 79M_R1.fastp-trim.fq.gz to 79M_R1.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 79M_R1.fastp-trim.fq.gz (10001 sequences in total) gzip: stdout: Broken pipe Processing reads up to sequence no. 10000 from ../data/79M_R2.fastp-trim.fq.gz Writing a G -> A converted version of the input file 79M_R2.fastp-trim.fq.gz to 79M_R2.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 79M_R2.fastp-trim.fq.gz (10001 sequences in total) Input files are 79M_R1.fastp-trim.fq.gz_C_to_T.fastq and 79M_R2.fastp-trim.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/project-mytilus-methylation/data/ with the specified options: -q --score-min L,0,-1.0 -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 79M_R1.fastp-trim.fq.gz_C_to_T.fastq and 79M_R2.fastp-trim.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-1.0 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: LH00469:254:22HGFVLT4:2:1101:1906:1028_1:N:0:GTCATCGA+GATCCACT/1 99 NC_086380.1_CT_converted 73522031 17 131M = 73522189 289 TTGAGTAATGAATTGTGAAAATAAGGTTATGGTTAAATAAAATTTGTGTGATTATTGTATAGATTATAAAATATTTTTATATATTTAATATAGTTTATTTATGGTGTATAGTATTTGATAAAATGATTAAA III9IIIIIIIIIIIIIIIIIII9IIIII-IIIIII9IIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIII9IIIIIIIIIIIII9IIIIII9IIIIIIIIIIIIIIIIIIIIIIII-I9IIIIIIII AS:i:-6 XS:i:-36 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:29A101 YS:i:0 YT:Z:CP LH00469:254:22HGFVLT4:2:1101:1906:1028_2:N:0:GTCATCGA+GATCCACT/2 147 NC_086380.1_CT_converted 73522189 17 131M = 73522031 -289 ATTATGAAAATAAGGTTAAGGTTAGATAATATTTGTTTATTAGATATGTTTATTTTTTAATAATTTTATATAATAAATATAGTAGATTTATTGTTTAAAGTATGAGAAAAATAGATTAAATATAAAAATTT IIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIII9I9IIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIII9IIIII9IIIII9IIIIIIIIII9IIIII9IIIII9IIIIIII AS:i:0 XS:i:-32 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:131 YS:i:-6 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 79M_R1.fastp-trim.fq.gz_C_to_T.fastq and 79M_R2.fastp-trim.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-1.0 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --nofw)) Found first alignment: LH00469:254:22HGFVLT4:2:1101:1906:1028_1:N:0:GTCATCGA+GATCCACT/1 83 NC_086376.1_GA_converted 19370512 22 131M = 19370354 -289 TTTAATCATTTTATCAAATACTATACACCATAAATAAACTATATTAAATATATAAAAATATTTTATAATCTATACAATAATCACACAAATTTTATTTAACCATAACCTTATTTTCACAATTCATTACTCAA IIIIIIII9I-IIIIIIIIIIIIIIIIIIIIIIII9IIIIII9IIIIIIIIIIIII9IIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIII9IIIIII-IIIII9IIIIIIIIIIIIIIIIIII9III AS:i:-6 XS:i:-120 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:101T29 YS:i:0 YT:Z:CP LH00469:254:22HGFVLT4:2:1101:1906:1028_2:N:0:GTCATCGA+GATCCACT/2 163 NC_086376.1_GA_converted 19370354 22 131M = 19370512 289 AAATTTTTATATTTAATCTATTTTTCTCATACTTTAAACAATAAATCTACTATATTTATTATATAAAATTATTAAAAAATAAACATATCTAATAAACAAATATTATCTAACCTTAACCTTATTTTCATAAT IIIIIII9IIIII9IIIII9IIIIIIIIII9IIIII9IIIII9IIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIII9I9IIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIII AS:i:0 XS:i:-50 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:131 YS:i:-6 YT:Z:CP >>> Writing bisulfite mapping results to 79M_L0-1.0_pe.bam <<< Reading in the sequence files ../data/79M_R1.fastp-trim.fq.gz and ../data/79M_R2.fastp-trim.fq.gz 10000 reads; of these: 10000 (100.00%) were paired; of these: 4180 (41.80%) aligned concordantly 0 times 2038 (20.38%) aligned concordantly exactly 1 time 3782 (37.82%) aligned concordantly >1 times 58.20% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 4252 (42.52%) aligned concordantly 0 times 1971 (19.71%) aligned concordantly exactly 1 time 3777 (37.77%) aligned concordantly >1 times 57.48% 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 79M_R1.fastp-trim.fq.gz_C_to_T.fastq and 79M_R2.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 10000 Final Cytosine Methylation Report ================================= Total number of C's analysed: 248718 Total methylated C's in CpG context: 3240 Total methylated C's in CHG context: 358 Total methylated C's in CHH context: 1768 Total methylated C's in Unknown context: 58 Total unmethylated C's in CpG context: 25744 Total unmethylated C's in CHG context: 39459 Total unmethylated C's in CHH context: 178149 Total unmethylated C's in Unknown context: 1384 C methylated in CpG context: 11.2% C methylated in CHG context: 0.9% C methylated in CHH context: 1.0% C methylated in unknown context (CN or CHN): 4.0% Bismark completed in 0d 0h 0m 19s ==================== Bismark run complete ==================== gzip: stdout: Broken pipe gzip: stdout: Broken pipe gzip: stdout: Broken pipe