I've come across a bit of a mystery in my Core and I was wondering if anyone might have an idea as to what's going on.
I have a customer who prepared an exosomal RNA library using a kit from System Biosciences (SBI), specifically their XRNA Exosome RNA-Seq Library Kit. There was much consultation and whatnot but in the end they generated what appeared to be a proper library.
Upon submitting their library to my Core we did our standard Tapestation check and Kapa qPCR. Tapestation gives a single peak at ~152bp, right where the kit says it should be. qPCR says the library concentration is 2nM, exactly what I asked the submitting lab to normalize to (they quantified with Qubit).
We load the library on our Miseq for a v3 PE75 run with 20% phiX (an amount requested by the customer). The Miseq errors out on the focus step, no cluster detected. Having a look at the focus images shows they are completely black. In full disclosure this kit uses a special primer for read 2, but that is spiked into the read 2 primer in the Miseq cartridge at the appropriate concentration and shouldn't be a factor in clustering.
We call up TS and tell them what's going on and we run through all of the system checks we can run without an FS rep coming in. Everything checks out. Must have been a fluke right? Maybe bad reagent kit? Whatever's happening, the customer is in a hurry and needs the data. So....
We load up the library again, this time bumping phiX up to 30% as suggested by TS. Again, run errors out on focus with no clusters detected. All images black again as well.
Something's obviously going terribly wrong somewhere. Re-qPCR, re-Tapestation, still looks the same. Qubit quantification agrees with qPCR. As a test of the machine we load up the next person in the queue. It clusters beautifully and runs to completion without issue. Granted this was a PE250 v2 kit so we can't say it's a complete apples-to-apples comparison reagent-wise but I've never seen two kits fail back-to-back.
In my years of Illumina sequencing I've seen libraries that won't cluster and you only see phiX, but I've never come across a library that will not cluster AND will also keep phiX from clustering. Especially at 20 and 30% loading!
The customer contacted SBI and they claimed they only repackage reagents and never actually did any of the science to develop the kit. (Uh, what?)
Thoughts or suggestions would be appreciated.
I have a customer who prepared an exosomal RNA library using a kit from System Biosciences (SBI), specifically their XRNA Exosome RNA-Seq Library Kit. There was much consultation and whatnot but in the end they generated what appeared to be a proper library.
Upon submitting their library to my Core we did our standard Tapestation check and Kapa qPCR. Tapestation gives a single peak at ~152bp, right where the kit says it should be. qPCR says the library concentration is 2nM, exactly what I asked the submitting lab to normalize to (they quantified with Qubit).
We load the library on our Miseq for a v3 PE75 run with 20% phiX (an amount requested by the customer). The Miseq errors out on the focus step, no cluster detected. Having a look at the focus images shows they are completely black. In full disclosure this kit uses a special primer for read 2, but that is spiked into the read 2 primer in the Miseq cartridge at the appropriate concentration and shouldn't be a factor in clustering.
We call up TS and tell them what's going on and we run through all of the system checks we can run without an FS rep coming in. Everything checks out. Must have been a fluke right? Maybe bad reagent kit? Whatever's happening, the customer is in a hurry and needs the data. So....
We load up the library again, this time bumping phiX up to 30% as suggested by TS. Again, run errors out on focus with no clusters detected. All images black again as well.
Something's obviously going terribly wrong somewhere. Re-qPCR, re-Tapestation, still looks the same. Qubit quantification agrees with qPCR. As a test of the machine we load up the next person in the queue. It clusters beautifully and runs to completion without issue. Granted this was a PE250 v2 kit so we can't say it's a complete apples-to-apples comparison reagent-wise but I've never seen two kits fail back-to-back.
In my years of Illumina sequencing I've seen libraries that won't cluster and you only see phiX, but I've never come across a library that will not cluster AND will also keep phiX from clustering. Especially at 20 and 30% loading!
The customer contacted SBI and they claimed they only repackage reagents and never actually did any of the science to develop the kit. (Uh, what?)
Thoughts or suggestions would be appreciated.
Comment