[vtosha]

Bam-format: http://bioinformatics.oxfordjournals.../26/5/676.full

[joskee]

Can anyone explain to me whether step 2 is correctly shown in this figure?

I have always been told that the DNA you want to sequence is ligated to 2 adaptors and that 1 adaptor of those 2 will bind a complementary (primer) piece that is attached to the glas plate. And then the bridge PCR can start from the primer piece attached to the plate. Thus meaning that the DNA piece with both adaptors will be released after denaturation (it does not "stick" to the plate).

Rather then that the adaptor itself will bind the plate (like shown in this figure).


So if it right what I think, then the figure is a bit wrong. It should show how the adaptor piece binds with a primer piece and then get elongated with polymerase.

[vtosha]

Yes, you are right. Adaptors bind to oligos on glass of flowcell. These oligos are absent on picture of step 2.

[joskee]

Yes, you are right. Adaptors bind to oligos on glass of flowcell. These oligos are absent on picture of step 2.

Ok thanks.

I find it a bit weird that solexa/illumina itself does not state this clearly on their website/information documents.
If no-one would have told me this before, I would have simply believed that the adaptors themself would stick to the glass plate.

[vtosha]

See Bentley at al., 2008 (http://www.ncbi.nlm.nih.gov/pubmed?t...or%20chemistry).
Supplementary gives many valuable information for beginners (oligos, adaptors, primers, reagents). For example, how to bind oligos to flowcell.
Now Illumina use another system (longer adaptors with index), but article is very useful to understanding of whole process.

[joskee]

See Bentley at al., 2008 (http://www.ncbi.nlm.nih.gov/pubmed?t...or%20chemistry).
Supplementary gives many valuable information for beginners (oligos, adaptors, primers, reagents). For example, how to bind oligos to flowcell.
Now Illumina use another system (longer adaptors with index), but article is very useful to understanding of whole process.

I'll check it.
Thanks a lot.

[langjidong]

Very very useful, Thank you very much!

[akachigerry]

Hi,
Am a new member on. Like the stuff put out on this forum. They are richly informative and helpful. Keep it up!

[Oftalmic]

Thanks

Thank you very much! I've read your post with great pleasure. After reading I have a major comprehension of the technology at last.

[mms]

I have a question actually a couple of them. Has anyone ever observed difference in terms of nanomolar concentration of exome libraries when estimated by qPCR and Qubit. In my case qPCR generally reads high. In such situations which is the method to go with for cluster generation. Also I would like to know the possible reason. In addition to this I would like to know why there is no qPCR step at the end of sample preparation for exome as taht would tell us the nanomolar concentration of molecules ligated to adaptors.

[s_s71]

A question

It was very useful for me as I was confused how bridges form or over all how solid phase could be prepared . My question refers to the fragments which are chosen with different size!then how can ones know where is the location of these sequences.
Thank you.

[Jonathan87]

Hi, there are 2 things I don't understand about Illumina sequencing:

1)
What if two reads attach to the flow cell in immediate neighborhood ? I mean when they get bridge-amplified, you will have two signals at that spot?

2)
Check this part of the video:

http://www.youtube.com/watch?v=l99aKKHcxC4#t=1m34s

"The reverse strands are cleaved and washed away"
How do you know make sure that all yellow-adaptor-reads (or green-adaptor-reads) come from the same strand (so that you can be sure that when cleaving and washing off e.g. the green-adaptor-ends you only wash away reads from the same strand) ?

[avilella]

1) They can form two clusters next to each other. The BaseCalling algorithms in the machine will determine if they are clonal or not based on the signal coming from each cycle and consider them as (a) one single cluster or (b) two different clusters or (c) either one, or the other, or both too noisy to basecall, and discard them.

[ogep]

I'm waiting for my run to finish right now and meanwhile I'm looking for a suitable software. | Oli Motor Terbaik – TOTAL Hi-Perf | Any suggestion would be of great help to me. Thank you.

[julieBV]

Thank you very much!