Use of uninitialized value $path_to_bowtie in concatenation (.) or string at /gscratch/srlab/programs/Bismark-0.21.0/bismark line 6893. Bowtie 2 seems to be working fine (tested command 'bowtie2 --version' [2.1.0]) Output format is BAM (default) Alignments will be written out in BAM format. Samtools found here: '/gscratch/srlab/programs/samtools-1.4/bin/samtools' Reference genome folder provided is /gscratch/srlab/sr320/data/caligus/genome/ (absolute path is '/gscratch/srlab/sr320/data/caligus/genome/)' FastQ format assumed (by default) Each Bowtie 2 instance is going to be run with 4 threads. Please monitor performance closely and tune down if necessary! Input files to be analysed (in current folder '/gscratch/scrubbed/sr320/1219'): /gscratch/srlab/sr320/data/caligus/reads/Sealice_F1_S20_R1_001_trimmed.5bp_3prime.fq.gz /gscratch/srlab/sr320/data/caligus/reads/Sealice_F1_S20_R2_001_trimmed.5bp_3prime.fq.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) Summary of all aligner options: -q --score-min L,0,-0.6 -p 4 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 Current working directory is: /gscratch/scrubbed/sr320/1219 Now reading in and storing sequence information of the genome specified in: /gscratch/srlab/sr320/data/caligus/genome/ chr Cr_scaffold0000001 (36897007 bp) chr Cr_scaffold0000002 (35124817 bp) chr Cr_scaffold0000003 (33138466 bp) chr Cr_scaffold0000004 (31456035 bp) chr Cr_scaffold0000005 (30612693 bp) chr Cr_scaffold0000006 (30259679 bp) chr Cr_scaffold0000007 (29872177 bp) chr Cr_scaffold0000008 (27803016 bp) chr Cr_scaffold0000009 (25126635 bp) chr Cr_scaffold0000010 (24815513 bp) chr Cr_scaffold0000011 (35794548 bp) chr Cr_scaffold0000012 (22957860 bp) chr Cr_scaffold0000013 (21502856 bp) chr Cr_scaffold0000014 (21148584 bp) chr Cr_scaffold0000015 (19091682 bp) chr Cr_scaffold0000016 (18525937 bp) chr Cr_scaffold0000017 (17562062 bp) chr Cr_scaffold0000018 (14680182 bp) chr Cr_scaffold0000019 (14290452 bp) chr Cr_scaffold0000020 (8286978 bp) chr Cr_scaffold0000021 (8023048 bp) Single-core mode: setting pid to 1 Paired-end alignments will be performed ======================================= The provided filenames for paired-end alignments are /gscratch/srlab/sr320/data/caligus/reads/Sealice_F1_S20_R1_001_trimmed.5bp_3prime.fq.gz and /gscratch/srlab/sr320/data/caligus/reads/Sealice_F1_S20_R2_001_trimmed.5bp_3prime.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file Sealice_F1_S20_R1_001_trimmed.5bp_3prime.fq.gz to Sealice_F1_S20_R1_001_trimmed.5bp_3prime.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file Sealice_F1_S20_R1_001_trimmed.5bp_3prime.fq.gz (226106788 sequences in total) Writing a G -> A converted version of the input file Sealice_F1_S20_R2_001_trimmed.5bp_3prime.fq.gz to Sealice_F1_S20_R2_001_trimmed.5bp_3prime.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file Sealice_F1_S20_R2_001_trimmed.5bp_3prime.fq.gz (225857063 sequences in total) [FATAL ERROR]: Number of bisulfite transformed reads are not equal between Read 1 (#226106788) and Read 2 (#225857063). Possible causes: file truncation, or as a result of specifying read pairs that do not belong to each other?! Please re-specify file names! Exiting... xargs: /gscratch/srlab/programs/Bismark-0.21.0/bismark: exited with status 255; aborting find: ‘*.bam’: No such file or directory basename: missing operand Try 'basename --help' for more information. *** Bismark methylation extractor version v0.21.0 *** Trying to determine the type of mapping from the SAM header line of file *deduplicated.bam [FATAL ERROR]: File >>*deduplicated.bam<< did not exist. Please re-specify file names and try again... Found no potential alignment reports in the current directory. Please specify a single Bismark alignment report file using the option '--alignment_report FILE' SYNOPSIS: This script uses a Bismark alignment report to generate a graphical HTML report page. Optionally, further reports of the Bismark suite such as deduplication, methylation extractor splitting or M-bias reports can be specified as well. If several Bismark reports are found in the same folder, a separate report will be generated for each of these, whereby the output filename will be derived from the Bismark alignment report file. bismark2report attempts to find optional reports automatically based on the file basename. USAGE: bismark2report [options] -o/--output Name of the output file (optional). If not specified explicitly, the output filename will be derived from the Bismark alignment report file. Specifying an output filename only works if the HTML report is to be generated for a single Bismark alignment report (and potentially additional reports). --dir Output directory. Output is written to the current directory if not specified explicitly. --alignment_report FILE If not specified explicitly, bismark2report attempts to find Bismark report file(s) in the current directory and produces a separate HTML report for each mapping report file. Based on the basename of the Bismark mapping report, bismark2report will also attempt to find the other Bismark reports (see below) for inclusion into the HTML report. Specifying a Bismark alignment report file is mandatory. --dedup_report FILE If not specified explicitly, bismark2report attempts to find a deduplication report file with the same basename as the Bismark mapping report (generated by deduplicate_bismark) in the current working directory. Including a deduplication report is optional, and using the FILE 'none' will skip this step entirely. --splitting_report FILE If not specified explicitly, bismark2report attempts to find a splitting report file with the same basename as the Bismark mapping report (generated by the Bismark methylation extractor) in the current working directory. Including a splitting report is optional, and using the FILE 'none' will skip this step entirely. --mbias_report FILE If not specified explicitly, bismark2report attempts to find a single M-bias report file with the same basename as the Bismark mapping report (generated by the Bismark methylation extractor) in the current working directory. Including an M-Bias report is optional, and using the FILE 'none' will skip this step entirely. --nucleotide_report FILE If not specified explicitly, bismark2report attempts to find a single nucleotide coverage report file with the same basename as the Bismark mapping report (generated by Bismark with the option '--nucleotide_coverage') in the current working directory. Including a nucleotide coverage statistics report is optional, and using the FILE 'none' will skip this report entirely. Script last modified: 07 August 2018 No Bismark/Bowtie2 single-end BAM files detected No Bismark/Bowtie2 paired-end BAM files detected No Bismark/HISAT2 single-end BAM files detected No Bismark/HISAT2 paired-end BAM files detected Error: No Bismark BAM files found to generate a Bismark project summary. Please respecify... USAGE: bismark2summary (*.bam), or bismark2summary --help for more information at /gscratch/srlab/programs/Bismark-0.21.0/bismark2summary line 201. find: ‘*deduplicated.bam’: No such file or directory basename: missing operand Try 'basename --help' for more information. find: ‘*.sorted.bam’: No such file or directory basename: missing operand Try 'basename --help' for more information. find: ‘*deduplicated.bismark.cov.gz’: No such file or directory basename: missing operand Try 'basename --help' for more information. cat: merged_CpG_evidence.cov: No such file or directory cat: *merged_CpG_evidence.cov: No such file or directory