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-TP4_R1_001.fastp-trim.fq.gz ../data/03-Peve-bismark/POR-245-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-245-TP4_R1_001.fastp-trim.fq.gz and ../data/03-Peve-bismark/POR-245-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-245-TP4_R1_001.fastp-trim.fq.gz Writing a C -> T converted version of the input file POR-245-TP4_R1_001.fastp-trim.fq.gz to POR-245-TP4_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file POR-245-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-245-TP4_R2_001.fastp-trim.fq.gz Writing a G -> A converted version of the input file POR-245-TP4_R2_001.fastp-trim.fq.gz to POR-245-TP4_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file POR-245-TP4_R2_001.fastp-trim.fq.gz (10001 sequences in total) Input files are POR-245-TP4_R1_001.fastp-trim.fq.gz_C_to_T.fastq and POR-245-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-245-TP4_R1_001.fastp-trim.fq.gz_C_to_T.fastq and POR-245-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: LH00526:197:22KFKVLT4:3:1101:49440:1056_1:N:0:CCTTCCAT+ATTGCGTG/1 77 * 0 0 * * 0 0 GTGGTAATTAAATTGTGTTATGATGTTTTTATTTAGAGTAGTTTATTTAAAGATTAATTTTTTAGTATGTTTTGATTTTGAAATATTTTTTATAAATTAGAAAGTAAAAGTTGGAGATTTTAAAATAGTTTGATG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-II9II-9III-I-I-IIIII-II99I9III9III--IIIIII-I9IIII99II-IIIIIIIIIIIII9IIIIIIIIIIIIII-II99I999I YT:Z:UP LH00526:197:22KFKVLT4:3:1101:49440:1056_2:N:0:CCTTCCAT+ATTGCGTG/2 141 * 0 0 * * 0 0 CCCTAATAACCAAAATATCCAACTCCCATACTTAATCCATACAACAACACTTCCACAAATCAATTTATTTTTAACATCAATTTAAAATACTTTCTCCCACCAAAATAAAAACACCATCTCATATATATACACATC IIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-I9IIII9IIIIIIIII9III YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from POR-245-TP4_R1_001.fastp-trim.fq.gz_C_to_T.fastq and POR-245-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: LH00526:197:22KFKVLT4:3:1101:49440:1056_1:N:0:CCTTCCAT+ATTGCGTG/1 83 Porites_evermani_scaffold_31_GA_converted 309485 42 135M = 309307 -313 CATCAAACTATTTTAAAATCTCCAACTTTTACTTTCTAATTTATAAAAAATATTTCAAAATCAAAACATACTAAAAAATTAATCTTTAAATAAACTACTCTAAATAAAAACATCATAACACAATTTAATTACCAC I999I99II-IIIIIIIIIIIIII9IIIIIIIIIIIII-II99IIII9I-IIIIII--III9III9I99II-IIIII-I-I-III9-II9II-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-6 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:57T77 YS:i:0 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:49440:1056_2:N:0:CCTTCCAT+ATTGCGTG/2 163 Porites_evermani_scaffold_31_GA_converted 309307 42 135M = 309485 313 CCCTAATAACCAAAATATCCAACTCCCATACTTAATCCATACAACAACACTTCCACAAATCAATTTATTTTTAACATCAATTTAAAATACTTTCTCCCACCAAAATAAAAACACCATCTCATATATATACACATC IIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-I9IIII9IIIIIIIII9III 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 >>> Writing bisulfite mapping results to POR-245-TP4_R1_001.fastp-trim_bismark_bt2_pe.bam <<< Reading in the sequence files ../data/03-Peve-bismark/POR-245-TP4_R1_001.fastp-trim.fq.gz and ../data/03-Peve-bismark/POR-245-TP4_R2_001.fastp-trim.fq.gz Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1101:35238:6309_1:N:0:CCTTCCAT+ATTGCGTG Porites_evermani_scaffold_2018 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1101:35254:6309_1:N:0:CCTTCCAT+ATTGCGTG Porites_evermani_scaffold_2018 1 10000 reads; of these: 10000 (100.00%) were paired; of these: 5351 (53.51%) aligned concordantly 0 times 2694 (26.94%) aligned concordantly exactly 1 time 1955 (19.55%) aligned concordantly >1 times 46.49% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 5576 (55.76%) aligned concordantly 0 times 2441 (24.41%) aligned concordantly exactly 1 time 1983 (19.83%) aligned concordantly >1 times 44.24% 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-TP4_R1_001.fastp-trim.fq.gz_C_to_T.fastq and POR-245-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: 318983 Total methylated C's in CpG context: 4645 Total methylated C's in CHG context: 300 Total methylated C's in CHH context: 1124 Total methylated C's in Unknown context: 3 Total unmethylated C's in CpG context: 45319 Total unmethylated C's in CHG context: 55764 Total unmethylated C's in CHH context: 211831 Total unmethylated C's in Unknown context: 365 C methylated in CpG context: 9.3% C methylated in CHG context: 0.5% C methylated in CHH context: 0.5% C methylated in unknown context (CN or CHN): 0.8% Bismark completed in 0d 0h 0m 16s ==================== Bismark run complete ==================== gzip: stdout: Broken pipe gzip: stdout: Broken pipe gzip: stdout: Broken pipe