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-69-TP4_R1_001.fastp-trim.fq.gz ../data/03-Peve-bismark/POR-69-TP4_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-69-TP4_R1_001.fastp-trim.fq.gz and ../data/03-Peve-bismark/POR-69-TP4_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-69-TP4_R1_001.fastp-trim.fq.gz Writing a C -> T converted version of the input file POR-69-TP4_R1_001.fastp-trim.fq.gz to POR-69-TP4_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file POR-69-TP4_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-69-TP4_R2_001.fastp-trim.fq.gz Writing a G -> A converted version of the input file POR-69-TP4_R2_001.fastp-trim.fq.gz to POR-69-TP4_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file POR-69-TP4_R2_001.fastp-trim.fq.gz (10001 sequences in total) Input files are POR-69-TP4_R1_001.fastp-trim.fq.gz_C_to_T.fastq and POR-69-TP4_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-69-TP4_R1_001.fastp-trim.fq.gz_C_to_T.fastq and POR-69-TP4_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: LH00526:197:22KFKVLT4:3:1101:49181:1056_1:N:0:GTCCTAAG+TGGTAGCT/1 99 Porites_evermani_scaffold_185_CT_converted 385102 17 135M = 385181 214 GATTTAAATTAAATTTTTAATTATTATTTTTTTATGATGTTTGTGGTAAAATATTGTTTTATTATTTGGTTAAATATATTTATTGATAATTAAAAAATAATAATTTGTTTTTGAAAAGTAAATTAGAATTAAATT IIIIIIIIII-IIIIIIIIIIIIIIII-IIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIII9IIIIII9 AS:i:-6 XS:i:-30 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:27A107 YS:i:0 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:49181:1056_2:N:0:GTCCTAAG+TGGTAGCT/2 147 Porites_evermani_scaffold_185_CT_converted 385181 17 135M = 385102 -214 TTATTGATAATTAAAAAATAATAATTTGTTTTTGAAAAGTAAATTAGAATTAAATTTTTGTTTAATTATATTGGTAAAGAATTTGGTTTAATGTTTTTAGTGATGAATTGTAGATATGTTGGTTTGGGTGTTTTG IIIIII-IIIIIIIIIIIII9IIIIIIIIIII9IIIIII9IIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIII9IIIIIIIIIIIII9I AS:i:0 XS:i:-12 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:135 YS:i:-6 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from POR-69-TP4_R1_001.fastp-trim.fq.gz_C_to_T.fastq and POR-69-TP4_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: LH00526:197:22KFKVLT4:3:1101:49181:1056_1:N:0:GTCCTAAG+TGGTAGCT/1 83 Porites_evermani_scaffold_4261_GA_converted 1578 42 135M = 1499 -214 AATTTAATTCTAATTTACTTTTCAAAAACAAATTATTATTTTTTAATTATCAATAAATATATTTAACCAAATAATAAAACAATATTTTACCACAAACATCATAAAAAAATAATAATTAAAAATTTAATTTAAATC 9IIIIII9IIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIII-IIIIIIIIIIIIIIII-IIIIIIIIII AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:74C32T27 YS:i:-6 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:49181:1056_2:N:0:GTCCTAAG+TGGTAGCT/2 163 Porites_evermani_scaffold_4261_GA_converted 1499 42 135M = 1578 214 CAAAACACCCAAACCAACATATCTACAATTCATCACTAAAAACATTAAACCAAATTCTTTACCAATATAATTAAACAAAAATTTAATTCTAATTTACTTTTCAAAAACAAATTATTATTTTTTAATTATCAATAA I9IIIIIIIIIIIII9IIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIII9IIIIII9IIIIIIIIIII9IIIIIIIIIIIII-IIIIII AS:i:-6 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:36C98 YS:i:-12 YT:Z:CP >>> Writing bisulfite mapping results to POR-69-TP4_R1_001.fastp-trim_bismark_bt2_pe.bam <<< Reading in the sequence files ../data/03-Peve-bismark/POR-69-TP4_R1_001.fastp-trim.fq.gz and ../data/03-Peve-bismark/POR-69-TP4_R2_001.fastp-trim.fq.gz Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1101:3007:1364_1:N:0:GTCCTAAG+TGGTAGCT Porites_evermani_scaffold_491 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1101:50387:1714_1:N:0:GTCCTAAG+TGGTAGCT Porites_evermani_scaffold_1840 101358 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1101:28012:1728_1:N:0:GTCCTAAG+TGGTAGCT Porites_evermani_scaffold_2920 59147 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1101:28028:1728_1:N:0:GTCCTAAG+TGGTAGCT Porites_evermani_scaffold_2920 59147 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1101:48429:2835_1:N:0:GTCCTAAG+TGGTAGCT Porites_evermani_scaffold_7175 1 10000 reads; of these: 10000 (100.00%) were paired; of these: 5290 (52.90%) aligned concordantly 0 times 2590 (25.90%) aligned concordantly exactly 1 time 2120 (21.20%) aligned concordantly >1 times 47.10% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 5263 (52.63%) aligned concordantly 0 times 2706 (27.06%) aligned concordantly exactly 1 time 2031 (20.31%) aligned concordantly >1 times 47.37% 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-69-TP4_R1_001.fastp-trim.fq.gz_C_to_T.fastq and POR-69-TP4_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: 326433 Total methylated C's in CpG context: 4108 Total methylated C's in CHG context: 331 Total methylated C's in CHH context: 1189 Total methylated C's in Unknown context: 7 Total unmethylated C's in CpG context: 48316 Total unmethylated C's in CHG context: 56310 Total unmethylated C's in CHH context: 216179 Total unmethylated C's in Unknown context: 455 C methylated in CpG context: 7.8% C methylated in CHG context: 0.6% C methylated in CHH context: 0.5% C methylated in unknown context (CN or CHN): 1.5% Bismark completed in 0d 0h 0m 16s ==================== Bismark run complete ==================== gzip: stdout: Broken pipe gzip: stdout: Broken pipe gzip: stdout: Broken pipe