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/72M_R1.fastp-trim.fq.gz ../data/72M_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/72M_L0-0.6/ Setting parallelization to single-threaded (default) Summary of all aligner options: -q --score-min L,0,-0.6 -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/72M_R1.fastp-trim.fq.gz and ../data/72M_R2.fastp-trim.fq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from ../data/72M_R1.fastp-trim.fq.gz Writing a C -> T converted version of the input file 72M_R1.fastp-trim.fq.gz to 72M_R1.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 72M_R1.fastp-trim.fq.gz (10001 sequences in total) gzip: stdout: Broken pipe Processing reads up to sequence no. 10000 from ../data/72M_R2.fastp-trim.fq.gz Writing a G -> A converted version of the input file 72M_R2.fastp-trim.fq.gz to 72M_R2.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 72M_R2.fastp-trim.fq.gz (10001 sequences in total) Input files are 72M_R1.fastp-trim.fq.gz_C_to_T.fastq and 72M_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,-0.6 -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 72M_R1.fastp-trim.fq.gz_C_to_T.fastq and 72M_R2.fastp-trim.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-0.6 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: LH00469:254:22HGFVLT4:2:1101:3128:1014_1:N:0:CAACACCT+AGCTACCA/1 99 NC_086375.1_CT_converted 24177712 2 126M = 24177694 -144 ATGTTGGGGTGTGTTATTTTATTTGTATGTTTTGTATTTGATAGTTGTATTATTAAATGGTGTATTTTATAGGATTTTGTTATGTATATGGGTTATAATTATAGGGTTAGATTGTTTTTTTTTTTT IIIII9IIIIIIIIIII9II-IIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-30 XS:i:-36 XN:i:0 XM:i:5 XO:i:0 XG:i:0 NM:i:5 MD:Z:44G38A36A2G1G0 YS:i:-23 YT:Z:CP LH00469:254:22HGFVLT4:2:1101:3128:1014_2:N:0:CAACACCT+AGCTACCA/2 147 NC_086375.1_CT_converted 24177694 2 9M2I115M = 24177712 144 TTATGGATTTGTGTGATATTATGTTGGGGTGTGTTATTTTATTTGTATGTTTTGTATTTGATAGTTGTATTATTAAATGGTGTATTTTATAGGATTTTGTTATGTATATGGGTTATAATTATAGGG 9IIIIIIIIIIIIIIIIIIIII9IIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIII9IIIIIIIIIIIIIIIIIIIIIIIII AS:i:-23 XS:i:-29 XN:i:0 XM:i:2 XO:i:1 XG:i:2 NM:i:4 MD:Z:62G38A22 YS:i:-30 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 72M_R1.fastp-trim.fq.gz_C_to_T.fastq and 72M_R2.fastp-trim.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-0.6 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --nofw)) Found first alignment: LH00469:254:22HGFVLT4:2:1101:3128:1014_1:N:0:CAACACCT+AGCTACCA/1 83 NC_086385.1_GA_converted 49520160 0 126M = 49520180 144 AAAAAAAAAAAACAATCTAACCCTATAATTATAACCCATATACATAACAAAATCCTATAAAATACACCATTTAATAATACAACTATCAAATACAAAACATACAAATAAAATAACACACCCCAACAT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIII-II9IIIIIIIIIII9IIIII AS:i:-48 XS:i:-60 XN:i:0 XM:i:8 XO:i:0 XG:i:0 NM:i:8 MD:Z:0C1C2T36T7T11A8C9C44 YS:i:-41 YT:Z:CP LH00469:254:22HGFVLT4:2:1101:3128:1014_2:N:0:CAACACCT+AGCTACCA/2 163 NC_086385.1_GA_converted 49520180 0 111M2I13M = 49520160 -144 CCCTATAATTATAACCCATATACATAACAAAATCCTATAAAATACACCATTTAATAATACAACTATCAAATACAAAACATACAAATAAAATAACACACCCCAACATAATATCACACAAATCCATAA IIIIIIIIIIIIIIIIIIIIIIIII9IIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIII9IIIIIIIIIIIIIIIIIIIII9 AS:i:-41 XS:i:-53 XN:i:0 XM:i:5 XO:i:1 XG:i:2 NM:i:7 MD:Z:22T7T11A8C9C62 YS:i:-48 YT:Z:CP >>> Writing bisulfite mapping results to 72M_L0-0.6_pe.bam <<< Reading in the sequence files ../data/72M_R1.fastp-trim.fq.gz and ../data/72M_R2.fastp-trim.fq.gz 10000 reads; of these: 10000 (100.00%) were paired; of these: 4874 (48.74%) aligned concordantly 0 times 1833 (18.33%) aligned concordantly exactly 1 time 3293 (32.93%) aligned concordantly >1 times 51.26% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 4897 (48.97%) aligned concordantly 0 times 1785 (17.85%) aligned concordantly exactly 1 time 3318 (33.18%) aligned concordantly >1 times 51.03% 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 72M_R1.fastp-trim.fq.gz_C_to_T.fastq and 72M_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: 212621 Total methylated C's in CpG context: 2738 Total methylated C's in CHG context: 311 Total methylated C's in CHH context: 1219 Total methylated C's in Unknown context: 13 Total unmethylated C's in CpG context: 23125 Total unmethylated C's in CHG context: 34524 Total unmethylated C's in CHH context: 150704 Total unmethylated C's in Unknown context: 636 C methylated in CpG context: 10.6% C methylated in CHG context: 0.9% C methylated in CHH context: 0.8% C methylated in unknown context (CN or CHN): 2.0% Bismark completed in 0d 0h 0m 19s ==================== Bismark run complete ==================== gzip: stdout: Broken pipe gzip: stdout: Broken pipe gzip: stdout: Broken pipe