[pmiguel]

Matt,

Sorry I can't be of much help. I'm sending my samples to a core facility and they flat out refuse to mix chemistries on a flow cell. I had been led to believe that it wasn't even possible to do what you're describing due to limitations of the cBot, but it seems you've found a strategy Illumina endorses. Not sure what the problem would be, unless there is some inherent different in the hybridization affinities between the two adapter types or some difference in the insert sizes that resulted in differential clustering of the two library types due to different concentrations of ends at a given pM concentration. It's late so I'm not even sure if that makes sense...

I do know Illumina says the MiSeq clusters more efficiently than the HiSeq, but you probably know that already.

Well, maybe for certain definitions of the word "efficiently".
The MiSeq produces a higher cluster density for a given concentration of library. However, consider the absolute amount of library consumed by the MiSeq per cluster generated:

600 ul of 10 pM library = 6 fmol of library to generate 15 million clusters on a MiSeq v2 run.

120 ul of 15 pM library = 1.8 fmol of library to generate >200 million clusters on the cBot running v3 cluster chemistry.

MiSeq 2.5 million clusters/fmol
cBot/HiSeq 111 million clusters/fmol

Sorry I can't be of much help... if you figure out a solution post an update because I'm curious.

Best,
DocBio

How about using Rapid chemistry? Doesn't that come with the Nextera primers already in the mix? Also does on instrument clustering.

--
Phillip

[matth431]

Well, maybe for certain definitions of the word "efficiently".
The MiSeq produces a higher cluster density for a given concentration of library. However, consider the absolute amount of library consumed by the MiSeq per cluster generated:

600 ul of 10 pM library = 6 fmol of library to generate 15 million clusters on a MiSeq v2 run.

120 ul of 15 pM library = 1.8 fmol of library to generate >200 million clusters on the cBot running v3 cluster chemistry.

MiSeq 2.5 million clusters/fmol
cBot/HiSeq 111 million clusters/fmol




How about using Rapid chemistry? Doesn't that come with the Nextera primers already in the mix? Also does on instrument clustering.

--
Phillip

That's good to know - unfortunately, we can't do rapid chemistry as yet due to only having a HiScanSQ. We are looking at upgrading in the next few months to a 2500, but the user is a bit more desperate for the data than that.

As far as Docbio's core facility not offering mixed runs, I suspect this is more down to the hassle rather than the actual ability to do it. Swapping out the cluster generation and indexing primers from the standard kit for those supplied in the Nextera Sequencing Primer kit is not an issue - these worked well enough on the TruSeq samples we had on that run so there should be no issue having some lanes TruSeq and some Nextera.

The "hassle" comes at the bioinformatics stage - because the sample sheets only give you the option of TruSeq OR Nextera (not both) and are applied across the whole flow-cell, I was told that we would essentially have to create two sample sheets (one TruSeq, one Nextera) and run the BaseCalltoFASTQ script twice in order to pull out the indices correctly.

That said, you also are essentially "wasting" cycles doing a second index read on the TruSeq samples when they only have a single index (unless using the new HT kits). Splitting SBS reagents for 101|8|8|101 indexing is a bit of a chore as well (a 200-cycle kit doesn't have enough spare reagents for the Nextera dual indexing, so you have to pool four 50-cycle kits). I can see larger facilities with high workloads not wanting the extra time to deal with this.

[pmiguel]

That's good to know - unfortunately, we can't do rapid chemistry as yet due to only having a HiScanSQ. We are looking at upgrading in the next few months to a 2500, but the user is a bit more desperate for the data than that.

Yes, we also used to run a HiScanSQ. Man, that machine was a headache compare to the HiSeq.

The "hassle" comes at the bioinformatics stage - because the sample sheets only give you the option of TruSeq OR Nextera (not both) and are applied across the whole flow-cell, I was told that we would essentially have to create two sample sheets (one TruSeq, one Nextera) and run the BaseCalltoFASTQ script twice in order to pull out the indices correctly.

I know they say this. It is untrue and bone-headed though. Single index libraries generate legitimate dual 8 base index sequences. Just stick those in your sample sheet and you can just run CASAVA once. Works on the MiSeq too. So, I don't see any impediment for mixing dual and single index libaries in the same lane, should the need arise.

That said, you also are essentially "wasting" cycles doing a second index read on the TruSeq samples when they only have a single index (unless using the new HT kits). Splitting SBS reagents for 101|8|8|101 indexing is a bit of a chore as well (a 200-cycle kit doesn't have enough spare reagents for the Nextera dual indexing, so you have to pool four 50-cycle kits). I can see larger facilities with high workloads not wanting the extra time to deal with this.

Yeah, this I am dreading. But hopefully we'll have 2 flowcells and we'll order two 200-cycle kits and one 50-cycle kit as well. Then just split the 50 cycle kit between the two 200-cycle kits.

Or maybe Illumina will turn on the 3rd swath for Rapid flowcells and then we don't need to use the slow chemistry any more.

--
Phillip

[koadman]

Eh? Why did the first index work then?
I mean does that make sense to you? NaOH sits around for days in the instrument during read1. Then it successfully denatures the first read to allow annealing of the index1 primer. But after the additional several hours it takes to do 8 cycles then it doesn't work?

--
Phillip

To be honest very little of it ever made sense to me. I don't run the instruments, the core facility does, and I don't communicate with Illumina tech support, the core facility does. I'm an informatics guy with only a cartoon view of how sequencing works! I am merely trying to communicate more broadly with others what Illumina claimed the problem was. It would be super interesting and extremely valuable for future runs to learn that the problem is something more temperamental and the fact that it worked when we (e.g. the core people here) loaded fresh NaOH was just a fluke.