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-72-TP1_R1_001.fastp-trim.fq.gz ../data/03-Peve-bismark/POR-72-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-72-TP1_R1_001.fastp-trim.fq.gz and ../data/03-Peve-bismark/POR-72-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-72-TP1_R1_001.fastp-trim.fq.gz Writing a C -> T converted version of the input file POR-72-TP1_R1_001.fastp-trim.fq.gz to POR-72-TP1_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file POR-72-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-72-TP1_R2_001.fastp-trim.fq.gz Writing a G -> A converted version of the input file POR-72-TP1_R2_001.fastp-trim.fq.gz to POR-72-TP1_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file POR-72-TP1_R2_001.fastp-trim.fq.gz (10001 sequences in total) Input files are POR-72-TP1_R1_001.fastp-trim.fq.gz_C_to_T.fastq and POR-72-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-72-TP1_R1_001.fastp-trim.fq.gz_C_to_T.fastq and POR-72-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: LH00526:197:22KFKVLT4:3:1101:38621:1070_1:N:0:GGCAAGTT+CTTCTGAG/1 99 Porites_evermani_scaffold_66_CT_converted 326779 42 135M = 327092 448 ATAATAATAAAATAATAATAAAATATTTATTATAATTAATTTTTTTTGAGGTAGATAATGTTAAGATTGATTAAAGAATGATAGGGATAAATTTAAGTATATTTATTTTAGGAGGTTATTTGTTTTAAAGAGGTG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-II9IIIIIIIIIIIIIIIIIIIIII9II9IIIIIIIIIIIII9IIIIIII9I9IIIIIIII-IIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:135 YS:i:-6 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:38621:1070_2:N:0:GGCAAGTT+CTTCTGAG/2 147 Porites_evermani_scaffold_66_CT_converted 327092 42 135M = 326779 -448 TATTATTAATGTTTTTGAGATTTTGAGATTTTGATAAAATTTTTTAAGATTTATGTTTTATGAGGTATTATTTGTTATTTTTATAAGGTTTAGAAATATATTATATATGTTTGAAAAAATTTAAGGATTTTTTTT III-IIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-6 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:30G104 YS:i:0 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from POR-72-TP1_R1_001.fastp-trim.fq.gz_C_to_T.fastq and POR-72-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: LH00526:197:22KFKVLT4:3:1101:38621:1070_1:N:0:GGCAAGTT+CTTCTGAG/1 77 * 0 0 * * 0 0 ATAATAATAAAATAATAATAAAATATTTATTATAATTAATTTTTTTTGAGGTAGATAATGTTAAGATTGATTAAAGAATGATAGGGATAAATTTAAGTATATTTATTTTAGGAGGTTATTTGTTTTAAAGAGGTG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-II9IIIIIIIIIIIIIIIIIIIIII9II9IIIIIIIIIIIII9IIIIIII9I9IIIIIIII-IIIIII YT:Z:UP LH00526:197:22KFKVLT4:3:1101:38621:1070_2:N:0:GGCAAGTT+CTTCTGAG/2 141 * 0 0 * * 0 0 AAAAAAAATCCTTAAATTTTTTCAAACATATATAATATATTTCTAAACCTTATAAAAATAACAAATAATACCTCATAAAACATAAATCTTAAAAAATTTTATCAAAATCTCAAAATCTCAAAAACATTAATAATA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIII-III YT:Z:UP >>> Writing bisulfite mapping results to POR-72-TP1_R1_001.fastp-trim_bismark_bt2_pe.bam <<< Reading in the sequence files ../data/03-Peve-bismark/POR-72-TP1_R1_001.fastp-trim.fq.gz and ../data/03-Peve-bismark/POR-72-TP1_R2_001.fastp-trim.fq.gz Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1101:9327:4376_1:N:0:GGCAAGTT+CTTCTGAG Porites_evermani_scaffold_3076 2 10000 reads; of these: 10000 (100.00%) were paired; of these: 5421 (54.21%) aligned concordantly 0 times 2545 (25.45%) aligned concordantly exactly 1 time 2034 (20.34%) aligned concordantly >1 times 45.79% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 5396 (53.96%) aligned concordantly 0 times 2564 (25.64%) aligned concordantly exactly 1 time 2040 (20.40%) aligned concordantly >1 times 46.04% 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-72-TP1_R1_001.fastp-trim.fq.gz_C_to_T.fastq and POR-72-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: 316329 Total methylated C's in CpG context: 4586 Total methylated C's in CHG context: 296 Total methylated C's in CHH context: 998 Total methylated C's in Unknown context: 4 Total unmethylated C's in CpG context: 45404 Total unmethylated C's in CHG context: 54942 Total unmethylated C's in CHH context: 210103 Total unmethylated C's in Unknown context: 396 C methylated in CpG context: 9.2% C methylated in CHG context: 0.5% C methylated in CHH context: 0.5% C methylated in unknown context (CN or CHN): 1.0% Bismark completed in 0d 0h 0m 15s ==================== Bismark run complete ==================== gzip: stdout: Broken pipe gzip: stdout: Broken pipe gzip: stdout: Broken pipe