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_v070/ (absolute path is '/gscratch/scrubbed/samwhite/data/P_generosa/Pgenerosa_v070/)' 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_v070_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_v070_100000 Now reading in and storing sequence information of the genome specified in: /gscratch/scrubbed/samwhite/data/P_generosa/Pgenerosa_v070/ 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_v070/ 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_scaffold25235__1_contigs__length_1316_CT_converted 580 1 50M = 580 -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:5:2301:11623:2069_1:N:0:ATCACG PGA_scaffold32732__1_contigs__length_2240 3 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:15086:2208_1:N:0:ATCACG PGA_scaffold41319__1_contigs__length_1135 1 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:17208:2040_1:N:0:ATCACG PGA_scaffold71178__1_contigs__length_4197 1 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:13969:2647_1:N:0:ATCACG PGA_scaffold23407__1_contigs__length_2192 1 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:5:2301:4047:4144_1:N:0:ATCACG PGA_scaffold45488__1_contigs__length_1333 1237 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:4971:4235_1:N:0:ATCACG PGA_scaffold52424__1_contigs__length_3527 3429 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:12517:4131_1:N:0:ATCACG PGA_scaffold20616__1_contigs__length_1183 1088 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:5:2301:15997:4377_1:N:0:ATCACG PGA_scaffold67771__1_contigs__length_6483 2 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:5:2301:3873:7275_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:6490:8347_1:N:0:ATCACG PGA_scaffold11450__1_contigs__length_1253 2 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:5:2301:6448:9781_1:N:0:ATCACG PGA_scaffold19151__1_contigs__length_2082 2 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:12198:10107_1:N:0:ATCACG PGA_scaffold14256__1_contigs__length_1778 1704 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:4811:11102_1:N:0:ATCACG PGA_scaffold30804__1_contigs__length_2641 2 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:12417:11389_1:N:0:ATCACG PGA_scaffold37296__1_contigs__length_1012 2 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:14680:11988_1:N:0:ATCACG PGA_scaffold34510__1_contigs__length_1314 1225 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:5:2301:13701:12308_1:N:0:ATCACG PGA_scaffold45254__1_contigs__length_1291 1 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:19459:12814_1:N:0:ATCACG PGA_scaffold25165__1_contigs__length_2707 2611 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:20626:13308_1:N:0:ATCACG PGA_scaffold61583__1_contigs__length_13650 2 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:9364:13583_1:N:0:ATCACG PGA_scaffold19863__1_contigs__length_1761 1669 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:20762:13651_1:N:0:ATCACG PGA_scaffold1564__1_contigs__length_1666 2 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:5:2301:16275:14515_1:N:0:ATCACG PGA_scaffold20616__1_contigs__length_1183 1085 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:19467:15122_1:N:0:ATCACG PGA_scaffold36490__1_contigs__length_5243 3 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:20447:15349_1:N:0:ATCACG PGA_scaffold9226__1_contigs__length_1034 1 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:14907:16248_1:N:0:ATCACG PGA_scaffold56753__1_contigs__length_3010 2 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:5:2301:7616:16737_1:N:0:GTCACG PGA_scaffold24917__1_contigs__length_1507 1415 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:13314:17737_1:N:0:ATCACG PGA_scaffold32803__1_contigs__length_2645 2555 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:5:2301:7001:20835_1:N:0:ATCACG PGA_scaffold323__1_contigs__length_1799026 1798929 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:12053:21375_1:N:0:ATCACG PGA_scaffold34208__1_contigs__length_2237 2144 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:4699:21694_1:N:0:ATCACG PGA_scaffold20616__1_contigs__length_1183 1087 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:20362:21666_1:N:0:ATCACG PGA_scaffold71472__1_contigs__length_4043 1 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:3979:21903_1:N:0:ATCACG PGA_scaffold50821__1_contigs__length_2343 1 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:5:2301:6749:23611_1:N:0:ATCACG PGA_scaffold51538__1_contigs__length_9641 3 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:11861:23521_1:N:0:ATCACG PGA_scaffold71988__1_contigs__length_4570 4471 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:5886:24241_1:N:0:ATCACG PGA_scaffold30867__1_contigs__length_1516 1 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:11558:24481_1:N:0:ATCACG PGA_scaffold43189__1_contigs__length_2819 2 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:7504:24810_1:N:0:ATCACG PGA_scaffold22606__1_contigs__length_4570 1 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:5:2301:12371:26365_1:N:0:ATCACG PGA_scaffold955__1_contigs__length_1266 2 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:5:2301:1977:27635_1:N:0:ATCACG PGA_scaffold34477__1_contigs__length_6039 1 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:5:2301:10862:27989_1:N:0:ATCACG PGA_scaffold2516__1_contigs__length_2719 2 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:5:2301:6874:31573_1:N:0:ATCACG PGA_scaffold14495__1_contigs__length_1124 1025 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:5:2301:13870:31760_1:N:0:ATCACG PGA_scaffold71873__1_contigs__length_8186 1 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:5:2301:7972:32861_1:N:0:AACACG PGA_scaffold25592__1_contigs__length_1155 1059 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:5:2301:18692:34381_1:N:0:ATCACG PGA_scaffold59977__1_contigs__length_1141 1043 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:5:2301:14829:36769_1:N:0:ATCACG PGA_scaffold48911__1_contigs__length_1808 1707 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:5:2301:1316:38991_1:N:0:ATCACG PGA_scaffold259769__1_contigs__length_1744 1658 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:17418:40568_1:N:0:ATCACG PGA_scaffold56216__1_contigs__length_1445 1354 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:17480:40958_1:N:0:ATCACG PGA_scaffold38848__1_contigs__length_1094 2 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:8443:41491_1:N:0:ATCACG PGA_scaffold31185__1_contigs__length_1433 2 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:15029:41867_1:N:0:ATCACG PGA_scaffold2416__1_contigs__length_3481 3391 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:13775:42754_1:N:0:ATCACG PGA_scaffold35412__1_contigs__length_2376 2284 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:4591:43018_1:N:0:ATCACG PGA_scaffold304912__1_contigs__length_1731 1630 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:5:2301:1419:44486_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:5:2301:4873:44657_1:N:0:ATCACG PGA_scaffold12622__1_contigs__length_2849 2750 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:5:2301:4184:47037_1:N:0:ATCACG PGA_scaffold5839__1_contigs__length_1041 2 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:10957:47064_1:N:0:ATCACG PGA_scaffold71478__1_contigs__length_4197 1 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:14591:47734_1:N:0:ATCACG PGA_scaffold73092__1_contigs__length_8564 1 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:17699:47602_1:N:0:ATCACG PGA_scaffold61692__1_contigs__length_6562 6465 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:16038:48486_1:N:0:ATCACG PGA_scaffold17225__1_contigs__length_1255 2 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:18012:48374_1:N:0:ATCACG PGA_scaffold71790__1_contigs__length_1978 1 Chromosomal sequence could not be extracted for SL-HBW:624:CAAWVANXX:5:2301:21106:48323_1:N:0:ATCACG PGA_scaffold47144__1_contigs__length_1542 1 100000 reads; of these: 100000 (100.00%) were paired; of these: 46879 (46.88%) aligned concordantly 0 times 20137 (20.14%) aligned concordantly exactly 1 time 32984 (32.98%) aligned concordantly >1 times 53.12% overall alignment rate 100000 reads; of these: 100000 (100.00%) were paired; of these: 47293 (47.29%) aligned concordantly 0 times 19953 (19.95%) aligned concordantly exactly 1 time 32754 (32.75%) aligned concordantly >1 times 52.71% 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: 1803055 Total methylated C's in CpG context: 54428 Total methylated C's in CHG context: 7752 Total methylated C's in CHH context: 13821 Total methylated C's in Unknown context: 292 Total unmethylated C's in CpG context: 207701 Total unmethylated C's in CHG context: 377061 Total unmethylated C's in CHH context: 1142292 Total unmethylated C's in Unknown context: 3516 C methylated in CpG context: 20.8% C methylated in CHG context: 2.0% C methylated in CHH context: 1.2% C methylated in unknown context (CN or CHN): 7.7% Bismark completed in 0d 0h 2m 5s ==================== Bismark run complete ====================