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/deep-dive-expression/D-Apul/data/)' FastQ format assumed (by default) 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 10 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/deep-dive-expression/D-Apul/code'): ../data/22-Apul-meth/ACR-150-TP2_R1.fastp-trim.fq.gz ../data/22-Apul-meth/ACR-150-TP2_R2.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/deep-dive-expression/D-Apul/output/22-Apul-methylation/ Setting parallelization to single-threaded (default) Summary of all aligner options: -q --score-min L,0,-1.0 -p 10 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 Current working directory is: /home/shared/8TB_HDD_03/sr320/github/deep-dive-expression/D-Apul/code Now reading in and storing sequence information of the genome specified in: /home/shared/8TB_HDD_03/sr320/github/deep-dive-expression/D-Apul/data/ Single-core mode: setting pid to 1 Paired-end alignments will be performed ======================================= The provided filenames for paired-end alignments are ../data/22-Apul-meth/ACR-150-TP2_R1.fastp-trim.fq.gz and ../data/22-Apul-meth/ACR-150-TP2_R2.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file ACR-150-TP2_R1.fastp-trim.fq.gz to ACR-150-TP2_R1.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file ACR-150-TP2_R1.fastp-trim.fq.gz (233529125 sequences in total) Writing a G -> A converted version of the input file ACR-150-TP2_R2.fastp-trim.fq.gz to ACR-150-TP2_R2.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file ACR-150-TP2_R2.fastp-trim.fq.gz (233529125 sequences in total) Input files are ACR-150-TP2_R1.fastp-trim.fq.gz_C_to_T.fastq and ACR-150-TP2_R2.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/deep-dive-expression/D-Apul/data/ with the specified options: -q --score-min L,0,-1.0 -p 10 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 Now starting a Bowtie 2 paired-end alignment for CTread1GAread2CTgenome (reading in sequences from ACR-150-TP2_R1.fastp-trim.fq.gz_C_to_T.fastq and ACR-150-TP2_R2.fastp-trim.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-1.0 -p 10 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: LH00260:131:22L7V3LT4:3:1101:1979:1014_1:N:0:CCGTGAAG+ANCCACTG/1 77 * 0 0 * * 0 0 AGTGAAATGTTGGATTGATGAATGTTGAGGATGTTTGAATTGTTTTGTGTGTTTGTTTGTTAA IIIIIIIIIIIIIIIIIIII99I9III9IIIIIIIII-II99IIII99III9I-II9IIIIII YT:Z:UP LH00260:131:22L7V3LT4:3:1101:1979:1014_1:N:0:CCGTGAAG+ANCCACTG/2 141 * 0 0 * * 0 0 AATAAAATATTAAATTAATAAATACTAAAAATATCTAAATTATTCTATATATTCATTTATTAA IIIIIIIIIIIIIIIIIIII99I9III9IIIIIIIII-II99IIII99III9I-II9IIIIII YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from ACR-150-TP2_R1.fastp-trim.fq.gz_C_to_T.fastq and ACR-150-TP2_R2.fastp-trim.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-1.0 -p 10 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --nofw)) Found first alignment: LH00260:131:22L7V3LT4:3:1101:1979:1014_1:N:0:CCGTGAAG+ANCCACTG/1 77 * 0 0 * * 0 0 AGTGAAATGTTGGATTGATGAATGTTGAGGATGTTTGAATTGTTTTGTGTGTTTGTTTGTTAA IIIIIIIIIIIIIIIIIIII99I9III9IIIIIIIII-II99IIII99III9I-II9IIIIII YT:Z:UP LH00260:131:22L7V3LT4:3:1101:1979:1014_1:N:0:CCGTGAAG+ANCCACTG/2 141 * 0 0 * * 0 0 AATAAAATATTAAATTAATAAATACTAAAAATATCTAAATTATTCTATATATTCATTTATTAA IIIIIIIIIIIIIIIIIIII99I9III9IIIIIIIII-II99IIII99III9I-II9IIIIII YT:Z:UP >>> Writing bisulfite mapping results to ACR-150-TP2_pe.bam <<< Reading in the sequence files ../data/22-Apul-meth/ACR-150-TP2_R1.fastp-trim.fq.gz and ../data/22-Apul-meth/ACR-150-TP2_R2.fastp-trim.fq.gz Processed 1000000 sequence pairs so far Processed 2000000 sequence pairs so far Processed 3000000 sequence pairs so far Processed 4000000 sequence pairs so far Processed 5000000 sequence pairs so far Processed 6000000 sequence pairs so far Processed 7000000 sequence pairs so far Processed 8000000 sequence pairs so far Processed 9000000 sequence pairs so far Processed 10000000 sequence pairs so far Processed 11000000 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227000000 sequence pairs so far Processed 228000000 sequence pairs so far Processed 229000000 sequence pairs so far Processed 230000000 sequence pairs so far Processed 231000000 sequence pairs so far Processed 232000000 sequence pairs so far Processed 233000000 sequence pairs so far 233529125 reads; of these: 233529125 (100.00%) were paired; of these: 233094312 (99.81%) aligned concordantly 0 times 206297 (0.09%) aligned concordantly exactly 1 time 228516 (0.10%) aligned concordantly >1 times 0.19% overall alignment rate 233529125 reads; of these: 233529125 (100.00%) were paired; of these: 233091073 (99.81%) aligned concordantly 0 times 203511 (0.09%) aligned concordantly exactly 1 time 234541 (0.10%) aligned concordantly >1 times 0.19% overall alignment rate Processed 233529125 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 934116500. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 934116500. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 934116500. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 934116500. Successfully deleted the temporary files ACR-150-TP2_R1.fastp-trim.fq.gz_C_to_T.fastq and ACR-150-TP2_R2.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 233529125 Final Cytosine Methylation Report ================================= Total number of C's analysed: 9465098 Total methylated C's in CpG context: 721196 Total methylated C's in CHG context: 547496 Total methylated C's in CHH context: 4267634 Total methylated C's in Unknown context: 55005 Total unmethylated C's in CpG context: 662934 Total unmethylated C's in CHG context: 690342 Total unmethylated C's in CHH context: 2575496 Total unmethylated C's in Unknown context: 94368 C methylated in CpG context: 52.1% C methylated in CHG context: 44.2% C methylated in CHH context: 62.4% C methylated in unknown context (CN or CHN): 36.8% Bismark completed in 0d 4h 24m 56s ==================== Bismark run complete ====================