I've tried several times with many ideas but have not been successful yet. In this post I will describe one idea I was sure was going to be the winning one, although in the end someone else had beaten me to it (gues who).
The problem: Illumina NGS systems require very careful quantification of the final sequencing library to generate cluster densities that give the highest yield from a flowcell. Getting QT wrong means over-or under clustering both of which lead to a drop in yield. And over-clustering can mean zero yield or lower quality data. Illumina recommends qPCR (although the method suggested has a few flaws) and many labs have had success wth qPCR, Bioanalyser, QuBit and other QT methods.
|Standard Illumina clustering
The idea: basically I wanted to make sure only a certain number of discrete library molecules could hybridise to the flowcell surface for the initial hyb, but allow clustering to proceed normally afterwards. There are currently two oligos in the lawn on the flowcell surface, complementary to the ends of the Illumina adapters. I proposed adding a third “cluster seeding” oligo at a lower concentration. This oligo would have the same sequence as the current flowcell oligos but would be longer and include slightly more of the adapter sequence. Flowcells would be created with a similar lawn of primers such that the seed oligo is present at a spacing consistent with maximal yield and cluster requirements. Additionally the lawn oligos would be blocked by short complementary primers leaving only the 5’ end of the seed oligo unblocked for hybridization with library molecules. A covalently attached library molecule would be produced by polymerase extension. The first round of denaturation would remove the original library molecule and all blocking oligos. Bridge amplification would then proceed as normal.
This method would hopefully allow high, low and/or variable concentration of libraries to be entered into the flowcell without quantification as only a certain fraction would be able to hybridise. The yield of the flowcells would be significantly less variable.
This would remove the need for Illumina customers (you and I) to perform quantification and significantly increase yields from runs.
|Seed-oligo clustering idea
Who got there first: Unfortunately when searching through patents to see if anyone else had a similar idea we found this patent US20090226975 by Illumina. They describe a similar method with a clever use of a hairpin oligo so the blocking is removed enzymatically. Fundamentally the same outcome is achievable and quantification can be considered a thing of the past.
|US20090226975 figure 3
Why are we all running lots of qPCR: I don't understand why this has not made it out into the current generation of kits. All the users I talk to agree that removing quantification would be a good thing. Even if it is done perfectly it still takes an hour to do so time can be saved.
Come on Illumina, where is it?
Hopefully reading this has spurred you on to move ahead with the good idea you had a couple of months ago and left on the "back burner". I'll follow this post with another one about a completely different idea that is about to be realised by Life Technologies instead!
Ho hum, spending my millions will just have to wait!