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_virginica/genomes/GCF_002022765.2_C_virginica-3.0/ (absolute path is '/gscratch/srlab/sam/data/C_virginica/genomes/GCF_002022765.2_C_virginica-3.0/)' 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/20190312_cvir_gonad_bismark/total_reads_bismark'): /gscratch/scrubbed/samwhite/outputs/20190312_cvir_gonad_bismark/cvir_bsseq_all_pe_R1.fastq.gz /gscratch/scrubbed/samwhite/outputs/20190312_cvir_gonad_bismark/cvir_bsseq_all_pe_R2.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: /gscratch/scrubbed/samwhite/outputs/20190312_cvir_gonad_bismark/total_reads_bismark Now reading in and storing sequence information of the genome specified in: /gscratch/srlab/sam/data/C_virginica/genomes/GCF_002022765.2_C_virginica-3.0/ Single-core mode: setting pid to 1 Paired-end alignments will be performed ======================================= The provided filenames for paired-end alignments are /gscratch/scrubbed/samwhite/outputs/20190312_cvir_gonad_bismark/cvir_bsseq_all_pe_R1.fastq.gz and /gscratch/scrubbed/samwhite/outputs/20190312_cvir_gonad_bismark/cvir_bsseq_all_pe_R2.fastq.gz Input files are in FastQ format Writing a C -> T converted version of the input file cvir_bsseq_all_pe_R1.fastq.gz to cvir_bsseq_all_pe_R1.fastq.gz_C_to_T.fastq Writing a G -> A converted version of the input file cvir_bsseq_all_pe_R1.fastq.gz to cvir_bsseq_all_pe_R1.fastq.gz_G_to_A.fastq Created C -> T as well as G -> A converted versions of the FastQ file cvir_bsseq_all_pe_R1.fastq.gz (275914272 sequences in total) Writing a C -> T converted version of the input file cvir_bsseq_all_pe_R2.fastq.gz to cvir_bsseq_all_pe_R2.fastq.gz_C_to_T.fastq Writing a G -> A converted version of the input file cvir_bsseq_all_pe_R2.fastq.gz to cvir_bsseq_all_pe_R2.fastq.gz_G_to_A.fastq Created C -> T as well as G -> A converted versions of the FastQ file cvir_bsseq_all_pe_R2.fastq.gz (275914272 sequences in total) Input files are cvir_bsseq_all_pe_R1.fastq.gz_C_to_T.fastq and cvir_bsseq_all_pe_R1.fastq.gz_G_to_A.fastq and cvir_bsseq_all_pe_R2.fastq.gz_C_to_T.fastq and cvir_bsseq_all_pe_R2.fastq.gz_G_to_A.fastq (FastQ) Now running 4 individual instances of Bowtie 2 against the bisulfite genome of /gscratch/srlab/sam/data/C_virginica/genomes/GCF_002022765.2_C_virginica-3.0/ with the specified options: -q --score-min L,0,-0.6 -p 28 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 Now starting a Bowtie 2 paired-end alignment for CTread1GAread2CTgenome (reading in sequences from cvir_bsseq_all_pe_R1.fastq.gz_C_to_T.fastq and cvir_bsseq_all_pe_R2.fastq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-0.6 -p 28 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: HWI-C00124:321:CC781ANXX:2:1101:1232:2155_1:N:0:GATCAG/1 77 * 0 0 * * 0 0 ATTGTATTAATTATAAAAAATATNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNATTTATTATTAAAAATATTAAATA FFFFFFFFFBFFFFFFFFFFFFF#################################<>> Writing bisulfite mapping results to cvir_bsseq_all_pe_R1_bismark_bt2_pe.bam <<< Reading in the sequence files /gscratch/scrubbed/samwhite/outputs/20190312_cvir_gonad_bismark/cvir_bsseq_all_pe_R1.fastq.gz and /gscratch/scrubbed/samwhite/outputs/20190312_cvir_gonad_bismark/cvir_bsseq_all_pe_R2.fastq.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 sequence pairs so far Processed 12000000 sequence pairs so far Processed 13000000 sequence pairs so far Processed 14000000 sequence pairs so far Processed 15000000 sequence pairs so far Processed 16000000 sequence pairs so far Processed 17000000 sequence pairs so far Processed 18000000 sequence pairs so far Processed 19000000 sequence pairs so far Processed 20000000 sequence pairs so far Processed 21000000 sequence pairs so far Processed 22000000 sequence pairs so far Processed 23000000 sequence pairs so far Processed 24000000 sequence pairs so far Processed 25000000 sequence pairs so far Processed 26000000 sequence pairs so far Processed 27000000 sequence pairs so far Processed 28000000 sequence pairs so far Processed 29000000 sequence pairs so far Processed 30000000 sequence pairs so far Processed 31000000 sequence pairs so far Processed 32000000 sequence pairs so far Processed 33000000 sequence pairs so far Processed 34000000 sequence pairs so far Processed 35000000 sequence pairs so far Processed 36000000 sequence pairs so far Processed 37000000 sequence pairs so far Processed 38000000 sequence pairs so far Processed 39000000 sequence pairs so far Processed 40000000 sequence pairs so far Processed 41000000 sequence pairs so far Processed 42000000 sequence pairs so far Processed 43000000 sequence pairs so far Processed 44000000 sequence pairs so far Processed 45000000 sequence pairs so far Processed 46000000 sequence pairs so far Processed 47000000 sequence pairs so far Processed 48000000 sequence pairs so far Processed 49000000 sequence pairs so far Processed 50000000 sequence pairs so far Processed 51000000 sequence pairs so far Processed 52000000 sequence pairs so far Processed 53000000 sequence pairs so far Chromosomal sequence could not be extracted for HWI-C00124:321:CC781ANXX:1:1210:4310:17386_1:N:0:TGACCA NC_035780.1 1 Processed 54000000 sequence pairs so far Processed 55000000 sequence pairs so far Processed 56000000 sequence pairs so far Processed 57000000 sequence pairs so far Processed 58000000 sequence pairs so far Processed 59000000 sequence pairs so far Processed 60000000 sequence pairs so far Processed 61000000 sequence pairs so far Chromosomal sequence could not be extracted for HWI-C00124:321:CC781ANXX:1:1316:4047:74652_1:N:0:TGACCA NC_007175.2 17170 Processed 62000000 sequence pairs so far Processed 63000000 sequence pairs so far Processed 64000000 sequence pairs so far Processed 65000000 sequence pairs so far Processed 66000000 sequence pairs so far Processed 67000000 sequence pairs so far Processed 68000000 sequence pairs so far Processed 69000000 sequence pairs so far Processed 70000000 sequence pairs so far Processed 71000000 sequence pairs so far Processed 72000000 sequence pairs so far Processed 73000000 sequence pairs so far Processed 74000000 sequence pairs so far Processed 75000000 sequence pairs so far Processed 76000000 sequence pairs so far Processed 77000000 sequence pairs so far Processed 78000000 sequence pairs so far Processed 79000000 sequence pairs so far Processed 80000000 sequence pairs so far Processed 81000000 sequence pairs so far Processed 82000000 sequence pairs so far Processed 83000000 sequence pairs so far Processed 84000000 sequence pairs so far Processed 85000000 sequence pairs so far Processed 86000000 sequence pairs so far Processed 87000000 sequence pairs so far Processed 88000000 sequence pairs so far Processed 89000000 sequence pairs so far Processed 90000000 sequence pairs so far Processed 91000000 sequence pairs so far Processed 92000000 sequence pairs so far Processed 93000000 sequence pairs so far Processed 94000000 sequence pairs so far Processed 95000000 sequence pairs so far Processed 96000000 sequence pairs so far Processed 97000000 sequence pairs so far Processed 98000000 sequence pairs so far Processed 99000000 sequence pairs so far Processed 100000000 sequence pairs so far Processed 101000000 sequence pairs so far Processed 102000000 sequence pairs so far Processed 103000000 sequence pairs so far Processed 104000000 sequence pairs so far Processed 105000000 sequence pairs so far Processed 106000000 sequence pairs so far Processed 107000000 sequence pairs so far Processed 108000000 sequence pairs so far Processed 109000000 sequence pairs so far Processed 110000000 sequence pairs so far Processed 111000000 sequence pairs so far Processed 112000000 sequence pairs so far Processed 113000000 sequence pairs so far Processed 114000000 sequence pairs so far Processed 115000000 sequence pairs so far Processed 116000000 sequence pairs so far Processed 117000000 sequence pairs so far Processed 118000000 sequence pairs so far Processed 119000000 sequence pairs so far Processed 120000000 sequence pairs so far Processed 121000000 sequence pairs so far Processed 122000000 sequence pairs so far Processed 123000000 sequence pairs so far Chromosomal sequence could not be extracted for HWI-C00124:321:CC781ANXX:1:2206:10235:74664_1:N:0:GCCAAT NC_007175.2 17189 Processed 124000000 sequence pairs so far Processed 125000000 sequence pairs so far Processed 126000000 sequence pairs so far Processed 127000000 sequence pairs so far Processed 128000000 sequence pairs so far Processed 129000000 sequence pairs so far Processed 130000000 sequence pairs so far Processed 131000000 sequence pairs so far Processed 132000000 sequence pairs so far Processed 133000000 sequence pairs so far Processed 134000000 sequence pairs so far Processed 135000000 sequence pairs so far Processed 136000000 sequence pairs so far Processed 137000000 sequence pairs so far Processed 138000000 sequence pairs so far Processed 139000000 sequence pairs so far Processed 140000000 sequence pairs so far Processed 141000000 sequence pairs so far Processed 142000000 sequence pairs so far Processed 143000000 sequence pairs so far Processed 144000000 sequence pairs so far Processed 145000000 sequence pairs so far Processed 146000000 sequence pairs so far Processed 147000000 sequence pairs so far Processed 148000000 sequence pairs so far Processed 149000000 sequence pairs so far Processed 150000000 sequence pairs so far Chromosomal sequence could not be extracted for HWI-C00124:321:CC781ANXX:1:2116:16897:28912_1:N:0:CAGATC NC_035780.1 1 Processed 151000000 sequence pairs so far Processed 152000000 sequence pairs so far Processed 153000000 sequence pairs so far Processed 154000000 sequence pairs so far Processed 155000000 sequence pairs so far Processed 156000000 sequence pairs so far Processed 157000000 sequence pairs so far Processed 158000000 sequence pairs so far Processed 159000000 sequence pairs so far Processed 160000000 sequence pairs so far Processed 161000000 sequence pairs so far Processed 162000000 sequence pairs so far Processed 163000000 sequence pairs so far Processed 164000000 sequence pairs so far Processed 165000000 sequence pairs so far Chromosomal sequence could not be extracted for HWI-C00124:321:CC781ANXX:1:1201:7258:96604_1:N:0:CTTGTA NC_035780.1 1 Processed 166000000 sequence pairs so far Processed 167000000 sequence pairs so far Processed 168000000 sequence pairs so far Processed 169000000 sequence pairs so far Processed 170000000 sequence pairs so far Processed 171000000 sequence pairs so far Processed 172000000 sequence pairs so far Processed 173000000 sequence pairs so far Processed 174000000 sequence pairs so far Processed 175000000 sequence pairs so far Processed 176000000 sequence pairs so far Processed 177000000 sequence pairs so far Processed 178000000 sequence pairs so far Processed 179000000 sequence pairs so far Processed 180000000 sequence pairs so far Processed 181000000 sequence pairs so far Processed 182000000 sequence pairs so far Processed 183000000 sequence pairs so far Processed 184000000 sequence pairs so far Processed 185000000 sequence pairs so far Processed 186000000 sequence pairs so far Processed 187000000 sequence pairs so far Processed 188000000 sequence pairs so far Processed 189000000 sequence pairs so far Processed 190000000 sequence pairs so far Processed 191000000 sequence pairs so far Processed 192000000 sequence pairs so far Processed 193000000 sequence pairs so far Processed 194000000 sequence pairs so far Processed 195000000 sequence pairs so far Processed 196000000 sequence pairs so far Processed 197000000 sequence pairs so far Processed 198000000 sequence pairs so far Processed 199000000 sequence pairs so far Processed 200000000 sequence pairs so far Processed 201000000 sequence pairs so far Processed 202000000 sequence pairs so far Processed 203000000 sequence pairs so far Processed 204000000 sequence pairs so far Processed 205000000 sequence pairs so far Processed 206000000 sequence pairs so far Processed 207000000 sequence pairs so far Chromosomal sequence could not be extracted for HWI-C00124:321:CC781ANXX:1:2211:12726:18562_1:N:0:ATCACG NC_007175.2 17178 Processed 208000000 sequence pairs so far Processed 209000000 sequence pairs so far Processed 210000000 sequence pairs so far Processed 211000000 sequence pairs so far Processed 212000000 sequence pairs so far Processed 213000000 sequence pairs so far Processed 214000000 sequence pairs so far Processed 215000000 sequence pairs so far Processed 216000000 sequence pairs so far Processed 217000000 sequence pairs so far Processed 218000000 sequence pairs so far Processed 219000000 sequence pairs so far Processed 220000000 sequence pairs so far Processed 221000000 sequence pairs so far Processed 222000000 sequence pairs so far Processed 223000000 sequence pairs so far Processed 224000000 sequence pairs so far Processed 225000000 sequence pairs so far Processed 226000000 sequence pairs so far Processed 227000000 sequence pairs so far Chromosomal sequence could not be extracted for HWI-C00124:321:CC781ANXX:1:1311:7196:8646_1:N:0:TTAGGC NC_007175.2 1 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 Processed 234000000 sequence pairs so far Processed 235000000 sequence pairs so far Processed 236000000 sequence pairs so far Processed 237000000 sequence pairs so far Processed 238000000 sequence pairs so far Processed 239000000 sequence pairs so far Chromosomal sequence could not be extracted for HWI-C00124:321:CC781ANXX:1:2303:6577:56043_1:N:0:TTAGGC NC_007175.2 17170 Processed 240000000 sequence pairs so far Processed 241000000 sequence pairs so far Processed 242000000 sequence pairs so far Processed 243000000 sequence pairs so far Processed 244000000 sequence pairs so far Processed 245000000 sequence pairs so far Processed 246000000 sequence pairs so far Processed 247000000 sequence pairs so far Processed 248000000 sequence pairs so far Processed 249000000 sequence pairs so far Processed 250000000 sequence pairs so far Processed 251000000 sequence pairs so far Processed 252000000 sequence pairs so far Processed 253000000 sequence pairs so far Processed 254000000 sequence pairs so far Processed 255000000 sequence pairs so far Processed 256000000 sequence pairs so far Processed 257000000 sequence pairs so far Processed 258000000 sequence pairs so far Processed 259000000 sequence pairs so far Processed 260000000 sequence pairs so far Processed 261000000 sequence pairs so far Processed 262000000 sequence pairs so far Processed 263000000 sequence pairs so far Processed 264000000 sequence pairs so far Processed 265000000 sequence pairs so far Processed 266000000 sequence pairs so far Processed 267000000 sequence pairs so far Chromosomal sequence could not be extracted for HWI-C00124:321:CC781ANXX:2:2207:4799:56169_1:N:0:ACTTGA NC_007175.2 17167 Processed 268000000 sequence pairs so far Processed 269000000 sequence pairs so far Processed 270000000 sequence pairs so far Processed 271000000 sequence pairs so far Processed 272000000 sequence pairs so far Processed 273000000 sequence pairs so far Processed 274000000 sequence pairs so far Processed 275000000 sequence pairs so far 275914272 reads; of these: 275914272 (100.00%) were paired; of these: 226738044 (82.18%) aligned concordantly 0 times 22223208 (8.05%) aligned concordantly exactly 1 time 26953020 (9.77%) aligned concordantly >1 times 17.82% overall alignment rate 275914272 reads; of these: 275914272275914272 ( reads; of these: 275914272 (100.00%) were paired; of these: 100.00224584830% () were paired; of these:81.40 % ) aligned concordantly 0 times226740753 ( 82.1823175511% () aligned concordantly 0 times8.40 % ) aligned concordantly exactly 1 time22172592 ( 8.0428153931% () aligned concordantly exactly 1 time10.20 % ) aligned concordantly >1 times27000927 (18.609.79%% overall alignment rate) aligned concordantly >1 times 17.82% overall alignment rate 275914272 reads; of these: 275914272 (100.00%) were paired; of these: 224616541 (81.41%) aligned concordantly 0 times 23086354 (8.37%) aligned concordantly exactly 1 time 28211377 (10.22%) aligned concordantly >1 times 18.59% overall alignment rate Processed 275914272 sequences in total Successfully deleted the temporary files cvir_bsseq_all_pe_R1.fastq.gz_C_to_T.fastq, cvir_bsseq_all_pe_R1.fastq.gz_G_to_A.fastq, cvir_bsseq_all_pe_R2.fastq.gz_C_to_T.fastq and cvir_bsseq_all_pe_R2.fastq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 275914272 Final Cytosine Methylation Report ================================= Total number of C's analysed: 2447811202 Total methylated C's in CpG context: 252963945 Total methylated C's in CHG context: 10974793 Total methylated C's in CHH context: 38264164 Total methylated C's in Unknown context: 1418685 Total unmethylated C's in CpG context: 82162796 Total unmethylated C's in CHG context: 546133588 Total unmethylated C's in CHH context: 1517311916 Total unmethylated C's in Unknown context: 8077086 C methylated in CpG context: 75.5% C methylated in CHG context: 2.0% C methylated in CHH context: 2.5% C methylated in unknown context (CN or CHN): 14.9% Bismark completed in 2d 20h 32m 48s ==================== Bismark run complete ====================