Path to Bowtie 2 specified as: bowtie2 Bowtie seems to be working fine (tested command 'bowtie2 --version' [2.3.0]) Output format is BAM (default) Did not find Samtools on the system. Alignments will be compressed with GZIP instead (.sam.gz) Reference genome folder provided is Crassostrea_gigas.oyster_v9.dna_sm.toplevel/ (absolute path is '/Users/yaamini/Documents/project-gigas-oa-meth/analyses/2019-08-30-Bismark-Parameter-Testing/Crassostrea_gigas.oyster_v9.dna_sm.toplevel/)' Processing sequences up to read no. 10000 from the input file Input files to be analysed (in current folder '/Users/yaamini/Documents/project-gigas-oa-meth/analyses/2019-08-30-Bismark-Parameter-Testing'): YRVL_R1_001.fastq.gz YRVL_R2_001.fastq.gz Library was specified to be not strand-specific (non-directional), therefore alignments to all four possible bisulfite strands (OT, CTOT, OB and CTOB) will be reported Setting parallelization to single-threaded (default) Current working directory is: /Users/yaamini/Documents/project-gigas-oa-meth/analyses/2019-08-30-Bismark-Parameter-Testing Now reading in and storing sequence information of the genome specified in: /Users/yaamini/Documents/project-gigas-oa-meth/analyses/2019-08-30-Bismark-Parameter-Testing/Crassostrea_gigas.oyster_v9.dna_sm.toplevel/ Single-core mode: setting pid to 1 Paired-end alignments will be performed ======================================= The provided filenames for paired-end alignments are YRVL_R1_001.fastq.gz and YRVL_R2_001.fastq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from YRVL_R1_001.fastq.gz Writing a C -> T converted version of the input file YRVL_R1_001.fastq.gz to YRVL_R1_001.fastq.gz_C_to_T.fastq Writing a G -> A converted version of the input file YRVL_R1_001.fastq.gz to YRVL_R1_001.fastq.gz_G_to_A.fastq Created C -> T as well as G -> A converted versions of the FastQ file YRVL_R1_001.fastq.gz (10001 sequences in total) Processing reads up to sequence no. 10000 from YRVL_R2_001.fastq.gz Writing a C -> T converted version of the input file YRVL_R2_001.fastq.gz to YRVL_R2_001.fastq.gz_C_to_T.fastq Writing a G -> A converted version of the input file YRVL_R2_001.fastq.gz to YRVL_R2_001.fastq.gz_G_to_A.fastq Created C -> T as well as G -> A converted versions of the FastQ file YRVL_R2_001.fastq.gz (10001 sequences in total) Input files are YRVL_R1_001.fastq.gz_C_to_T.fastq and YRVL_R1_001.fastq.gz_G_to_A.fastq and YRVL_R2_001.fastq.gz_C_to_T.fastq and YRVL_R2_001.fastq.gz_G_to_A.fastq (FastQ) Now running 4 individual instances of Bowtie 2 against the bisulfite genome of /Users/yaamini/Documents/project-gigas-oa-meth/analyses/2019-08-30-Bismark-Parameter-Testing/Crassostrea_gigas.oyster_v9.dna_sm.toplevel/ with the specified options: -q --score-min L,0,-0.6 -p 4 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 Now starting a Bowtie 2 paired-end alignment for CTread1GAread2CTgenome (reading in sequences from YRVL_R1_001.fastq.gz_C_to_T.fastq and YRVL_R2_001.fastq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-0.6 -p 4 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: GWNJ-0901:413:GW1904042006th:8:1101:9120:1327_1:N:0:NGACCA/1 77 * 0 0 * * 0 0 NTATTTGAAATAAGTTTTGTAGTGTGTGTAAAAAATTGAAATAATGTTGTTTATGGTGTTAAGGTTTTTTGTGGAAATAAAAAGATATATATTTAAATATGGAGATTTTTTTTTTTATATAATTTTTTTTAGATTGTAAGTGTTTATGTT #AAFFJFJJJJJJJJJJ>> Writing bisulfite mapping results to YRVL_R1_001_bismark_bt2_pe.sam.gz <<< Reading in the sequence files YRVL_R1_001.fastq.gz and YRVL_R2_001.fastq.gz Chromosomal sequence could not be extracted for GWNJ-0901:413:GW1904042006th:8:1101:18264:3524_1:N:0:TGACCA scaffold36208 2 10000 reads; of these: 10000 (100.00%) were paired; of these: 9993 (99.93%) aligned concordantly 0 times 3 (0.03%) aligned concordantly exactly 1 time 4 (0.04%) aligned concordantly >1 times 0.07% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 9986 (99.86%) aligned concordantly 0 times 6 (0.06%) aligned concordantly exactly 1 time 8 (0.08%) aligned concordantly >1 times 0.14% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 6514 (65.14%) aligned concordantly 0 times 2005 (20.05%) aligned concordantly exactly 1 time 1481 (14.81%) aligned concordantly >1 times 34.86% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 6534 (65.34%) aligned concordantly 0 times 1972 (19.72%) aligned concordantly exactly 1 time 1494 (14.94%) aligned concordantly >1 times 34.66% overall alignment rate Processed 10000 sequences in total Successfully deleted the temporary files YRVL_R1_001.fastq.gz_C_to_T.fastq, YRVL_R1_001.fastq.gz_G_to_A.fastq, YRVL_R2_001.fastq.gz_C_to_T.fastq and YRVL_R2_001.fastq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 10000 Final Cytosine Methylation Report ================================= Total number of C's analysed: 222656 Total methylated C's in CpG context: 4408 Total methylated C's in CHG context: 1358 Total methylated C's in CHH context: 6232 Total methylated C's in Unknown context: 187 Total unmethylated C's in CpG context: 22424 Total unmethylated C's in CHG context: 35582 Total unmethylated C's in CHH context: 152652 Total unmethylated C's in Unknown context: 937 C methylated in CpG context: 16.4% C methylated in CHG context: 3.7% C methylated in CHH context: 3.9% C methylated in unknown context (CN or CHN): 16.6% Bismark completed in 0d 0h 0m 27s ==================== Bismark run complete ====================