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/scrubbed/samwhite/data/P_generosa/Pgenerosa_v071/ (absolute path is '/gscratch/scrubbed/samwhite/data/P_generosa/Pgenerosa_v071/)' Processing sequences up to read no. 100000 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_geo_rrbs_bismark/subset_Pgenerosa_v071_100000'): /gscratch/scrubbed/samwhite/data/P_generosa/BSeq/rrbs/pgen_bsseq_all_R1.fastq.gz /gscratch/scrubbed/samwhite/data/P_generosa/BSeq/rrbs/pgen_bsseq_all_R2.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_geo_rrbs_bismark/subset_Pgenerosa_v071_100000 Now reading in and storing sequence information of the genome specified in: /gscratch/scrubbed/samwhite/data/P_generosa/Pgenerosa_v071/ Single-core mode: setting pid to 1 Paired-end alignments will be performed ======================================= The provided filenames for paired-end alignments are /gscratch/scrubbed/samwhite/data/P_generosa/BSeq/rrbs/pgen_bsseq_all_R1.fastq.gz and /gscratch/scrubbed/samwhite/data/P_generosa/BSeq/rrbs/pgen_bsseq_all_R2.fastq.gz Input files are in FastQ format Processing reads up to sequence no. 100000 from /gscratch/scrubbed/samwhite/data/P_generosa/BSeq/rrbs/pgen_bsseq_all_R1.fastq.gz Writing a C -> T converted version of the input file pgen_bsseq_all_R1.fastq.gz to pgen_bsseq_all_R1.fastq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file pgen_bsseq_all_R1.fastq.gz (100001 sequences in total) Processing reads up to sequence no. 100000 from /gscratch/scrubbed/samwhite/data/P_generosa/BSeq/rrbs/pgen_bsseq_all_R2.fastq.gz Writing a G -> A converted version of the input file pgen_bsseq_all_R2.fastq.gz to pgen_bsseq_all_R2.fastq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file pgen_bsseq_all_R2.fastq.gz (100001 sequences in total) Input files are pgen_bsseq_all_R1.fastq.gz_C_to_T.fastq and pgen_bsseq_all_R2.fastq.gz_G_to_A.fastq (FastQ) Now running 2 instances of Bowtie 2 against the bisulfite genome of /gscratch/scrubbed/samwhite/data/P_generosa/Pgenerosa_v071/ 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 pgen_bsseq_all_R1.fastq.gz_C_to_T.fastq and pgen_bsseq_all_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: SL-HBW:624:CAAWVANXX:5:2301:1512:1956_1:N:0:ATCACG/1 99 PGA_scaffold71710__1_contigs__length_40502_CT_converted 40348 40 50M = 40348 -50 NGTTTATATGTATGTATTATATTTGTGTAGGTATTGTATTTGATAAGGAT #<<>> Writing bisulfite mapping results to pgen_bsseq_all_R1_bismark_bt2_pe.bam <<< Reading in the sequence files /gscratch/scrubbed/samwhite/data/P_generosa/BSeq/rrbs/pgen_bsseq_all_R1.fastq.gz and /gscratch/scrubbed/samwhite/data/P_generosa/BSeq/rrbs/pgen_bsseq_all_R2.fastq.gz Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:1609:3036_1:N:0:ATCACG PGA_scaffold65517__1_contigs__length_15888 1 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:14032:3053_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:7726:3624_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798928 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:3973:3828_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:17368:4030_1:N:0:ATCACG PGA_scaffold65517__1_contigs__length_15888 1 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:14831:4639_1:N:0:ATCACG PGA_scaffold65517__1_contigs__length_15888 1 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:10593:5178_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:3873:7275_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:12727:9121_1:N:0:ATCACG PGA_scaffold65325__1_contigs__length_12743 12649 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:15388:9040_1:N:0:ATCACG PGA_scaffold65517__1_contigs__length_15888 2 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:13361:10834_1:N:0:ATCACG PGA_scaffold60946__1_contigs__length_19950 19853 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:4623:12077_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:15228:13489_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:9736:14242_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798928 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:4542:15045_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:20327:15253_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:15434:16045_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798926 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:5113:17810_1:N:0:ATCACG PGA_scaffold48397__1_contigs__length_20908 2 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:13947:18376_1:N:0:ATCACG PGA_scaffold63075__1_contigs__length_16706 3 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:14648:18683_1:N:0:ATCACG PGA_scaffold71716__1_contigs__length_11894 2 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:7001:20835_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:7539:21586_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798928 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:6493:23041_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:18807:23475_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:19744:25783_1:N:0:ATCACG PGA_scaffold67542__1_contigs__length_23105 1 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:3323:26853_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:1834:27730_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:7779:27669_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:19571:31179_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798927 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:11200:31541_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:3159:31974_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:5855:32292_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798927 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:4156:34487_1:N:0:ATCACG PGA_scaffold718__1_contigs__length_989392 989315 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:10665:34864_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:11790:36217_1:N:0:ATCACG PGA_scaffold65517__1_contigs__length_15888 2 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:5437:36841_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:14324:37124_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798928 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:2101:37716_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:18845:40323_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798928 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:10610:41002_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798925 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:15018:42958_1:N:0:ATCACG PGA_scaffold69964__1_contigs__length_13211 1 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:15869:43273_1:N:0:ATCACG PGA_scaffold65517__1_contigs__length_15888 1 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:19579:43965_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:1419:44486_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:4210:44750_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:4479:45035_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:16746:47406_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798928 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:3739:47602_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:7807:48626_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:8461:48977_1:N:0:ATCACG PGA_scaffold65517__1_contigs__length_15888 1 100000 reads; of these: 100000 (100.00%) were paired; of these: 54124 (54.12%) aligned concordantly 0 times 20751 (20.75%) aligned concordantly exactly 1 time 25125 (25.12%) aligned concordantly >1 times 45.88% overall alignment rate 100000 reads; of these: 100000 (100.00%) were paired; of these: 53688 (53.69%) aligned concordantly 0 times 21046 (21.05%) aligned concordantly exactly 1 time 25266 (25.27%) aligned concordantly >1 times 46.31% overall alignment rate Processed 100000 sequences in total Successfully deleted the temporary files pgen_bsseq_all_R1.fastq.gz_C_to_T.fastq and pgen_bsseq_all_R2.fastq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 100000 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1694841 Total methylated C's in CpG context: 52999 Total methylated C's in CHG context: 7471 Total methylated C's in CHH context: 13214 Total methylated C's in Unknown context: 238 Total unmethylated C's in CpG context: 185806 Total unmethylated C's in CHG context: 350443 Total unmethylated C's in CHH context: 1084908 Total unmethylated C's in Unknown context: 3497 C methylated in CpG context: 22.2% C methylated in CHG context: 2.1% C methylated in CHH context: 1.2% C methylated in unknown context (CN or CHN): 6.4% Bismark completed in 0d 0h 1m 43s ==================== Bismark run complete ====================