Bowtie seems to be working fine (tested command '/gscratch/srlab/programs/bowtie2-2.3.4.1-linux-x86_64/bowtie2 --version' [2.3.4]) Output format is BAM (default) Alignments will be written out in BAM format. Samtools found here: '/gscratch/srlab/programs/samtools-1.9/samtools' Reference genome folder provided is /gscratch/srlab/sam/data/C_gigas/genomes/ (absolute path is '/gscratch/srlab/sam/data/C_gigas/genomes/)' Processing sequences up to read no. 500000 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 Input files to be analysed (in current folder '/gscratch/scrubbed/samwhite/outputs/20190222_cgigas_se_bismark/subset_500000'): /gscratch/scrubbed/samwhite/data/C_gigas/BSseq/cgig_bsseq_all_R1.fastq.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!) Setting parallelization to single-threaded (default) Current working directory is: /gscratch/scrubbed/samwhite/outputs/20190222_cgigas_se_bismark/subset_500000 Now reading in and storing sequence information of the genome specified in: /gscratch/srlab/sam/data/C_gigas/genomes/ Single-core mode: setting pid to 1 Single-end alignments will be performed ======================================= Input file is in FastQ format Processing reads up to sequence no. 500000 from /gscratch/scrubbed/samwhite/data/C_gigas/BSseq/cgig_bsseq_all_R1.fastq.gz Writing a C -> T converted version of the input file cgig_bsseq_all_R1.fastq.gz to cgig_bsseq_all_R1.fastq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file cgig_bsseq_all_R1.fastq.gz (500001 sequences in total) Input file is cgig_bsseq_all_R1.fastq.gz_C_to_T.fastq (FastQ) Now running 2 instances of Bowtie 2 against the bisulfite genome of /gscratch/srlab/sam/data/C_gigas/genomes/ with the specified options: -q --score-min L,0,-0.6 -p 28 --reorder --ignore-quals Now starting the Bowtie 2 aligner for CTreadCTgenome (reading in sequences from cgig_bsseq_all_R1.fastq.gz_C_to_T.fastq with options -q --score-min L,0,-0.6 -p 28 --reorder --ignore-quals --norc) Using Bowtie 2 index: /gscratch/srlab/sam/data/C_gigas/genomes/Bisulfite_Genome/CT_conversion/BS_CT Found first alignment: HWI-D00743:54:C8MOMACXX:1:1101:1599:2153_1:N:0:TAGCTT 4 * 0 0 * * 0 0 TNGGGGTAGGTTGAATTTGTATTTTTTATGTAGTTTTTTGAGAAAGATTGA C#1AD;2@;D>> Writing bisulfite mapping results to cgig_bsseq_all_R1_bismark_bt2.bam <<< Reading in the sequence file /gscratch/scrubbed/samwhite/data/C_gigas/BSseq/cgig_bsseq_all_R1.fastq.gz Chromosomal sequence could not be extracted for HWI-D00743:54:C8MOMACXX:1:1101:1773:34563_1:N:0:TAGCTT scaffold33742 2 Chromosomal sequence could not be extracted for HWI-D00743:54:C8MOMACXX:1:1102:3577:18659_1:N:0:TAGCTT C23010 2001 Chromosomal sequence could not be extracted for HWI-D00743:54:C8MOMACXX:1:1103:12835:3587_1:N:0:TAGCTT C23010 2002 Chromosomal sequence could not be extracted for HWI-D00743:54:C8MOMACXX:1:1104:10421:24530_1:N:0:TAGCTT scaffold38030 1 Chromosomal sequence could not be extracted for HWI-D00743:54:C8MOMACXX:1:1104:18665:62350_1:N:0:TAGCTT C34600 19430 Chromosomal sequence could not be extracted for HWI-D00743:54:C8MOMACXX:1:1104:2988:100874_1:N:0:TAGCTT scaffold811 2 Chromosomal sequence could not be extracted for HWI-D00743:54:C8MOMACXX:1:1105:13327:43238_1:N:0:TAGCTT C28016 2 500000 reads; of these: 500000 (100.00%) were unpaired; of these: 224886 (44.98%) aligned 0 times 126918 (25.38%) aligned exactly 1 time 148196 (29.64%) aligned >1 times 55.02% overall alignment rate 500000 reads; of these: 500000 (100.00%) were unpaired; of these: 224669 (44.93%) aligned 0 times 127294 (25.46%) aligned exactly 1 time 148037 (29.61%) aligned >1 times 55.07% overall alignment rate Successfully deleted the temporary file cgig_bsseq_all_R1.fastq.gz_C_to_T.fastq Final Alignment report ====================== Sequences analysed in total: 500000 Number of alignments with a unique best hit from the different alignments: 275445 Mapping efficiency: 55.1% Final Cytosine Methylation Report ================================= Total number of C's analysed: 2323933 Total methylated C's in CpG context: 90514 Total methylated C's in CHG context: 14428 Total methylated C's in CHH context: 27427 Total methylated C's in Unknown context: 399 Total unmethylated C's in CpG context: 262264 Total unmethylated C's in CHG context: 476749 Total unmethylated C's in CHH context: 1452551 Total unmethylated C's in Unknown context: 3906 C methylated in CpG context: 25.7% C methylated in CHG context: 2.9% C methylated in CHH context: 1.9% C methylated in Unknown context (CN or CHN): 9.3% Bismark completed in 0d 0h 2m 50s ==================== Bismark run complete ====================