Split reads along the genome in my samples

Question

[paulaotero.sanchez]

Hello,

I am working with DNA sequencing libraries and I have found out that there are a lot of split reads randomly distributed (not clustered) along the genome in my samples. When I do a Blast of the sequence of these reads, I always find this behaviour:

For some reason, the split region corresponds to the same DNA locus but it has with a different orientation.

Any clue? Thanks!

[GenoMax]

Are you working on regular sanger sequence data or some form of high throughput data? Remember BLAST is a local aligner so it is always going to find these local HSP's.

[paulaotero.sanchez]

This is illumina Pair End, 350bp (150bp each read). However I detected my DNA fragments are smaller than expected for this library, do you think it is related?

[GenoMax]

This is illumina Pair End, 350bp (150bp each read). However I detected my DNA fragments are smaller than expected for this library,

Even if the inserts are small, if this is DNAseq, then the alignments should not split for a single read (unless you have some chimeric library fragments). Are you showing the alignment from one of the read pairs above?

Please use a proper NGS aligner rather than blast+ (unless you have a specific reason to do so).

[paulaotero.sanchez]

The data was aligned with BWA-mem, then I used IGV and saw these split reads. To know the origin of the split region of the read, I used blast. As you say, the image corresponds to the alignment of one read pair.

As you suggest, I also thought about chimeras in my data but, if I understand this ok, I might think that chimeras would happen because of the union of any DNA fragment (even fragments from different chromosomes or very distant loci). However, I always see that the two parts of the same read belong to the same DNA region.