Low QC for R2 from beginning

s_farraj · 11-12-2018, 08:57 AM

Hi all,

I made 2 runs on Miseq using 300cycles (2X150). I used 2 different library preparation kits. One of them KAPA RNA Hyper prep and the second one Nextera dna flex library prep. The both libraries were normalized manually after I checked their quality by bioanalyzer. the first library was 10pM conc. and the other one 12pM (like the kit recommend). In both runs I got the same problem. The reads of R2 became low from beginning (the pic. attached will clarify the problem).
Plz if anyone has an explanation for this result, tell me.

Many thanks

GSviral · 11-13-2018, 05:56 AM

Hi,

It looks to me that your run is borderline overclustered from the second image. V2 chemistry recommends a clustering range of 1000 - 1200 K/mm2. This is probably why you are seeing a drop in read quality in R2. You might find the same libraries, loaded at 8 - 9 pM, would bring your clustering within range and produce better quality data.

I've had some success with slight overclustering using MiSeq V2 chemistry (~1350 K/mm2) still returning really great data but this seems to be dependent on the type of library being sequenced.

s_farraj · 11-13-2018, 07:26 AM

Hi GSviral,

Thanks for your reply.

I also have successful runs with 300cycle where the cluster density overpassed 1400K/mm2 but it is the first time for me to see the quality of R2 like that from beginning.

GSviral · 11-13-2018, 11:32 PM

Good to hear that those higher cluster densities can still produce good quality data.

Unfortunately it's hard to predict, one library can produce completely different metrics to another at those densities (and indeed within the specified density range). Since it's only the last 2 runs you have seen this quality drop in R2 I can only say to reduce the cluster density and see if the problem still exists. I would be more concerned if you had seen this quality drop in the last 10 runs consistently, then it may be a fault with library preparation or the machine itself.

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11-13-2018, 05:56 AM	#2
GSviral Member Location: UK Join Date: Dec 2014 Posts: 37	Hi, It looks to me that your run is borderline overclustered from the second image. V2 chemistry recommends a clustering range of 1000 - 1200 K/mm2. This is probably why you are seeing a drop in read quality in R2. You might find the same libraries, loaded at 8 - 9 pM, would bring your clustering within range and produce better quality data. I've had some success with slight overclustering using MiSeq V2 chemistry (~1350 K/mm2) still returning really great data but this seems to be dependent on the type of library being sequenced.

11-13-2018, 07:26 AM	#3
s_farraj Junior Member Location: Saudi Arabia Join Date: May 2012 Posts: 7	Hi GSviral, Thanks for your reply. I also have successful runs with 300cycle where the cluster density overpassed 1400K/mm2 but it is the first time for me to see the quality of R2 like that from beginning.

11-13-2018, 11:32 PM	#4
GSviral Member Location: UK Join Date: Dec 2014 Posts: 37	Good to hear that those higher cluster densities can still produce good quality data. Unfortunately it's hard to predict, one library can produce completely different metrics to another at those densities (and indeed within the specified density range). Since it's only the last 2 runs you have seen this quality drop in R2 I can only say to reduce the cluster density and see if the problem still exists. I would be more concerned if you had seen this quality drop in the last 10 runs consistently, then it may be a fault with library preparation or the machine itself.