Problem with ONT sequencing pipeline. Probably in the Samtools part

[EmilioKolo]

Hi everyone. I have two sets of .fastq files from two ONT sequencing runs and one of them isn't working after generating .bam files.

The .bam files are generated and recognised by samtools, and they have reasonable sizes given the number of sequences they have, but they seem to not be mapped to the custom amplicon sequence I'm using as a reference, even though samtools tells me both sets of files are mapped to the same reference.

I ran "samtools view -H" on files from either experiment and both have the same reference (799yT32), plus they both seem to not have any other differences in versions nor upstream scripts.

$ samtools view -H ./file_from_exp1.bam
@HD     VN:1.6  SO:coordinate
@SQ     SN:799yT32      LN:8546
@PG     ID:minimap2     PN:minimap2     VN:2.26-r1175   CL:minimap2 -ax map-ont -t 12 /[path]/ref_seq/779yT32.fa /[path]/barcode03_concat.fastq
@PG     ID:samtools     PN:samtools     PP:minimap2     VN:1.10 CL:samtools view -H ./file_from_exp1.bam
$ samtools view -H ./file_from_exp2.bam
@HD     VN:1.6  SO:coordinate
@SQ     SN:799yT32      LN:8546
@PG     ID:minimap2     PN:minimap2     VN:2.26-r1175   CL:minimap2 -ax map-ont -t 12 /[path]/ref_seq/779yT32.fa /[path]/barcode06_concat.fastq
@PG     ID:samtools     PN:samtools     PP:minimap2     VN:1.10 CL:samtools view -H ./file_from_exp2.bam​

Despite that, when I run "samtools view -c" on them to see the number of reads, it seems to stop recognising the reference sequence, though it still shows the file as having 100k reads in it.

$ samtools view -c ./file_from_exp1.bam 799yT32
2944​
$ samtools view -c ./file_from_exp2.bam
114145
$ samtools view -c ./file_from_exp2.bam 799yT32
[main_samview] region "779yT32" specifies an invalid region or unknown reference. Continue anyway.
0​

I'm guessing the problem stems from not recognising the custom reference sequence name, but I don't know how can it work with one set of reads and not the other.

I also tried using IGV, which shows no reads and a "Junctions" row on the .bam file from experiment 2 which hadn't appeared previously in other .bam files I opened before (screencap attached).

The .fastq files come from two different experiments with different chemistries (experiment 1 was done with r9.4.1 while experiment 2 was done with r10), but look functionally the same. I don't think that can cause any problems but I point it out in case it might be relevant.

Do you know anything about this problem? Is there any way to solve this?

Thanks in advance.