10x genomics 3'RNA-seq dual indexing

[martin2801]

Hello,

so I had a discussion with the 10x Genomics support people about sequencing the v3 chemistry 3'RNAseq libraries on a HiSeq3000/4000 system with dual indexing. They don't recommend dual indexed sequencing as in some cases the top surface of the flow cell lane went completely dark, while the bottom surface was fine.

This seems rather odd to me, as I can't imagine why it would be surface dependent?! I was wondering if anyone has ever tried it or experienced this. Dual indexed sequencing would be helpful for us as it would mean higher turn around time for our samples. Do you have any explanation of why this is happening?

Thanks a lot!

[JoeKutch]

We had this issue when trying to sequence 10x libraries alongside 150/100 PE samples on our HiSeq 4000. I spoke to Illumina and they weren't able to give me a clear answer, but basically said that after the intensity drop after the first 28 cycles the focus point drifts. It refocuses during the indexing cycles but then moves back to focus point for R1 (which is now out of focus) for R2 without refocusing. I think the bottom surface focuses better because it's a fixed distance from there to the stage, but the top surface is a little more variable.

This is how I understood it from my conversation, but at this point Illumina didn't really have much knowledge of the issue - if you access to support they may have more and clearer information now.

[seqsuave]

I have heard through the grapevine that 10x Genomics will be switiching to dual index for all their kits down the road. The new Visium kit has 10bp dual index, and the ATACseq kit already uses dual index that is clunky.

[martin2801]

Originally Posted by JoeKutch

We had this issue when trying to sequence 10x libraries alongside 150/100 PE samples on our HiSeq 4000. I spoke to Illumina and they weren't able to give me a clear answer, but basically said that after the intensity drop after the first 28 cycles the focus point drifts. It refocuses during the indexing cycles but then moves back to focus point for R1 (which is now out of focus) for R2 without refocusing. I think the bottom surface focuses better because it's a fixed distance from there to the stage, but the top surface is a little more variable.

This is how I understood it from my conversation, but at this point Illumina didn't really have much knowledge of the issue - if you access to support they may have more and clearer information now.

This actually makes a sense. Yeah we tried to talk to people at Illumina and 10x and none of them could really tell us whats going on or how to fix this issue except for not sequencing dual indexed. Thanks for your explanation!