Offtargeting in SureCall

Question

[mandar.bedse]

How to find if reads are offtargets and where are they aligned actually? I am using SureCall software.

[Ram]

What have you tried? What does your dataset look like? Why does your post only have the tag alignment when there tags like surecall that would also be appropriate and an easier target for subject matter experts?

[GenoMax]

Since this is commercial software from Agilent you may not find many people here who may have used it. Your best bet is to contact Agilent support.

Answer 1

[lakhujanivijay]

Hi mandar.bedse,

You should take the suggestions from RamRS and genomax very seriously. Anyways, "offtarget reads" essentially means that the targeted sequencing was unable to cover the regions it was supposed to cover; simple ! The best way to see where the reads mapped is to use IGV from Broad institue. You will require indexed Bam file + the design and the target bed file.

Moreover, it depends on the design size also. For SureSelect, small designs (<200 kb) tend to have large off-target percentage--if baits are in molar excess relative to the number of targets, the baits will bind non specifically.