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I have several long-read sequences, which when aligned to a human reference genome, aligns to multiple chromosomal fragment as a result of chromothripsis shattering the linear DNA and randomly piecing them together. This creates for a single primary alignment multiple supplementary alignments.

With a single example of a read (only showing parts of all the bamfile information) being

cdaf1fcb-27bf-4890-8ac7-a097dfa12c38 16 chr11 15151487 60

With the primary alignment starting at chr11 at 15151487

Then the supplementary alignment can be seen as follows SA:Z:chr13,49827969,+,1839S956M25D1584S,60,53;chr11,15152459,-,833S749M22D2797S,60,72;chrX,138324656,-,2540S404M2D1435S,60,20;chr11,15152066,+,278M11D4101S,60,20;

Where the alignments are to chr13, 11, X and 11. With both regions on chr11 being close to the initial region from the primary alignment, making the regions on chromosome 11 the same (I know I haven't calculated the full chromosomal interval using the Cigar string for this example).

I assume the order of that supplementary information above is in the actual order. But the problem is that my DNA should be circular which doesn't fit with the order of the alignments since the circle would then be chr 11 (primary, start of circle) -> chr 13 -> chr11 (same as primary) -> chrX -> chr11 (back to the start of circle)

My question is: Q) So is the order of the supplementary information just pieces together randomly or is it true that I can assume the order is the true order of the sequencing read?

So thanks a lot for your help.

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There is nothing in the SAMtags specification that dictates the order of the entries in an SA tag. It may be entirely dependent on the aligner that you used.

Looking at my own data that was aligned with bwa, I cannot see a definite pattern. Longer matches tend to be first, but not always.

Your aligner documentation may have more information on how it uses SA tags.

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