Number of reads processed by modkit

[ZhihanNUS]

Hi, it is my first time to use Modkit to extract modified base signals but have some problems.

I use Dorado v0.7.1 to do SUP + modification calling (m6A,pseU).
I use minimap2 to align the unaligned_mod_bam file
Number of read aligned to 'Mus_musculus.GRCm39.dna.primary_assembly.fa' is 19,446,790
However, when I use modkit to extract m6A, it says Processed ~7505886 reads and skipped zero reads

I wonder why modkit filtered the other reads?
Here is what I did,
modkit pileup input.bam output.bed --ref Mus_musculus.GRCm39.dna.primary_assembly.fa --motif A 0 -t 36

I notice it says Threshold of 0.6464844 for base A is low. Consider increasing the filter-percentile or specifying a higher threshold.
Is it related to my problem?

Thank you very much!

[ArtRand]

Hello @ZhihanNUS,

The pileup command will only use primary alignments to generate the bedMethyl table. If there are errors with records in the modBAM file you can inspect the reasons by using the --log-filepath $log_file option. The threshold is not related to the number of reads that are used, skipped, or error. Let me take a look at what threshold values I get on similar (mouse) data and get back to you.

[ZhihanNUS]

Hi @ArtRand ,

I have checked my aligned bam file and the number of reads of primary alignment is 19M, I tried to realign the bam file and run modkit, but I still find the mismatch. Since there is no skipped reads, does it mean modkit pocessed all reads in bamfile, or there is any other rules to filter some reads?
This mismatch between number of reads from aligned bam file and number of reads processed by modkit happened in all samples. Do you have any other suggestions to check the reason for this?

Thank you very much!

[ArtRand]

Hello @ZhihanNUS,

Since there is no skipped reads, does it mean modkit pocessed all reads in bamfile, or there is any other rules to filter some reads?

Can you check the output of the --log-filepath? It will tell you if a read isn't used (usually because there isn't any modified base information).

The counter in modkit pileup that you see on the command line isn't completely perfect (thus why it says "Processed approx X reads"). The information in the log file will be more accurate. If you're not seeing any errors in the log file, all of the primary alignments are being used. If you have a log file I'd be happy to review it.

[ZhihanNUS]

m6Alog.txt
Hello @ArtRand ,
Here it is the log file. And may I know if you have a recommend threshold value for mouse data?