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-73-TP3_R1_001.fastp-trim.fq.gz ../data/03-Peve-bismark/POR-73-TP3_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-73-TP3_R1_001.fastp-trim.fq.gz and ../data/03-Peve-bismark/POR-73-TP3_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-73-TP3_R1_001.fastp-trim.fq.gz Writing a C -> T converted version of the input file POR-73-TP3_R1_001.fastp-trim.fq.gz to POR-73-TP3_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file POR-73-TP3_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-73-TP3_R2_001.fastp-trim.fq.gz Writing a G -> A converted version of the input file POR-73-TP3_R2_001.fastp-trim.fq.gz to POR-73-TP3_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file POR-73-TP3_R2_001.fastp-trim.fq.gz (10001 sequences in total) Input files are POR-73-TP3_R1_001.fastp-trim.fq.gz_C_to_T.fastq and POR-73-TP3_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-73-TP3_R1_001.fastp-trim.fq.gz_C_to_T.fastq and POR-73-TP3_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: LH00526:197:22KFKVLT4:3:1101:52143:1056_1:N:0:GTACCACA+TGTTGTGG/1 99 Porites_evermani_scaffold_40_CT_converted 757126 42 135M = 757167 176 TTTTGAAAAATTTGTTAGTATTGTGTTTGGTTGAGTTTGAGATAATATTAAAATTTTTTAGGATTATAGATTAATGATATATTGTTTTTGTTGAGTTAGTGTTATTTTTATTGTTGTTGGTTGGAGGATTAAAAG IIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIII9I9IIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:49T39T45 YS:i:-12 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:52143:1056_2:N:0:GTACCACA+TGTTGTGG/2 147 Porites_evermani_scaffold_40_CT_converted 757167 42 135M = 757126 -176 ATAATATTAAAATTTTTTAGGATTATAGATTAATGATATATTGTTTTTGTTGAGTTAGTGTTATTTTTATTGTTGTTGGTTGGAGGATTAAAAGATGGGAGGTGTTTTTATGTTGATTGAAGGTTTATTTTTTTT IIII-I9IIIIIIIIIIIIIII9II-IIIII99III99-IIIIIIIIIIIIIIIII9IIIIIIIIIII9III9IIIIIIIIIIIIII9IIIIIIIIIIIIIII9IIIIIIIIIII9IIIIIIIIIII9I9IIIII AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:8T39T86 YS:i:-12 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from POR-73-TP3_R1_001.fastp-trim.fq.gz_C_to_T.fastq and POR-73-TP3_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: LH00526:197:22KFKVLT4:3:1101:52143:1056_1:N:0:GTACCACA+TGTTGTGG/1 77 * 0 0 * * 0 0 TTTTGAAAAATTTGTTAGTATTGTGTTTGGTTGAGTTTGAGATAATATTAAAATTTTTTAGGATTATAGATTAATGATATATTGTTTTTGTTGAGTTAGTGTTATTTTTATTGTTGTTGGTTGGAGGATTAAAAG IIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIII9I9IIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII YT:Z:UP LH00526:197:22KFKVLT4:3:1101:52143:1056_2:N:0:GTACCACA+TGTTGTGG/2 141 * 0 0 * * 0 0 AAAAAAAATAAACCTTCAATCAACATAAAAACACCTCCCATCTTTTAATCCTCCAACCAACAACAATAAAAATAACACTAACTCAACAAAAACAATATATCATTAATCTATAATCCTAAAAAATTTTAATATTAT IIIII9I9IIIIIIIIIII9IIIIIIIIIII9IIIIIIIIIIIIIII9IIIIIIIIIIIIII9III9IIIIIIIIIII9IIIIIIIIIIIIIIIII-99III99IIIII-II9IIIIIIIIIIIIIII9I-IIII YT:Z:UP >>> Writing bisulfite mapping results to POR-73-TP3_R1_001.fastp-trim_bismark_bt2_pe.bam <<< Reading in the sequence files ../data/03-Peve-bismark/POR-73-TP3_R1_001.fastp-trim.fq.gz and ../data/03-Peve-bismark/POR-73-TP3_R2_001.fastp-trim.fq.gz Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1101:41469:4488_1:N:0:GTACCACA+TGTTGTGG Porites_evermani_scaffold_507 3 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1101:46333:4698_1:N:0:GTACCACA+TGTTGTGG Porites_evermani_scaffold_6241 6473 10000 reads; of these: 10000 (100.00%) were paired; of these: 5325 (53.25%) aligned concordantly 0 times 2584 (25.84%) aligned concordantly exactly 1 time 2091 (20.91%) aligned concordantly >1 times 46.75% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 5222 (52.22%) aligned concordantly 0 times 2713 (27.13%) aligned concordantly exactly 1 time 2065 (20.65%) aligned concordantly >1 times 47.78% 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-73-TP3_R1_001.fastp-trim.fq.gz_C_to_T.fastq and POR-73-TP3_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: 322368 Total methylated C's in CpG context: 4485 Total methylated C's in CHG context: 370 Total methylated C's in CHH context: 1536 Total methylated C's in Unknown context: 9 Total unmethylated C's in CpG context: 47630 Total unmethylated C's in CHG context: 56712 Total unmethylated C's in CHH context: 211635 Total unmethylated C's in Unknown context: 392 C methylated in CpG context: 8.6% C methylated in CHG context: 0.6% C methylated in CHH context: 0.7% C methylated in unknown context (CN or CHN): 2.2% Bismark completed in 0d 0h 0m 16s ==================== Bismark run complete ==================== gzip: stdout: Broken pipe gzip: stdout: Broken pipe gzip: stdout: Broken pipe