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'): YRVA_R1_001.fastq.gz YRVA_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 YRVA_R1_001.fastq.gz and YRVA_R2_001.fastq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from YRVA_R1_001.fastq.gz Writing a C -> T converted version of the input file YRVA_R1_001.fastq.gz to YRVA_R1_001.fastq.gz_C_to_T.fastq Writing a G -> A converted version of the input file YRVA_R1_001.fastq.gz to YRVA_R1_001.fastq.gz_G_to_A.fastq Created C -> T as well as G -> A converted versions of the FastQ file YRVA_R1_001.fastq.gz (10001 sequences in total) Processing reads up to sequence no. 10000 from YRVA_R2_001.fastq.gz Writing a C -> T converted version of the input file YRVA_R2_001.fastq.gz to YRVA_R2_001.fastq.gz_C_to_T.fastq Writing a G -> A converted version of the input file YRVA_R2_001.fastq.gz to YRVA_R2_001.fastq.gz_G_to_A.fastq Created C -> T as well as G -> A converted versions of the FastQ file YRVA_R2_001.fastq.gz (10001 sequences in total) Input files are YRVA_R1_001.fastq.gz_C_to_T.fastq and YRVA_R1_001.fastq.gz_G_to_A.fastq and YRVA_R2_001.fastq.gz_C_to_T.fastq and YRVA_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,-0.9 -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 YRVA_R1_001.fastq.gz_C_to_T.fastq and YRVA_R2_001.fastq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-0.9 -p 4 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: GWNJ-0901:413:GW1904042006th:8:1101:12510:1327_1:N:0:NGATGT/1 99 scaffold1878_CT_converted 10355 0 150M = 10402 197 NTAGTGGTTTTTTTATTAATTTGAATTTATTTTTAGGATAAAATAGAAATTTTGATTTGTAAATTATATTTGTAGATAAGGTAAGAAGTTTTAAAAATGTTAATATATGTATTAAATTATTATTTTTAGAATGATATTGTTTTTTAATTG #AAA-AFFJJJJJJJJJAF-JJJFJJJJFJJJJJJJJFJFFFFFFJJ-AJJJJJFJJJJJ->> Writing bisulfite mapping results to YRVA_R1_001_bismark_bt2_pe.sam.gz <<< Reading in the sequence files YRVA_R1_001.fastq.gz and YRVA_R2_001.fastq.gz Chromosomal sequence could not be extracted for GWNJ-0901:413:GW1904042006th:8:1101:7902:3964_1:N:0:CGATGT C25882 1 10000 reads; of these: 10000 (100.00%) were paired; of these: 5381 (53.81%) aligned concordantly 0 times 2314 (23.14%) aligned concordantly exactly 1 time 2305 (23.05%) aligned concordantly >1 times 46.19% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 9888 (98.88%) aligned concordantly 0 times 82 (0.82%) aligned concordantly exactly 1 time 30 (0.30%) aligned concordantly >1 times 1.12% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 9884 (98.84%) aligned concordantly 0 times 85 (0.85%) aligned concordantly exactly 1 time 31 (0.31%) aligned concordantly >1 times 1.16% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 5442 (54.42%) aligned concordantly 0 times 2246 (22.46%) aligned concordantly exactly 1 time 2312 (23.12%) aligned concordantly >1 times 45.58% overall alignment rate Processed 10000 sequences in total Successfully deleted the temporary files YRVA_R1_001.fastq.gz_C_to_T.fastq, YRVA_R1_001.fastq.gz_G_to_A.fastq, YRVA_R2_001.fastq.gz_C_to_T.fastq and YRVA_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: 283139 Total methylated C's in CpG context: 5592 Total methylated C's in CHG context: 1734 Total methylated C's in CHH context: 8583 Total methylated C's in Unknown context: 421 Total unmethylated C's in CpG context: 29193 Total unmethylated C's in CHG context: 45569 Total unmethylated C's in CHH context: 192468 Total unmethylated C's in Unknown context: 1721 C methylated in CpG context: 16.1% C methylated in CHG context: 3.7% C methylated in CHH context: 4.3% C methylated in unknown context (CN or CHN): 19.7% Bismark completed in 0d 0h 0m 29s ==================== Bismark run complete ====================