ONT sequencing pipeline

[GrinMinded]

I have two sets of .fastq files from ONT sequencing runs (one using R9.4.1 and the other R10), after generating .bam files, one set isnt working correctly. Both .bam files are recognized by samtools, mapped to the same custom reference (799yT32), and have similar read counts and sizes. Though, the second set doesn't seem to map correctly, samtools view -c doesn't recognize the reference despite showing 100k reads, and IGV displays no reads and an unexpected "Junctions" row. I'm guessing the issue is with the reference sequence not being recognized, but I'm not nsure why it affects just one set. Help please.

 

[Emali_day]

The junctions row in IGV usually pops up when it thinks you're dealing with spliced RNA, so maybe the BAM from the second set got flagged as RNA-seq somehow? check if the reference names in the BAM header match exactly with your 799yT32 reference.

 

[NoRules]

I have two sets of .fastq files from ONT sequencing runs (one using R9.4.1 and the other R10), after generating .bam files, one set isnt working correctly. Both .bam files are recognized by samtools, mapped to the same custom reference (799yT32), and have similar read counts and sizes. Though, the second set doesn't seem to map correctly, samtools view -c doesn't recognize the reference despite showing 100k reads, and IGV displays no reads and an unexpected "Junctions" row. I'm guessing the issue is with the reference sequence not being recognized, but I'm not nsure why it affects just one set. Help please.

try with samtools index <file.bam> so the index will regenerate and see if the problem resolves.

 

[SnipSniper]

It sounds like your BAM file and reference might not match up. This could be due to differences in the reference header or sequence names.

First, make sure both BAM files use the same reference FASTA, checking the header format too. Also, confirm the problematic BAM has a .bai index file, as IGV needs this to show reads correctly.

To check, use samtools view -H on both BAMs to compare headers, and use samtools index to confirm indexing. if it still does not work, remap the dataset.

Do you have the reference FASTA available for checking?

 

[fastqfanatic0761]

Oh man, we had a total nightmare like that last fall! One of our BAMs was using a reference file where the contig names were just a bit off, like one set had those annoying 'chr' prefixes while the other totally didn't. IGV didn't even catch it until we figured it out. It was so frustrating. Reindexing didn't solve anything until we finally fixed those headers. Lesson learned, right?