[Fri Oct  9 10:55:46 PDT 2026] Pum1 (Pum/Pumalin)

 *** Bismark methylation extractor version v0.25.1 ***

Output will be written into the directory: /mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/
Setting option '--no_overlap' since this is (normally) the right thing to do for paired-end data

Summarising Bismark methylation extractor parameters:
===============================================================
Bismark paired-end SAM format specified (default)
Number of cores to be used: 8
First 5 bp will be disregarded when processing the methylation call string of Read 2
Strand-specific outputs will be skipped. Separate output files for cytosines in CpG, CHG and CHH context will be generated
Merge CHG and CHH context to non-CpG context specified
Output path specified as: /mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/


Summarising bedGraph parameters:
===============================================================
Generating additional output in bedGraph and coverage format
bedGraph format:	<Chromosome> <Start Position> <End Position> <Methylation Percentage>
coverage format:	<Chromosome> <Start Position> <End Position> <Methylation Percentage> <count methylated> <count non-methylated>

Using a cutoff of 1 read(s) to report cytosine positions
Reporting and sorting cytosine methylation information in CpG context only (default)
The bedGraph UNIX sort command will use the following memory setting:	'40G'. Temporary directory used for sorting is the output directory

Checking file >>output/13-wgbs/dedup/Pum1_pe.deduplicated.bam<< for signs of file truncation...


Now testing Bismark result file >output/13-wgbs/dedup/Pum1_pe.deduplicated.bam< for positional sorting (which would be bad...)	...passed!
Writing result file containing methylation information for C in CpG context to /mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/CpG_context_Pum1_pe.deduplicated.txt.gz
Writing result file containing methylation information for C in any other context to /mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/Non_CpG_context_Pum1_pe.deduplicated.txt.gz

Now reading in Bismark result file output/13-wgbs/dedup/Pum1_pe.deduplicated.bam


Now reading in Bismark result file output/13-wgbs/dedup/Pum1_pe.deduplicated.bam


Now reading in Bismark result file output/13-wgbs/dedup/Pum1_pe.deduplicated.bam


Now reading in Bismark result file output/13-wgbs/dedup/Pum1_pe.deduplicated.bam


Now reading in Bismark result file output/13-wgbs/dedup/Pum1_pe.deduplicated.bam


Now reading in Bismark result file output/13-wgbs/dedup/Pum1_pe.deduplicated.bam

Now reading in Bismark result file output/13-wgbs/dedup/Pum1_pe.deduplicated.bam

Now reading in Bismark result file output/13-wgbs/dedup/Pum1_pe.deduplicated.bam

skipping SAM header line:	@HD	VN:1.0	SO:unsorted
skipping SAM header line:	@SQ	SN:Chromosome_10A	LN:41401248
skipping SAM header line:	@SQ	SN:Chromosome_2A	LN:98156180
skipping SAM header line:	@SQ	SN:Chromosome_4A	LN:93973257
skipping SAM header line:	@SQ	SN:Chromosome_6A	LN:76092957
skipping SAM header line:	@SQ	SN:Chromosome_1B	LN:231619483
skipping SAM header line:	@SQ	SN:Chromosome_5B	LN:125625032
skipping SAM header line:	@SQ	SN:Chromosome_6B	LN:129267145
skipping SAM header line:	@SQ	SN:Chromosome_7B	LN:183991082
skipping SAM header line:	@SQ	SN:Chromosome_8B	LN:108828118
skipping SAM header line:	@SQ	SN:Chromosome_9B	LN:91966420
skipping SAM header line:	@PG	ID:Bismark	VN:v0.25.1	CL:"bismark --genome data/13-wgbs-genome -1 output/13-wgbs/trimmed/Pum1_R1.fq.gz -2 output/13-wgbs/trimmed/Pum1_R2.fq.gz --score_min L,0,-0.6 --parallel 8 -p 2 --temp_dir /tmp/13-wgbs-Pum1-3530457 --output_dir output/13-wgbs/bismark"
skipping SAM header line:	@PG	ID:samtools	PN:samtools	PP:Bismark	VN:1.22.1	CL:/mmfs1/gscratch/srlab/sr320/miniforge3/envs/myflow/bin/samtools view -bSh -
skipping SAM header line:	@PG	ID:samtools.1	PN:samtools	PP:samtools	VN:1.22.1	CL:/mmfs1/gscratch/srlab/sr320/miniforge3/envs/myflow/bin/samtools view -h /mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/bismark/Pum1_R1.fq.gz.temp.1_bismark_bt2_pe.bam
skipping SAM header line:	@PG	ID:samtools.2	PN:samtools	PP:samtools.1	VN:1.22.1	CL:/mmfs1/gscratch/srlab/sr320/miniforge3/envs/myflow/bin/samtools view --threads 4 -bSh -
skipping SAM header line:	@PG	ID:samtools.3	PN:samtools	PP:samtools.2	VN:1.22.1	CL:/mmfs1/gscratch/srlab/sr320/miniforge3/envs/myflow/bin/samtools view -h --threads 1 output/13-wgbs/bismark/Pum1_pe.bam
skipping SAM header line:	@PG	ID:samtools.4	PN:samtools	PP:samtools.3	VN:1.22.1	CL:/mmfs1/gscratch/srlab/sr320/miniforge3/envs/myflow/bin/samtools view -bS --threads 1 -
skipping SAM header line:	@PG	ID:samtools.5	PN:samtools	PP:samtools.4	VN:1.22.1	CL:/mmfs1/gscratch/srlab/sr320/miniforge3/envs/myflow/bin/samtools view -h output/13-wgbs/dedup/Pum1_pe.deduplicated.bam
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Finished processing child process. Exiting..
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All child process successfully finished.
Merging individual splitting reports into overall report: '/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/Pum1_pe.deduplicated_splitting_report.txt'
Merging from these individual files:
/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/Pum1_pe.deduplicated_splitting_report.txt.1
/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/Pum1_pe.deduplicated_splitting_report.txt.2
/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/Pum1_pe.deduplicated_splitting_report.txt.3
/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/Pum1_pe.deduplicated_splitting_report.txt.4
/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/Pum1_pe.deduplicated_splitting_report.txt.5
/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/Pum1_pe.deduplicated_splitting_report.txt.6
/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/Pum1_pe.deduplicated_splitting_report.txt.7
/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/Pum1_pe.deduplicated_splitting_report.txt.8


Processed 19975663 lines in total
Total number of methylation call strings processed: 39951326

Final Cytosine Methylation Report
=================================
Total number of C's analysed:	706674834

Total methylated C's in CpG context:	16692933
Total methylated C's in CHG context:	588226
Total methylated C's in CHH context:	2643226

Total C to T conversions in CpG context:	69440998
Total C to T conversions in CHG context:	120026487
Total C to T conversions in CHH context:	497282964

C methylated in CpG context:	19.4%
C methylated in non-CpG context:	0.5%



Merging individual M-bias reports into overall M-bias statistics from these 8 individual files:
/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/Pum1_pe.deduplicated_splitting_report.txt.1.mbias
/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/Pum1_pe.deduplicated_splitting_report.txt.2.mbias
/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/Pum1_pe.deduplicated_splitting_report.txt.3.mbias
/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/Pum1_pe.deduplicated_splitting_report.txt.4.mbias
/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/Pum1_pe.deduplicated_splitting_report.txt.5.mbias
/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/Pum1_pe.deduplicated_splitting_report.txt.6.mbias
/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/Pum1_pe.deduplicated_splitting_report.txt.7.mbias
/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/Pum1_pe.deduplicated_splitting_report.txt.8.mbias

Determining maximum read lengths for M-Bias plots
Maximum read length of Read 1: 141
Maximum read length of Read 2: 136

Perl module GD::Graph::lines is not installed, skipping drawing M-bias plots (only writing out M-bias plot table)
Determining maximum read lengths for M-Bias plots
Maximum read length of Read 1: 141
Maximum read length of Read 2: 136

Perl module GD::Graph::lines is not installed, skipping drawing M-bias plots (only writing out M-bias plot table)
Deleting unused files ...

/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/CpG_context_Pum1_pe.deduplicated.txt.gz contains data ->	kept
/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/Non_CpG_context_Pum1_pe.deduplicated.txt.gz contains data ->	kept


Using these input files: /mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/CpG_context_Pum1_pe.deduplicated.txt.gz /mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/Non_CpG_context_Pum1_pe.deduplicated.txt.gz

Summary of parameters for bismark2bedGraph conversion:
======================================================
bedGraph output:		Pum1_pe.deduplicated.bedGraph.gz
output directory:		>/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/<
remove whitespaces:		no
CX context:			no (CpG context only, default)
No-header selected:		no
Sorting method:			Unix sort-based (smaller memory footprint, but slower)
Sort buffer size:		40G
Coverage threshold:		1
=============================================================================
Methylation information will now be written into a bedGraph and coverage file
=============================================================================

Using the following files as Input:
/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/CpG_context_Pum1_pe.deduplicated.txt.gz

Writing bedGraph to file: Pum1_pe.deduplicated.bedGraph.gz
Also writing out a coverage file including counts methylated and unmethylated residues to file: Pum1_pe.deduplicated.bismark.cov.gz

Changed directory to /mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/
Now writing methylation information for file >>CpG_context_Pum1_pe.deduplicated.txt.gz<< to individual files for each chromosome
Finished writing out individual chromosome files for CpG_context_Pum1_pe.deduplicated.txt.gz

Collecting temporary chromosome file information... Processing the following input file(s):
CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_8B.methXtractor.temp
CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_2A.methXtractor.temp
CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_7B.methXtractor.temp
CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_5B.methXtractor.temp
CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_1B.methXtractor.temp
CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_6A.methXtractor.temp
CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_9B.methXtractor.temp
CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_6B.methXtractor.temp
CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_10A.methXtractor.temp
CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_4A.methXtractor.temp

Sorting input file CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_10A.methXtractor.temp by positions (using -S of 40G)
Successfully deleted the temporary input file CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_10A.methXtractor.temp

Sorting input file CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_1B.methXtractor.temp by positions (using -S of 40G)
Successfully deleted the temporary input file CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_1B.methXtractor.temp

Sorting input file CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_2A.methXtractor.temp by positions (using -S of 40G)
Successfully deleted the temporary input file CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_2A.methXtractor.temp

Sorting input file CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_4A.methXtractor.temp by positions (using -S of 40G)
Successfully deleted the temporary input file CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_4A.methXtractor.temp

Sorting input file CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_5B.methXtractor.temp by positions (using -S of 40G)
Successfully deleted the temporary input file CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_5B.methXtractor.temp

Sorting input file CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_6A.methXtractor.temp by positions (using -S of 40G)
Successfully deleted the temporary input file CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_6A.methXtractor.temp

Sorting input file CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_6B.methXtractor.temp by positions (using -S of 40G)
Successfully deleted the temporary input file CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_6B.methXtractor.temp

Sorting input file CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_7B.methXtractor.temp by positions (using -S of 40G)
Successfully deleted the temporary input file CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_7B.methXtractor.temp

Sorting input file CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_8B.methXtractor.temp by positions (using -S of 40G)
Successfully deleted the temporary input file CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_8B.methXtractor.temp

Sorting input file CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_9B.methXtractor.temp by positions (using -S of 40G)
Successfully deleted the temporary input file CpG_context_Pum1_pe.deduplicated.txt.gz.chrChromosome_9B.methXtractor.temp

Finished BedGraph conversion ...

Output will be written into the directory: /mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/
Summary of parameters for genome-wide cytosine report:
==============================================================================
Coverage infile:				output/13-wgbs/methylation/Pum1_pe.deduplicated.bismark.cov.gz
Output directory:				>/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/<
Parent directory:				>/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/<
Genome directory:				>/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/data/13-wgbs-genome/<
Coverage threshold				0 (all positions will be reported, default)
CX context:					no (CpG context only, default)
Pooling CpG top/bottom strand evidence:		yes
Genome coordinates used:			1-based (default)
GZIP compression:				yes
Split by chromosome:				no


Now reading in and storing sequence information of the genome specified in: /mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/data/13-wgbs-genome/

Couldn't find files ending in .fa, trying .fa.gz instead
chr Chromosome_10A (41401248 bp)
chr Chromosome_2A (98156180 bp)
chr Chromosome_4A (93973257 bp)
chr Chromosome_6A (76092957 bp)
chr Chromosome_1B (231619483 bp)
chr Chromosome_5B (125625032 bp)
chr Chromosome_6B (129267145 bp)
chr Chromosome_7B (183991082 bp)
chr Chromosome_8B (108828118 bp)
chr Chromosome_9B (91966420 bp)

Stored sequence information of 10 chromosomes/scaffolds in total

==============================================================================
Methylation information will now be written into a genome-wide cytosine report
==============================================================================

Adding context-specific methylation summaries

>>> Writing genome-wide cytosine report to: Pum1.CpG_report.txt.gz <<<

>>> Writing all cytosine context summary file to: Pum1.cytosine_context_summary.txt <<<

Storing all covered cytosine positions for chromosome: Chromosome_10A
Writing cytosine report for chromosome Chromosome_10A (stored 1404528 different covered positions)
Writing cytosine report for chromosome Chromosome_1B (stored 1804688 different covered positions)
Writing cytosine report for chromosome Chromosome_2A (stored 3092653 different covered positions)
Writing cytosine report for chromosome Chromosome_4A (stored 2999370 different covered positions)
Writing cytosine report for chromosome Chromosome_5B (stored 2935352 different covered positions)
Writing cytosine report for chromosome Chromosome_6A (stored 2569802 different covered positions)
Writing cytosine report for chromosome Chromosome_6B (stored 2852217 different covered positions)
Writing cytosine report for chromosome Chromosome_7B (stored 1475406 different covered positions)
Writing cytosine report for chromosome Chromosome_8B (stored 2869458 different covered positions)
Writing cytosine report for last chromosome Chromosome_9B (stored 2717488 different covered positions)
Finished writing out cytosine report for covered chromosomes (processed 10 chromosomes/scaffolds in total)

Now processing chromosomes that were not covered by any methylation calls in the coverage file...
All chromosomes in the genome were covered by at least some reads. coverage2cytosine processing complete.

Now merging top and bottom strand CpGs into a single CG dinucleotide entity (reading from file >>Pum1.CpG_report.txt.gz<<, in output directory '/mmfs1/gscratch/scrubbed/sr320/github/project-ostrea-chil/output/13-wgbs/methylation/')
>>> Writing a new coverage file with top and bottom strand CpG methylation evidence merged to Pum1.CpG_report.merged_CpG_evidence.cov.gz <<<

CpG merging complete. Good luck finding DMRs with bsseq!

[Fri Oct  9 11:26:37 PDT 2026] Pum1 done