creating bam files for 6mA and 4mC

[Qvdauwer]

Dear,

I would like to use nanopolish to call methylation for 5mC, 6mA, and 4mC.

When I try it using following commands, it works and seems to produce a normal output:

nanopolish call-methylation -v -t 14 -q cpg -r /projects/methylation_work/isolates/Isolates/isolates_29_09_part_2/barcoded/reads/barcode17.fastq.gz -b barcode17_reads_nanopolish_sorted.bam -g /projects/methylation_work/isolates/Isolates/isolates_29_09_part_2/barcoded/assemblies/barcode17_trycycler.fasta > methylation_calls_nanopolish_bc17_29_09_5mC.tsv

nanopolish call-methylation -v -t 14 -q dam -r /projects/methylation_work/isolates/Isolates/isolates_29_09_part_2/barcoded/reads/barcode17.fastq.gz -b barcode17_reads_nanopolish_sorted.bam -g /projects/methylation_work/isolates/Isolates/isolates_29_09_part_2/barcoded/assemblies/barcode17_trycycler.fasta > methylation_calls_nanopolish_bc17_29_09_6mA.tsv

nanopolish call-methylation -v -t 14 -q dcm -r /projects/methylation_work/isolates/Isolates/isolates_29_09_part_2/barcoded/reads/barcode17.fastq.gz -b barcode17_reads_nanopolish_sorted.bam -g /projects/methylation_work/isolates/Isolates/isolates_29_09_part_2/barcoded/assemblies/barcode17_trycycler.fasta > methylation_calls_nanopolish_bc17_29_09_4mC.tsv

But when I add the --modbam-output-name tag, only 5mC works:

nanopolish call-methylation -v -t 14 -q cpg -r /projects/methylation_work/isolates/Isolates/isolates_29_09_part_2/barcoded/rea.gz -b barcode17_reads_nanopolish_sorted.bam -g /projects/methylation_work/isolates/Isolates/isolates_29_09_part_2/barcoded/assemblies/barcode17_trycycler.fasta --modbam-output-name  methyolish_bc17_29_09_5mC.bam
[post-run summary] total reads: 67258, unparseable: 0, qc fail: 58, could not calibrate: 67, no alignment: 2106, bad fast5: 0

nanopolish call-methylation -v -t 14 -q dam -r /projects/methylation_work/isolates/Isolates/isolates_29_09_part_2/barcoded/rea.gz -b barcode17_reads_nanopolish_sorted.bam -g /projects/methylation_work/isolates/Isolates/isolates_29_09_part_2/barcoded/assemblies/barcode17_trycycler.fasta --modbam-output-name  methyolish_bc17_29_09_6mA.bam
nanopolish: src/basemods/nanopolish_basemods.cpp:135: bam1_t* create_modbam_record(const bam1_t*, const std::map<int, ScoredSite>&, const MethylationCallingParameters&): Assertion `callinget->get_name() == "cpg"' failed.
Aborted

nanopolish call-methylation -v -t 14 -q dcm -r /projects/methylation_work/isolates/Isolates/isolates_29_09_part_2/barcoded/rea.gz -b barcode17_reads_nanopolish_sorted.bam -g /projects/methylation_work/isolates/Isolates/isolates_29_09_part_2/barcoded/assemblies/barcode17_trycycler.fasta --modbam-output-name  methyolish_bc17_29_09_4mC.bam
nanopolish: src/basemods/nanopolish_basemods.cpp:37: void get_modification_symbols(const Alphabet*, char&, char): Assertion `alphabet->num_recognition_sites() == 1' failed.
Aborted

Does to conversion to bam output only work for the cpg pattern?

Thanks in advance,
Quentin

[jts]

Hi,

Yes, the modbam support only works for CpG methylation. If you need modbams for other modifications and contexts I recommend using ONT's callers (guppy/dorado/remora).

Jared