5500xl starting questions

[rtpon]

Hi
Our lab has just received a 5500xl upgrade for our SOLiD 4 and I have some newbie questions that I haven't found answers for in our 5500 documentation.

1. Can we re-run libraries, i.e. a set of exome sequence captures, which were prepared for the SOLiD 4 on the 5500. I know that 5500 libraries have a new A-tailing step, but does this affect the sequencing? We would make fresh bead preps using our EZ-Bead system.

2. How many beads need to be deposited per lane? How many are actually sequenced? If we switch to nanobeads, how much improvement will there be?

3. How many panels are there in each lane? How many are imaged? I've seen three different numbers mentioned: 670 panels/lane is assumed in the reagent cost calculator; 708 panels/lane was provided by my FAS; and 1200 panels/lane are mentioned in the user guide (when calculating the number of beads to be deposited, i.e. 1200 panels x 250k beads/panel). I want to estimate how much sequence will be produced from each lane.

4. How long do sequencing runs take on the 5500xl? I know that they can do different types of runs at the same time, but assume all 12 lanes (2 flowchips) are either all single 75 bp reads or all 75+35 bp PE reads for simplicity. I want to estimate how many runs a year we can perform.


Finally, how well have these new machines been working for everyone? We're looking forward to all the promised improvements.

Thanks everyone

[colindaven]

Hi,

I can only provide information about question 4.

It seems runs up to 50bp run quite quickly (i.e. as specified in the docs), but beyond that things slow down.
We recently got our 5500xl and have done one 60bp fragment run, which took around two weeks (also including a bit of downtime due to a software error). I think if you calculate for 50bp you can use the manufacturer's data.

[onehalfmex]

We just finished our first run so I am by no means an expert, but here's what I can tell you...

1. We reran a multiplex paired end S4 library. All libraries on the 5500 have to have an internal adapter (IA). The 5500 uses the IA to locate the user’s beads versus the control beads. This is slightly different than on S4 where the P2 adapter is used. So if your library has an IA it might work. Because of the A-tailing, we ran the library as an RNA library even though it was DNA. The RNA IA is the same as the S4 DNA IA. But the new DNA IA has an extra T for the tailing.

2. We deposited ~400 million beads per lane. At this concentration we were able to get ~210k beads per panel. I don't have any experience with the nanobeads but supposedly you should get a 1.4 fold increase or so.

3. The imager only uses 708 panels per lane. With our deposition concentration we got ~150 million reads per lane.

4. As far as time goes, I can't really attest to the speed at full capacity. It took us 6 days for 2 lanes with 10 bp barcodes, 50 bp FWD1 and 35 bp REV.

So far so good with the 5500. However, on our first run as well as on the test run the liquid handler dripped on the slide and impaired imaging of portions of the lanes... The FAS installed a shield to protect the slide during fluidics. I think it will do the trick. With that said, make sure you get the shielded installed ASAP.

Hope that helps.

[JPC]

Hi onehalfmex, your bead density seems a little low given you are loading 400M beads and there are 1200 panels per lane, wouldn't you expect a bead density of around 333k? or am I missing something in my calculations?

If you really are losing a third of you beads, maybe a longer 3' mod incubation or longer slide incubation might help?

JC

[mnandita]

What is the starting amount of library required for templated bead prep for Solid 5500xl?

The user manual recommends a 0.8pM titration point for unknown libraries. (Applied Biosystems SOLiD EZ Bead Emulsifier Getting started guide 441486 Rev E).

The ePCR_calculator_V5_25_08_2010 supplied to us by our FAS appears to use a Titration point of 0.459 picomoles in 11 ml (= 45pM ?).

Thanks

[JPC]

HI mnandita, it depends on the size of reaction, do you want to do an E20, 80 or 120?

JPC