bam file showing question mark

[Cen T]

Hi All,

I saw a similar question where the IGV alignment showing question mark symbol (?) but there was no follow-up https://groups.google.com/g/igv-help/c/AsvavQFOmME/m/WlI7GYb-CgAJ. So, I'm posting a new thread.

Here is what it looks like zoomed-in:

Like the original poster, I was using minimap2 to align. I hope someone can help me figure out the problem. I am attaching my bam file.

One of the problem region is this one:  

Read name = hg38_wgEncodeGencodeBasicV38_ENST00000344954.10_8_FLI1_intron4_of6
Read length = undefined
Flags = 256
----------------------
Mapping = Secondary @ MAPQ 0
Reference span = scaffold_8:7,930,294-7,932,041 (+) = 1,748bp
Cigar = 1748M
Clipping = None
----------------------
s1 = 1724
NM = 2
AS = 3484
cm = 317
nn = 0
tp = S
ms = 3484
dv = 0.0015Location = scaffold_8:7,930,294
Base = ? @ QV 126

Thank you in advance for your help.

[robin...@gmail.com]

Thanks for the BAM file, but I can't really use it without the fasta.   Do you have a link to the fasta file this was aligned against?

[robin...@gmail.com]

I'm fairly certain that the alignment in question is the one shown below,  as you can see there is no read sequence stored in the file for this record,  that is why you see ?,  it means the read base is not known.

hg38_wgEncodeGencodeBasicV38_ENST00000344954.10_8_FLI1_intron4_of6    256    scaffold_8    7930294    0    1748M    *    0    0    *    *    NM:i:2    ms:i:3484    AS:i:3484    nn:i:0    tp:A:S    cm:i:317    s1:i:1724    dv:f:0.0015

[robin...@gmail.com]

A quick web search yielded this.  You did not say what aligner you were using, but apparently minimap2 does not store read sequence or quality for secondary alignments,  which the alignment in question is, to save disk space.   

https://github.com/lh3/minimap2/issues/4

In any event if the read sequence is not stored in the BAM file for the alignment being viewed IGV cannot display it.

[Cen T]

Hi ,

Sorry for my late reply and thank you for looking into this. 

I did use minimap for alignment.

Do you know of any aligner that store read sequence for secondary alignments? I tried to using bowtie but it wasn't able to align the same "read".

Let me know if you still need my fasta file.

Thank you!

[igv-help]

I don't,  that would be a question for a more general forum, or the minimap2 git forum.