FelixKrueger
commented
4 weeks ago Hmm, something here doesn't seem right. -q 32 should either result in an error, or use 32 as the path to the reference genome. -q is the default anyway, so just drop it. In case you were trying to use -p 32, I would not recommend doing that as it almost certainly won't result in a performance gain, but use something like 70 cores in total.
Also, you seem to have gotten alignments to the OT strand only, so I am wondering if you have enough system resources available? How much RAM and how many cores do you have available, and how big is your genome?
I would suggest keeping things simple to start with: move into the directory where the files are located, and run the following:
bismark --genome /mnt/f/Referance_genomes/molino_reference_genome/ -1 Mol_2S_S152_L003_R1_001.fastq -2 Mol_2S_S152_L003_R2_001.fastq(these filenames do not look like Trim Galore trimmed files by the way). Can you see if that works?
KayaHarper
Hello, I am using bismark to prep my files before using Methylkit. I have run FastQC on all my data and then trimmed them using Trim Galore! I was able to successfully run the genome preparations as well when running the bismark command:
bismark -q 32 --samtools_path /usr/bin/samtools –bowtie2_path /usr/bin/bowtie2 --genome /mnt/f/Referance_genomes/molino_reference_genome/ -1 /mnt/f/RawData/methyl-seq_raw_data/methyl-seq/molino/Mol_2S_S152_L003_R1_001.fastq -2 /mnt/f/RawData/methyl-seq_raw_data/methyl-seq/molino/Mol_2S_S152_L003_R2_001.fastq -o /mnt/f/meth-seq_data_bismark_aligned/bismark_aligned/molino
I am hit with a repeating sequence of error messages Use of uninitialized value in substr at /home/lab/Bismark/bismark line 4302, <__ANONIO__> line 10195. substr outside of string at /home/lab/Bismark/bismark line 4302, <__ANONIO__> line 10195. Use of uninitialized value in concatenation (.) or string at /home/lab/Bismark/bismark line 4302, <__ANONIO__> line 10195. Use of uninitialized value in numeric ge (>=) at /home/lab/Bismark/bismark line 4362, <__ANONIO__> line 10195. Chromosomal sequence could not be extracted for SRR020138.15034313_SALK_2029:7:100:1793:953_length=86 15 87239651
This sequence repeats hundreds of times and then while it does eventually end and create the .bam and alignment report files, the report doesnt seem right:
Final Alignment report
Sequences analysed in total: 10000 Number of alignments with a unique best hit from the different alignments: 4912 Mapping efficiency: 49.1%
Sequences with no alignments under any condition: 4176 Sequences did not map uniquely: 912 Sequences which were discarded because genomic sequence could not be extracted: 4912
Number of sequences with unique best (first) alignment came from the bowtie output: CT/CT: 0 ((converted) top strand) CT/GA: 2450 ((converted) bottom strand) GA/CT: 0 (complementary to (converted) top strand) GA/GA: 0 (complementary to (converted) bottom strand)
Number of alignments to (merely theoretical) complementary strands being rejected in total: 0
Final Cytosine Methylation Report
Total number of C's analysed: 0
Total methylated C's in CpG context: 0 Total methylated C's in CHG context: 0 Total methylated C's in CHH context: 0 Total methylated C's in Unknown context: 0
Total unmethylated C's in CpG context: 0 Total unmethylated C's in CHG context: 0 Total unmethylated C's in CHH context: 0 Total unmethylated C's in Unknown context: 0
Can't determine percentage of methylated Cs in CpG context if value was 0 Can't determine percentage of methylated Cs in CHG context if value was 0 Can't determine percentage of methylated Cs in CHH context if value was 0 Can't determine percentage of methylated Cs in Unknown context (CN or CHN) if value was 0
I have redone the command using the test files available on Bismark GitHub and the human genome from NCBI , and the result was the same. Does anyone have any suggestions on how to resolve this?