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/69M_R1.fastp-trim.fq.gz ../data/69M_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/69M_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/69M_R1.fastp-trim.fq.gz and ../data/69M_R2.fastp-trim.fq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from ../data/69M_R1.fastp-trim.fq.gz Writing a C -> T converted version of the input file 69M_R1.fastp-trim.fq.gz to 69M_R1.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 69M_R1.fastp-trim.fq.gz (10001 sequences in total) gzip: stdout: Broken pipe Processing reads up to sequence no. 10000 from ../data/69M_R2.fastp-trim.fq.gz Writing a G -> A converted version of the input file 69M_R2.fastp-trim.fq.gz to 69M_R2.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 69M_R2.fastp-trim.fq.gz (10001 sequences in total) Input files are 69M_R1.fastp-trim.fq.gz_C_to_T.fastq and 69M_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 69M_R1.fastp-trim.fq.gz_C_to_T.fastq and 69M_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: LH00469:254:22HGFVLT4:2:1101:3646:1014_1:N:0:GTGAAGTG+GAGCAATC/1 99 NC_086373.1_CT_converted 93602713 21 131M = 93602697 -147 TTTGTAGTTGAGTAATTTGTGATAAAATTATGTTATATTTGAGAAAATTATTAAAAAGATTTTATTTATATATTTTGTTGTTATATTGGGATAATTGTAATTGTAGTTATGATTATTTTTTTAATTTTTTT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-18 XS:i:-74 XN:i:0 XM:i:3 XO:i:0 XG:i:0 NM:i:3 MD:Z:125A2A0G1 YS:i:0 YT:Z:CP LH00469:254:22HGFVLT4:2:1101:3646:1014_2:N:0:GTGAAGTG+GAGCAATC/2 147 NC_086373.1_CT_converted 93602697 21 131M = 93602713 147 AATGGTATAGAAAAATTTTGTAGTTGAGTAATTTGTGATAAAATTATGTTATATTTGAGAAAATTATTAAAAAGATTTTATTTATATATTTTGTTGTTATATTGGGATAATTGTAATTGTAGTTATGATTA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XS:i:-56 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:131 YS:i:-18 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 69M_R1.fastp-trim.fq.gz_C_to_T.fastq and 69M_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: LH00469:254:22HGFVLT4:2:1101:3646:1014_1:N:0:GTGAAGTG+GAGCAATC/1 83 NC_086385.1_GA_converted 33713748 2 88M1D43M = 33713764 148 AAAAAAATTAAAAAAATAATCATAACTACAATTACAATTATCCCAATATAACAACAAAATATATAAATAAAATCTTTTTAATAATTTTCTCAAATATAACATAATTTTATCACAAATTACTCAACTACAAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-68 XS:i:-68 XN:i:0 XM:i:10 XO:i:1 XG:i:1 NM:i:11 MD:Z:1C0T2T14A36C1A17A0C3C5^A15T27 YS:i:-50 YT:Z:CP LH00469:254:22HGFVLT4:2:1101:3646:1014_2:N:0:GTGAAGTG+GAGCAATC/2 163 NC_086385.1_GA_converted 33713764 2 72M1D59M = 33713748 -148 TAATCATAACTACAATTACAATTATCCCAATATAACAACAAAATATATAAATAAAATCTTTTTAATAATTTTCTCAAATATAACATAATTTTATCACAAATTACTCAACTACAAAATTTTTCTATACCATT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-50 XS:i:-62 XN:i:0 XM:i:7 XO:i:1 XG:i:1 NM:i:8 MD:Z:4A36C1A17A0C3C5^A15T43 YS:i:-68 YT:Z:CP >>> Writing bisulfite mapping results to 69M_L0-1.0_pe.bam <<< Reading in the sequence files ../data/69M_R1.fastp-trim.fq.gz and ../data/69M_R2.fastp-trim.fq.gz 10000 reads; of these: 10000 (100.00%) were paired; of these: 3744 (37.44%) aligned concordantly 0 times 1794 (17.94%) aligned concordantly exactly 1 time 4462 (44.62%) aligned concordantly >1 times 62.56% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 3831 (38.31%) aligned concordantly 0 times 1684 (16.84%) aligned concordantly exactly 1 time 4485 (44.85%) aligned concordantly >1 times 61.69% 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 69M_R1.fastp-trim.fq.gz_C_to_T.fastq and 69M_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: 246358 Total methylated C's in CpG context: 3506 Total methylated C's in CHG context: 376 Total methylated C's in CHH context: 1723 Total methylated C's in Unknown context: 54 Total unmethylated C's in CpG context: 27410 Total unmethylated C's in CHG context: 39602 Total unmethylated C's in CHH context: 173741 Total unmethylated C's in Unknown context: 1327 C methylated in CpG context: 11.3% C methylated in CHG context: 0.9% C methylated in CHH context: 1.0% C methylated in unknown context (CN or CHN): 3.9% Bismark completed in 0d 0h 0m 20s ==================== Bismark run complete ==================== gzip: stdout: Broken pipe gzip: stdout: Broken pipe gzip: stdout: Broken pipe