Sample tile images across a lane

[ScottC]

Hi Everyone,

We have had a GA I (or 'classic') installed recently and have had a lot of problems during set-up and training. I'd like to get an idea of how much variation there is between instrument in terms of the appearance of the tile images produced - evenness of illumination, vignetting, etc. Because we haven't really seen any 'good' tiles, I'm interested in seeing what others look like in general.

In particular, is it common to see a 'blank' area on the left side of a column 1 image, a good coverage of clusters in column two, then a blank area on the right side of column 3 (i.e. it looks as though there are fewer/no clusters at the edges of the lanes)?

So, if anyone is willing to show/send me a subset of images, particularly sets of three columns across a lane, please contact me.

Cheers,

Scott

[SillyPoint]

Yes, it is entirely common to see image illumination artifacts such as you describe with the GA1 instrument. If you are seeing only blank areas, that's a mild form of the problem. In the worst case, you also get reflections off the flowcell wall showing up along the edge of the images in columns 1 & 3. These for some reason are brightest in the 'A' channel, so they pass purity filtering and produce nice reads containing all A's. You have to throw those away.

Pay a visit to www.genographia.org. Go to Topics/Example Data Sets/Illumina. There are some images from Sanger there, including a good one, and one with flowcell wall reflections.

Lots of other good stuff on the site. You may have to register first: 'Enter' from the home page, then 'Register' in the upper right corner -- a little hard to spot.

Good luck with it. Post more questions if you have them.

--SillyPoint

[ScottC]

Hi,

Thanks for that infor and resource. That site looks good. I didn't know about it before.

I realise that some 'blank' area at each edge of the lanes is normal... I just want to get an idea of how large this area should be, and how event he illumination should be over the rest of the area of the tile. We've just had a problem 'fixed' in ours where nearly half the tile was blank. They tell us that they've 'done the best they can', which is fair enough, but as I said before, I'd like to see some examples from other machines to see how well (or badly) it compares!

EDIT: I've taken a bit more of a look at the site you linked to... that's excelent. Just what I wanted! Who owns/manages that site? I couldnt really find any affiliation details other than that they're supported by intel and genome research ltd. Does it belong to an institution or is it just a public information site like seqanswers?

Cheers,

Scott.

[clivey]

Hi - (formerly cgb here).

I set up the original as a public wiki (rather than a message board like this) paid for and sponsored by Sanger (GRL). The idea being to get people to share experiences, methods, data etc to a reasonable degree of depth. Effectivey creating a self-supporting community. After some discussions with Intel they agreed to set up a version of their own, migrating all of the old content, that would scale to more users. My former group at Sanger have posted a lot of stuff on there, but very few other people post, so they get tired of doing it. The site has no commercial undertones whatsoever.

[ScottC]

Oh ok... sounds good to me. I'd be happy to post there.