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/80M_R1.fastp-trim.fq.gz ../data/80M_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/80M_L-1-0.6/ Setting parallelization to single-threaded (default) Summary of all aligner options: -q --score-min L,-1,-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/80M_R1.fastp-trim.fq.gz and ../data/80M_R2.fastp-trim.fq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from ../data/80M_R1.fastp-trim.fq.gz Writing a C -> T converted version of the input file 80M_R1.fastp-trim.fq.gz to 80M_R1.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 80M_R1.fastp-trim.fq.gz (10001 sequences in total) gzip: stdout: Broken pipe Processing reads up to sequence no. 10000 from ../data/80M_R2.fastp-trim.fq.gz Writing a G -> A converted version of the input file 80M_R2.fastp-trim.fq.gz to 80M_R2.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 80M_R2.fastp-trim.fq.gz (10001 sequences in total) Input files are 80M_R1.fastp-trim.fq.gz_C_to_T.fastq and 80M_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,-1,-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 80M_R1.fastp-trim.fq.gz_C_to_T.fastq and 80M_R2.fastp-trim.fq.gz_G_to_A.fastq, with the options: -q --score-min L,-1,-0.6 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: LH00469:254:22HGFVLT4:2:1101:1397:1014_1:N:0:AGCACTTC+ACGTCCAA/1 77 * 0 0 * * 0 0 TTTTTTATGGTTTTTAAATTAAATTTATATAAAGTAATTTATATTAAATTAATTGAAATATTAAATTGTAATAAAAATATATTGTAGTAAAAATATATTTATTAAATTGATTAAAATAATTAATAATGTAT IIIIIIIIIIIIIII9IIIII9II-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9 YT:Z:UP LH00469:254:22HGFVLT4:2:1101:1397:1014_2:N:0:AGCACTTC+ACGTCCAA/2 141 * 0 0 * * 0 0 TTATTTTAATCAATTTAATAAATATATTTTTACTACAATATATTTTTATTACAATTTAATATTTCAATTAATTTAATATAAATTACTTTATATAAATTTAATTTAAAAACCATAAAAAACAAACAACTTAA IIIIIIIIII-I9III9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-9IIIIIIIII99IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 80M_R1.fastp-trim.fq.gz_C_to_T.fastq and 80M_R2.fastp-trim.fq.gz_G_to_A.fastq, with the options: -q --score-min L,-1,-0.6 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --nofw)) Found first alignment: LH00469:254:22HGFVLT4:2:1101:1397:1014_1:N:0:AGCACTTC+ACGTCCAA/1 83 NC_086375.1_GA_converted 75639756 40 131M = 75639768 143 ATACATTATTAATTATTTTAATCAATTTAATAAATATATTTTTACTACAATATATTTTTATTACAATTTAATATTTCAATTAATTTAATATAAATTACTTTATATAAATTTAATTTAAAAACCATAAAAAA 9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-II9IIIII9IIIIIIIIIIIIIII AS:i:-24 XN:i:0 XM:i:4 XO:i:0 XG:i:0 NM:i:4 MD:Z:0C8C27C59A33 YS:i:-18 YT:Z:CP LH00469:254:22HGFVLT4:2:1101:1397:1014_2:N:0:AGCACTTC+ACGTCCAA/2 163 NC_086375.1_GA_converted 75639768 40 131M = 75639756 -143 TTATTTTAATCAATTTAATAAATATATTTTTACTACAATATATTTTTATTACAATTTAATATTTCAATTAATTTAATATAAATTACTTTATATAAATTTAATTTAAAAACCATAAAAAACAAACAACTTAA IIIIIIIIII-I9III9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-9IIIIIIIII99IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-18 XN:i:0 XM:i:3 XO:i:0 XG:i:0 NM:i:3 MD:Z:25C59A33T11 YS:i:-24 YT:Z:CP >>> Writing bisulfite mapping results to 80M_L-1-0.6_pe.bam <<< Reading in the sequence files ../data/80M_R1.fastp-trim.fq.gz and ../data/80M_R2.fastp-trim.fq.gz 10000 reads; of these: 10000 (100.00%) were paired; of these: 5074 (50.74%) aligned concordantly 0 times 1852 (18.52%) aligned concordantly exactly 1 time 3074 (30.74%) aligned concordantly >1 times 49.26% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 5027 (50.27%) aligned concordantly 0 times 1874 (18.74%) aligned concordantly exactly 1 time 3099 (30.99%) aligned concordantly >1 times 49.73% 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 80M_R1.fastp-trim.fq.gz_C_to_T.fastq and 80M_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: 213204 Total methylated C's in CpG context: 2936 Total methylated C's in CHG context: 335 Total methylated C's in CHH context: 1411 Total methylated C's in Unknown context: 26 Total unmethylated C's in CpG context: 22167 Total unmethylated C's in CHG context: 34127 Total unmethylated C's in CHH context: 152228 Total unmethylated C's in Unknown context: 669 C methylated in CpG context: 11.7% C methylated in CHG context: 1.0% C methylated in CHH context: 0.9% C methylated in unknown context (CN or CHN): 3.7% Bismark completed in 0d 0h 0m 19s ==================== Bismark run complete ==================== gzip: stdout: Broken pipe gzip: stdout: Broken pipe gzip: stdout: Broken pipe