visualize my .BAM file in IGV

Question

[chrisgr]

Hi,

I got a little confused trying to visualize my bowtie2 mapped reads. I mapped some short (synthetic) RNA reads to a self-made (short) reference sequence with bowtie2, and I have aquired the .bam file.

Now I want to visualize this .bam file. I wanted to do this with IGV, however they want me to also provide a 'genome sequence' (which I understood is the reference sequence). Isn't the reference sequence already provided in the .bam file? Why do I need to provide a reference sequence again? I just want to visualize the mapping bowtie2 did.

I'd have to read more of the manuals of IGV but I can't seem to find an answer to this question. If there is an informaive page that could help me with this specific question I'd also be very happy!

Thanks in advance!

Chris

[cpad0112]

what is the reference organism? @ christiangriffioen

[chrisgr]

Not an organism, the reads mapped are synthetic, so I used a reference genome made up of these sequences pasted after each other.

[cpad0112]

IGV has few inbuilt reference genomes and allows to load external sequences as well. In your case, you may have to follow custom genome load instructions for IGV.

Answer 1

[GenoMax]

however they want me to also provide a 'genome sequence' (which I understood is the reference sequence)

No. The reference can be any sequence. In your case it will be self-made (short) reference sequence if that is what you used to create the bowtie index and for alignments. Create a custom genome (.genome file) in IGV with that.

Answer 2

[Pierre Lindenbaum]

Isn't the reference sequence already provided in the .bam

no, the bam only stores the positions and sequences of the reads.

The reference sequence is the fasta sequence you used to build the bowtie index. Most of the time, it must be indexed with samtools faidx.

[chrisgr]

Many thanks for the reply, I thought/read about this and don't understand yet, I really want to grasp this.

Like you said, the .bam file stores the positions and sequences of the reads on the reference sequence I provided right? I provided the reference sequence when I mapped them to the reference sequence using bowtie2, why is the reference sequence not included in the .bam file? If in IGV I select a reference genome and it would be slighty different, won't that mess with the .bam 'positions 'of the reads?

[Pierre Lindenbaum]

why is the reference sequence not included in the .bam

because the BAM format just don't do this.

If in IGV I select a reference genome and it would be slighty different, won't that mess with the .bam 'positions 'of the reads?

if slighty means a few subsitutions you'll see some mismatches in the reads.

if slighty means a few insertions/deletions : you'll get a mess.

[Pierre Lindenbaum]

why is the reference sequence not included in the .bam

imagine your mapping only one read to the human genome. The small bam would contains the 3G from the human genome ? no.