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Old 11-23-2009, 03:45 PM   #21
chayasue
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Default illumina bridge amplification

Can anyone explain how successive rounds of pcr are carried out in the bridge amplification step to form a cluster? what is the primer and is it separate or different from the oligos attached to the cell? are the unattached strands of dna discarded between successive pcr steps? if not, can anyone explain what happens to them?
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Old 01-10-2010, 05:14 AM   #22
tobiwan
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Default Fluorophore removal?

Quote:
Originally Posted by mccullou View Post
How does Illumina remove the fluorophore? They can't use AgNO3 (like SOLiD ) because of no Thio backbone. Are they using the Columbia IP that Intelligent BioSystems is using? http://www.intelligentbiosystems.com...20mod%201.html.

Any thoughts here?
This would be of interest to me as well!
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Old 01-10-2010, 07:36 AM   #23
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Quote:
Originally Posted by tobiwan View Post
This would be of interest to me as well!
You definitely need to read this review.
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Old 01-12-2010, 03:43 AM   #24
tobiwan
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Default Fluorophore removal?

Thanks for the hint! I am new to this field of work...
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Old 01-26-2010, 06:10 PM   #25
bio_emule
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thanks all. very useful information for me.
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Old 01-26-2010, 11:20 PM   #26
cgb
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the chemistry is proprietary.
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Old 01-27-2010, 07:39 AM   #27
mccullou
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So are most buffers if you ask qiagen or invitrogen, but we are all smart people here, right? It is either photo or chemical cleavage and looking at all the steps in the sequencing process there doesn't seem to be a hint. I am just curious because I have been having increasing background and phase issues and I think it might be inefficient removal of that flourophore/blocking agent, but I can't figure it out my issue if I don't know how the base is removed. . .
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Old 02-07-2010, 11:31 PM   #28
fafs26
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Hi All

I have just joined SeqAnswers and I am hoping to get some advise from those who are currently doing sequencing on the Illimina GA. I am hoping to do an infection profile for infected plants using the Paired-end muliplex system. Is this advisable? Or should I run each infection/control pair per lane?
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Old 03-25-2010, 08:41 AM   #29
Lorena82
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Thanks a lot!
Very interesting for me taking into cosideration my little experience!
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Old 05-25-2010, 12:14 PM   #30
anju
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Default comparison of illumina/ ABIsolid/ 454

simple comparison
Attached Files
File Type: pdf 454-illumina-ABIsolid.pdf (3.31 MB, 609 views)
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Old 05-31-2010, 01:01 AM   #31
vtosha
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There a small mistake on the picture. If we saw sequences of adaptors, primers and oligonucleotides
(Accurate whole human genome sequencing using reversible terminator chemistry.
David R. Bentley, Shankar Balasubramanian etc.
Nature, Vol 456 | 6 November 2008 | doi:10.1038/nature07517)
--- we can see that fragment links with oligonucleotide on the surface of flow cell not with adaptor but with additional sequences in primers, added by PCR.
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Old 06-20-2010, 07:34 AM   #32
MrGuy
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Quote:
Originally Posted by anju View Post
simple comparison
Pretty out of date, too. Anything with the latest HiSeq, Solid 4, and "1000-base" 454?

I'd love to see some info on useable read length, too.
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Old 06-23-2010, 11:19 AM   #33
pmclaughlin
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hello all I am very,very, very new to NGS. trying to learn as much as I can
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Old 09-16-2010, 10:16 AM   #34
BTS
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Default Bridge Amplification

Hi All,
I'm new to this whole NGS business. I will be using an Illumina GAII. In a recent article from Bio Techniques, I read that longer (about 600bp) fragments can increase the uniformity of coverage across a targeted region. In the paper, amplicons were fragmented to 200bp prior to adaptor ligation and subsequence sequencing. Does anyone know why this would increase uniformity? Also, does anyone know if it is possible to successfully generate clusters during bridge amplification with fragments larger than 200-300 bp? And if so, how large?

THANKS!!
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Old 09-24-2010, 05:09 PM   #35
rubenken
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Is this material applicable to Illumina's systems today, or does it need updating?
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Old 09-24-2010, 05:18 PM   #36
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Quote:
Originally Posted by rzhuo View Post
Hi there,

I have a question about the software I can use to analyze the Illumina sequencing data. I'm doing targeted resequencing of long PCR fragments to find some EMS mutations. I'm waiting for my run to finish right now and meanwhile I'm looking for a suitable software. Any suggestion would be of great help to me. Thank you.
Probably north of 95% is still perfectly applicable. The only major changes are to the chemistry robustness which increases yield and readlengths. I don't personally run any form of ILMN machine so I welcome correction from an Illumaniac*.


*New name for the Illumina forum!
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Old 09-27-2010, 04:45 AM   #37
krobison
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Will another forum be renamed to "On SOLiD Ground?"
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Old 01-29-2011, 06:26 PM   #38
li zhang
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Thanks for the useful information
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Old 02-08-2011, 01:36 PM   #39
paullaissue
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Hello, Im new in NGS. I want to sequence target regions using human genomic DNA. My purpose is to sequence de coding regions of 400 genes (around 1Mb). Which is the best approach?
In addition could you recommend me what software can be easily used to analyze data from exome assays.
Thank you.
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Old 02-09-2011, 06:56 AM   #40
krobison
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Wrong thread. You'll get more eyes & brains on your question if you post in a more appropriate area (and say how many samples you are working with & what sort of species this is for)
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