FLX+ upgrade

[ajthomas]

Anyone get their FLX+ instrument upgrades yet? Is it working as advertised for you?

Ours was supposed to be upgraded last week, but no one ever showed up to do it, and after talking to Roche it seems someone dropped the ball somewhere along the line. Now we're told that the upgrade parts are on backorder and we'll get the upgrade when they're available. (No idea when that will be.) Is anyone else waiting for upgrade parts and know anything about when they'll be available?

[RCJK]

Yes, ours was upgraded in Sep. The control test fragment run went well, and then I've run an XLR70 run which was as expected. If my enrichments today go well I'll do my first XL+ run on real samples on Monday. Fingers crossed!

[pmiguel]

Still waiting on ours. We were told the upgrades would be done basically in order of who had been the biggest reagent customers over a interval. Also that they were to be worked in between repairs.

--
Phillip

[AlexB]

We did our first 16-plex large plasmids (~100kb each) in 1/2 a XL+ run and are very happy with the data (both lenght, quality and coverage). Depending on sequence composition of course we get nice reads spanning 450+ till even 900.... roughly 700 averaged. It was also the 100th run so time for celebration.
Be sure to upgrade the Newbler assembler to the latest as well.
Enjoy!

[MissDNA]

Our upgrade was supposed to happen last week but it was delayed because parts are not in Brazil yet. Supposedly they will arrive sometime is November and our upgrade will be in Dec. According to Roche Brazil our upgrade is priority here, so we will see. They already sent us the kits for the Tf run.

[tokikake]

We had our upgrade some weeks ago and after the internal server crashed and had to be replaced, we had 3 runs on XL+ chemistry so far. Well...I am not satisfied with the performance.
With WGS libraries we got an average read length of around 500bp. The same with unidirectional sequenced Amplikons. Additionally, I am not happy with the high number of short reads AND the low number of passed wells.
As far as I know, we are not the only ones facing these problems and not the only ones having hardware troubles after the upgrade.

Cheers,
Sonja

[pmiguel]

Hi Sonja,
What is your total run yield? (In megabases?) What were you getting prior to the XL+ upgrade?
--
Phillip

[tokikake]

Hi Sonja,
What is your total run yield? (In megabases?) What were you getting prior to the XL+ upgrade?
--
Phillip

Hi Phillip,

here a some examples:

XL+
WGS: 64-68 Mb from a medium run; 26 Mbases from a M/S run.
Amplicons: varying between 18-27 from a M/S run and 34-52 from a medium run

FLX
WGS: 70-89 from a medium run
Amplicons: 60-76 Mb from a medium run

[pmiguel]

Thanks.
Did you use the new FLX+ emPCR protocols with the 10 minute extension (60 oC) steps?


--
Phillip

[tokikake]

Thanks.
Did you use the new FLX+ emPCR protocols with the 10 minute extension (60 oC) steps?


--
Phillip

Yep, I switched to the new protocols.
For the Amplicons we are evaluating which protocol works better, Titanium or XL+. Roche recommends to use the Titanium protocol for amplicons <650bp, but sequencing with XL+ chemistry (personal communication). After our first tests the results are not that clear, so we have to test some more samples.

[MissDNA]

We are still waiting for our upgrade. We already have accepted a project using the new chemistry. Since the kits take 60+ days to arrive in Brazil, I hope our upgrade should be ready by them.

We were told by Roche´s tech support that the new chemistry is still not recommended for amplicon runs. A software matter, I think.

Also, are the FLX+kits cheaper than FLX over there?

[tokikake]

We are sequencing unidirectional amplicons and processing them with the shotgun pipeline. Although it is not recomended from roche to use the Lib-A Kit we don't really see a difference in the moment to the Lib-L protocol for the unidrectional sequencing of amplicon libraries.
No, the price for XL+ and XLR70 Kits is the same.

[MissDNA]

Even when we do unidirecional sequencing we use amplicon pipeline. Our bioinformatics people analyzed the same data using amplicon and shotgun pipelines, and thought amplicon data was more reliable. I don´t know the specifics.

Kits in Brazil are way more expensive than else where, however the + kits will be about 1000 dollars cheaper than standard.

Have you guys paid for the instrument upgrade or not?

[RCJK]

So the first XL+ run here with customer samples failed as the PTP temperature was out of range. Apparently it's crucial for the run. Once they adjusted it again, the rerun worked well.
On 1/2 PTP, a gDNA sample yielded 715,763 reads, 395,284,650bp, avg length 552.26bp, modal length 723bp.

[MissDNA]

What would be the ideal PTP temperature? As far as I remember only the camera and the apyrase chiller have temperature control. Once we had a failed run because our camera reached -19ºC. Camera was replaced and was never an issue again.