test of Xgen blocking oligo

[hduborjal]

Did someone try the XGen blocking oligo from IDT instead of classical blocking oligos ? Is there a improvement of the off target ?

[hduborjal]

Hi, thank you for your answer.
I understand that you use dual indexing.
My first question is : Did you try specific Xgen blocker or the universal one for Illumina HT libraries ?
My second one : what was the on-target before using the Xgen blocker ? Is there an improvement by using it ?

[magarine]

Thanks superduper,

I'm doing to setup pre indexing hybridization method and test xGen IDT oligo blocks.
I'm happy to meet you here.

I am a little of bit surprised that on-target rate of perfect complementary blocking oligo was lower than xGen.

Conceptually some idea maybe was in synthesis of oligo for blocking.

[Ingeneious]

I was using one barcode per library.
1. I used the universal 8nt blocking oligo (mix of TS-p5 and TS-p7(8nt))

2. my on-targets were:
single capture; barcoding happens after hyb step (basically XT protocol): 70%
XGen blocking: 58-73%
Inosines instead of barcode location: up to 33%
perfect complement: 45%
random nucleotides: up to 35%

This is for PE37 sequencing. To compare samples where I had run reads longer, I clipped them back to PE37 to have a fair comparison between different conditions.
Your % on-target will increase the longer your read lengths are; I saw PE101 of 82% translate to 69% when I clipped it to PE37.

Did you add any kind of termination to the blocking oligo?