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General Category >> Help and How-to >> Alignment Visualization of bam.file on Unipro Ugene
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Message started by Johan on Mar 20th, 2023 at 8:59am

Title: Alignment Visualization of bam.file on Unipro Ugene
Post by Johan on Mar 20th, 2023 at 8:59am
Greetings,
I have a question about the alignment of ngs reads of bam.file visualized on unipro. I obtained a bam file for deep sequencing of a Cas9 mutant sample and visualized the alignment on unipro.

I found that there were few hundred reads with gaps highlighted in red around and on the cas9 target sites but become more abundant towards the end of the sequence and thought that these were the indels. However, when I did the variant calling, only the region towards the end of the sequence with high abuncance of the gaps was computed to have a 5 base insertion but not the region around the Cas9 target site. I also visualized the file on IGV and no gaps (indels) were identified. May I know what the gaps meant here that were only visible on Unipro gene?
indels.png (119 KB | 125 )

Title: Re: Alignment Visualization of bam.file on Unipro Ugene
Post by Dmitrii Sukhomlinov on Mar 20th, 2023 at 1:48pm
Hello,

In general, gap means, that character on this place wasn't sequenced successfully. If you point to any read you will see the line called "Cigar" (see attachment "cigar.png"). This line means the descripting of bases in a read. "M" means that the number of described bases has been successfully sequenced, "N" - that the number of described bases were not sequences, so we have a gap (defined as "-" in UGENE) in this spot. For example, in the example we have "10M32N38M", which means:
- 10 sequenced bases
- 32 gaps
- 38 sequenced bases

You may see this read on the attachment 2 (read.png).

UGENE parses SAM file correctly and takes into account gaps. I opened IGV and couldn't find any sequences with gaps too - I do not know how IGV parses SAM files, but, probably, it just skips reads with gaps. As you may see on your picture, gaps are inserted correctly - they "moves" character in reads and in different reads the same character locates under the same character.

Have I answered your question? If no, please, detail it a bit
cigar.png (20 KB | 83 )
read.png (37 KB | 77 )

Title: Re: Alignment Visualization of bam.file on Unipro Ugene
Post by Johan on Mar 22nd, 2023 at 6:18am
Woah, that is a great explanation! Now I completely understand how to interpret the alignment data of bam file on unipro gene. supercool! Thanks a lot once again! :) ;) :)

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