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/8TB_HDD_03/sr320/github/timeseries_molecular/E-Peve/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/8TB_HDD_03/sr320/github/timeseries_molecular/E-Peve/code'): ../data/03-Peve-bismark/POR-245-TP1_R1_001.fastp-trim.fq.gz ../data/03-Peve-bismark/POR-245-TP1_R2_001.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/8TB_HDD_03/sr320/github/timeseries_molecular/E-Peve/output/03-Peve-bismark/ 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/8TB_HDD_03/sr320/github/timeseries_molecular/E-Peve/code Now reading in and storing sequence information of the genome specified in: /home/shared/8TB_HDD_03/sr320/github/timeseries_molecular/E-Peve/data/ Single-core mode: setting pid to 1 Paired-end alignments will be performed ======================================= The provided filenames for paired-end alignments are ../data/03-Peve-bismark/POR-245-TP1_R1_001.fastp-trim.fq.gz and ../data/03-Peve-bismark/POR-245-TP1_R2_001.fastp-trim.fq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from ../data/03-Peve-bismark/POR-245-TP1_R1_001.fastp-trim.fq.gz Writing a C -> T converted version of the input file POR-245-TP1_R1_001.fastp-trim.fq.gz to POR-245-TP1_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file POR-245-TP1_R1_001.fastp-trim.fq.gz (10001 sequences in total) gzip: stdout: Broken pipe Processing reads up to sequence no. 10000 from ../data/03-Peve-bismark/POR-245-TP1_R2_001.fastp-trim.fq.gz Writing a G -> A converted version of the input file POR-245-TP1_R2_001.fastp-trim.fq.gz to POR-245-TP1_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file POR-245-TP1_R2_001.fastp-trim.fq.gz (10001 sequences in total) Input files are POR-245-TP1_R1_001.fastp-trim.fq.gz_C_to_T.fastq and POR-245-TP1_R2_001.fastp-trim.fq.gz_G_to_A.fastq (FastQ) Now running 2 instances of Bowtie 2 against the bisulfite genome of /home/shared/8TB_HDD_03/sr320/github/timeseries_molecular/E-Peve/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 POR-245-TP1_R1_001.fastp-trim.fq.gz_C_to_T.fastq and POR-245-TP1_R2_001.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: LH00652:104:22KG7JLT4:7:1101:7100:1084_1:N:0:GACGTCAT+TCAACTGG/1 99 Porites_evermani_scaffold_2402_CT_converted 18076 23 135M = 18268 326 TATAATAATATTATTATATTTAATGATTTTTTTTTTATTTTTGTTGTTTTTGTTTTGTAGATATTTTTGGTGAAGGTGATTTAGGTGAATTGTTAGATGATGATGATGATGAGGATGATGATGAAGATGAGGATG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-36 XN:i:0 XM:i:6 XO:i:0 XG:i:0 NM:i:6 MD:Z:15G40A25G8A2T32A7 YS:i:-38 YT:Z:CP LH00652:104:22KG7JLT4:7:1101:7100:1084_2:N:0:GACGTCAT+TCAACTGG/2 147 Porites_evermani_scaffold_2402_CT_converted 18268 23 67M1I67M = 18076 -326 TGTAGAAATGGTGGTTATTTTGTTATAATAGAATTGGTGGTATTTATTTTTAATAAAGAGAAATATATTTTTTTTTTTTTAATAGAAGATGAGAAGATGGAAATTATTGATTATATTGAAATAAATTTTGTTGTT I-I9I9II9III-III9-IIIIIIII9II-III-IIIIIIIIIII9IIIIIII-9IIIII-IIIII9-IIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIII9IIIIII9IIIIIIII9IIIIIIIIIIIIIII AS:i:-38 XN:i:0 XM:i:5 XO:i:1 XG:i:1 NM:i:6 MD:Z:21A18A16A0T0T74 YS:i:-36 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from POR-245-TP1_R1_001.fastp-trim.fq.gz_C_to_T.fastq and POR-245-TP1_R2_001.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: LH00652:104:22KG7JLT4:7:1101:7100:1084_1:N:0:GACGTCAT+TCAACTGG/1 83 Porites_evermani_scaffold_252_GA_converted 199505 24 135M = 199312 -328 CATCCTCATCTTCATCATCATCCTCATCATCATCATCATCTAACAATTCACCTAAATCACCTTCACCAAAAATATCTACAAAACAAAAACAACAAAAATAAAAAAAAAATCATTAAATATAATAATATTATTATA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:78T40A15 YS:i:-46 YT:Z:CP LH00652:104:22KG7JLT4:7:1101:7100:1084_2:N:0:GACGTCAT+TCAACTGG/2 163 Porites_evermani_scaffold_252_GA_converted 199312 24 55M1D50M5I25M = 199505 328 AACAACAAAATTTATTTCAATATAATCAATAATTTCCATCTTCTCATCTTCTATTAAAAAAAAAAAAATATATTTCTCTTTATTAAAAATAAATACCACCAATTCTATTATAACAAAATAACCACCATTTCTACA IIIIIIIIIIIIIII9IIIIIIII9IIIIII9IIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIII-9IIIII-IIIII9-IIIIIII9IIIIIIIIIII-III-II9IIIIIIII-9III-III9II9I9I-I AS:i:-46 XN:i:0 XM:i:3 XO:i:2 XG:i:6 NM:i:9 MD:Z:55^A21A0T15C36 YS:i:-12 YT:Z:CP >>> Writing bisulfite mapping results to POR-245-TP1_R1_001.fastp-trim_bismark_bt2_pe.bam <<< Reading in the sequence files ../data/03-Peve-bismark/POR-245-TP1_R1_001.fastp-trim.fq.gz and ../data/03-Peve-bismark/POR-245-TP1_R2_001.fastp-trim.fq.gz 10000 reads; of these: 10000 (100.00%) were paired; of these: 5861 (58.61%) aligned concordantly 0 times 2247 (22.47%) aligned concordantly exactly 1 time 1892 (18.92%) aligned concordantly >1 times 41.39% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 5890 (58.90%) aligned concordantly 0 times 2251 (22.51%) aligned concordantly exactly 1 time 1859 (18.59%) aligned concordantly >1 times 41.10% 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 POR-245-TP1_R1_001.fastp-trim.fq.gz_C_to_T.fastq and POR-245-TP1_R2_001.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: 280229 Total methylated C's in CpG context: 3704 Total methylated C's in CHG context: 317 Total methylated C's in CHH context: 1200 Total methylated C's in Unknown context: 5 Total unmethylated C's in CpG context: 40865 Total unmethylated C's in CHG context: 49359 Total unmethylated C's in CHH context: 184784 Total unmethylated C's in Unknown context: 424 C methylated in CpG context: 8.3% C methylated in CHG context: 0.6% C methylated in CHH context: 0.6% C methylated in unknown context (CN or CHN): 1.2% Bismark completed in 0d 0h 0m 16s ==================== Bismark run complete ==================== gzip: stdout: Broken pipe gzip: stdout: Broken pipe gzip: stdout: Broken pipe