Hi,

I did not understand if you have a IonProton machine or a IonTorrent, but I guess you ahve the IonTorrent given the chip you use. In my lab we have IonProton coupled with Ion One Touch machine, not the Ion Chef and we usually have loading percentage above 90.

And what about the percentage of usable reads? Is it high or it is low? I do not know if it is a high standard but we have usable reads around 70%

Anyway, I think it could be a problem with the amplification protocol in the OneTouch/IonChef instrument because its protocols are very restrictive and not so versatile: i.e. long amplicon could not be amplified with the wrong protocol. I think this could lead to a low percentage of loading.

I hope this helped

Dear Fabio,

Thanks for your inputs

We have a Ion PGM coupled with the Ion OneTouch2. Even though the chip loading is very low, amount of usable reads is around 68%.(For a 44% ISP loading density). However, I doubt that there is be an issue with Ion OneTouch amplification protocol, since the amplicon sequencing workflow gives satisfactory results on 400bp. We only have issues with 200bp amplicon sequencing.

In my experience with the Ion Torrent platform (we routinely run Ion Proton) I found that the only factor that actually make a difference in chip loading is an high amount of positive ISPs after onetouch 2.

Did you usually perform ISP quality check with the Life kit? If you have low % of positive ISP or low signal for the AF647 probe when read on Qubit (I found that < 1500 results in low chip loading on the PI chip, but I don't know if this stay true also for smaller PGM chips) this will results in a low number of ISP after One Touch ES enrichement. Take in mind that 20-40% of the positive ISP could be lost during the ES procedure.

If you don't do this usually I strongly suggest to perform this quality check step at least as diagnostic test to identify the source of your problems.

As you can imagine, the main factor influencing OneTouch 2 results is the amount of input library. It seems that you use Qubit for library quantification. Even if it is a rapid and cost effective choice for optimized protocols, it could lead to erroneous estimation of your library when dealing with custom made procedures. Maybe you may think to quantify your libraries using a realtime- based kit that will give you precise estimation of the amount of correctly ligated fragments that you are purring in the emPCR reaction. From my experience i found up to 4 times difference in quantification from Bioanalyzer and RealTime and switching to realtime saved me a lot of headache. So again we sill have a better idea on what is going on in your workflow.

All that said, given the various intrument failures that you experimented, it sould be that the problems are in the hardware part...Have you tried to re-run a library that performed good in the past to see if you can sequence it now? A colleague of mine using the PGM started to have some instrument troubles an it takes one year and several component substitution before it could have the machine full operating again, even if the exact cause of the problems couldn't be identified.  

Hi Edoardo, Thank you for your inputs. 

Talking about our template positive ISPs, it reaches about 68%.  We've been having similar percentages all throughout, and it used to work well for ampliseq libraries. Would you suggest that we try obtaining higher percentages? However, our library titration point is slightly high already.

We re-ran the E.coli library, it works perfect. Like I said, all our metagenomic tests are working fine,

We did another sequencing run today, with a slightly altered loading protocol as mentioned in Ion community, but the loading has still not improved.

Just out of curiosity, what exactly were the component substitutions your friend had to make to get his PGM back in operation?

PS- What is your opinion on multiplexing libraries?