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Old 11-03-2014, 06:46 AM   #41
GenoMax
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Illumina has a simplified animation for cluster generation/sequencing available here: http://www.youtube.com/watch?v=HMyCqWhwB8E
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Old 11-03-2014, 04:18 PM   #42
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Hi, Creeves, thank you for your reply. I understand that after the cluster generation, there would be a mixture of complementary strands grow from the p5 or p7 ends. Some documents suggested the strands with p5 ends attached to the flow cell would be stripped off for the first read and the i7 index read to ensures "that all copies are sequenced in the same direction". Then, after the turn around, the p7 grafted strands would be cleaved to allow all reads of read 2 come off the p5 tethered stands. Does this sound correct? If it is correct, I'd like to know how the p5 and p7 tethered stands were cut?

Here is the link where I got the information:

http://nextgen.mgh.harvard.edu/IlluminaChemistry.html
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Old 11-03-2014, 04:34 PM   #43
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Thank you, GenoMax. I am ashamed to say that I can't open any youtube link, because I am in China now. If anyone could explain to me how it works in simple words, it'll be greatly appreciated. I am sorry for being so ignorant.
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Old 11-04-2014, 04:01 AM   #44
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Indeed, my customized adapter and sequencing primer is the same for read1 and read2. So if the sequencing is done from one direction, it would be fine. But if the cluster is composed of two opposite strands when sequencing is done, then my sequencing primer could anneal to both stands and my reads would be a mess. I'd really like to find out if the opposite stands are clipped off from the cluster for both read 1 and read2? Thanks
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Old 11-04-2014, 04:45 AM   #45
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Since you can't see the video how about this link: http://nextgen.mgh.harvard.edu/IlluminaChemistry.html
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Old 11-04-2014, 04:58 AM   #46
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Quote:
If it is correct, I'd like to know how the p5 and p7 tethered stands were cut?
If you are looking for chemistry used for cleavage of strands during sequencing, I have got following from a rather old paper:

Immobilised oligos on paired end Flowcell surface:

Oligo ‘C’: 5’-PS-TTTTTTTTTTAATGATACGGCGACCACCGAGAUCTACAC-3’
(U = 2-deoxyuridine), Linearization of oligo C with “USER” enzyme retains strand 1, attached to “D” oligo.

Oligo ‘D’: 5’-PS-TTTTTTTTTTCAAGCAGAAGACGGCATACGAGoxoAT-3’,
(Goxo = 8-oxoguanine) Second strand is cleaved at the 8-oxoguanine in oligo 'D' using Fpg enzyme.
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Old 11-04-2014, 07:17 PM   #47
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GenoMax, thanks for the information. Nucacidhunter, thank for explaining the chemistry. This is really cool! Now feel more comfortable to try customized sequencing primers. Since each time there is only one strand interrogated for sequencing, even my sequencing primer could anneal to both strand, it should not cause any problems.

I still have some questions about the turn-around business though. After the i7 indexing read, the other strand would be synthesized along with i5 index sequenced after 7 dark cycles. Should the full length of the new strand be fully extended before the read 2 sequencing started, because the full strand is needed as sequencing template for read2? If so, where are the consumable (dNTPs etc.) come from? Are those also contained in the reagent cartridge? It shouldn't be part of the reagents for 200bp reads, should it? I feel I might sound silly, but those questions keep annoying me if I can't find an answer.
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Old 11-05-2014, 01:06 AM   #48
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Quote:
Originally Posted by cli View Post
I still have some questions about the turn-around business though. After the i7 indexing read, the other strand would be synthesized along with i5 index sequenced after 7 dark cycles. Should the full length of the new strand be fully extended before the read 2 sequencing started, because the full strand is needed as sequencing template for read2? If so, where are the consumable (dNTPs etc.) come from? Are those also contained in the reagent cartridge? It shouldn't be part of the reagents for 200bp reads, should it? I feel I might sound silly, but those questions keep annoying me if I can't find an answer.
After last cycle of 2nd index read, it is stripped away and new strand synthesis starts and reagents for it are in the sequencing kit components. If you want to make sure that your sequencing will be successful, you need to provide more information about your experiment design and protocol and ....

I wonder how you ended up having the same priming site in both strands.
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Old 11-05-2014, 10:56 AM   #49
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Originally Posted by TonyBrooks View Post
However, if you're generating library directly from PCR, why not remove those sequences all together and just use custom read primers. Your custom read primers will just be versions of your fwd and rev primer sequences. You'll also waste less reagent by not sequencing your primers. See my post further up.
Regarding this comment from a while back - if your target is a PCR amplicon product and the ends are all the same primer sequences, isn't the ONLY way to successfully sequence this is through a custom sequencing primer?

Otherwise you'd be sequencing a pool of homogeneous ends for the length of your PCR primers, right? (Unless your pool is made of products that came from multiple primer sets.)
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