solexa genomic adapter and pictures?

[seqgirl123]

I am trying to find slides from Illumina that depict these adapter structures along with mechanism of how they ligate to the genomic DNA after going through the end repair and 3' A extension steps. But Illumina's kept their slides pretty simple, not going into too many pictorials about the underlying mechanism.

Would anyone have illustrations of the exact adapter structure used for preparing genomic libraries? I know they are in some unusual structure (like a two pronged fork). Any pictures depiciting the entire mechanism of library construction would be good too (from end repair to ligation).

[kmcarr]

Seqgirl123,

I don't know of any images from Illumina (I've looked too) but I have attached a schematic of the genomic library prep (for single end sequencing libraries) which I created myself so I could understand how it worked. Hope it helps.

[seqgirl123]

kmcarr,

Thank you very much for sharing your diagram. I am also trying to learn. It's very informative and much better to look at than Illumina's colorful sticks to depict the mechanism!

I have another question if anyone knows. The other day, I had run a gel on PhiX DNA that went through the dA extension and ligation steps (my PhiX comes blunt ended so I skipped end repair). I also ran a negative control alongside where PhiX was ligated (no adapters, just PhiX ligated together using DNA ligase).

The negative control showed high molecular weight product while the other PhiX that went through extension & ligation steps was lower in molecular weight and ran a bit farther down the gel. My question is, why was the negative control in higher molecular weight than the other PhiX? I know it has something to do with the way it ligated, but I don't understand the underlying mechanism, if someone can explain? I would have thought that the dA extension and adaptor ligation would cause high molecular weight?

[kaichen]

I wish I saw this first~~~~~

[Sadia]

Hello
Can anybody tell me if I have a template having internal biotin(The biotin is linked to the internal dT via a 10-atom spacer arm) wether it will be amplified in PCR or polymerase will block at the biotinylated nucleotide

[Chen Rui]

It's useful. Thanks a lot!

[wzhang25]

It is very useful. Have another quetions about ChIP-Seq or genomic DNA-Seq library construction. Does anyone know the sequence details in adaptor oligo mix and primer 1.1 and 2.1 from the Kit for ChIP-seq or genomic DNA-seq preparation from Illumina.Thanks a lot. wzhang25

[bmcjc]

I want to sequence my PCR product on Solexa (it's a long story). Will it be feasible to PCR amplify with primers that has the Illumina's long PCR primer sequence extended to the 5'end so I don't have to do adaptor ligation again?

[weizhu]

Seqgirl123,

I don't know of any images from Illumina (I've looked too) but I have attached a schematic of the genomic library prep (for single end sequencing libraries) which I created myself so I could understand how it worked. Hope it helps.

I am a newcomer of this forum. could anyone tell me what I shall do to view the attachment?

Thanks,

Wei

[ECO]

I am a newcomer of this forum. could anyone tell me what I shall do to view the attachment?

Should be able to just click on it if you're logged in and confirmed (which you are!). What error are you getting?

[abelhj]

Hi,

I am trying to figure out Solexa paired end read output. Can someone please tell me if my toy example is correct?

1) One end of DNA is bound to substrate. Free end is sequenced in 5'-->3' direction.

#
5' #
------> #
GCCCxxxxxxATTT # ==> GCCC
CGGGxxxxxxTAAA #
#
#
#

2) Opposite end of DNA bound to substrate. Free end of *complementary* strand sequenced in 5'-->3' direction.

#
5' #
------> #
AAATxxxxxxGGGC # ==> AAAT
TTTAxxxxxxCCCG #
#
#
#

So the final output of the Solexa machine would be the pair (GCCC, AAAT) and then a program such as maq would worry about the details of orientation/taking complements.

Thanks,
hja

[abelhj]

Sorry my graphic got messed up. Here it is again.

Hi,

I am trying to figure out Solexa paired end read output. Can someone please tell me if my toy example is correct?

1) One end of DNA is bound to substrate. Free end is sequenced in 5'-->3' direction.

#
5' #
------> #
GCCCxxxxxxATTT # ==> GCCC
CGGGxxxxxxTAAA#
#
#
#

2) Opposite end of DNA bound to substrate. Free end of *complementary* strand sequenced in 5'-->3' direction.

#
5' #
------> #
AAATxxxxxxGGGC# ==> AAAT
TTTAxxxxxxCCCG #
#
#
#

So the final output of the Solexa machine would be the pair (GCCC, AAAT) and then a program such as maq would worry about the details of orientation/taking complements.

Thanks,
hja

[Anri]

I want to sequence my PCR product on Solexa (it's a long story). Will it be feasible to PCR amplify with primers that has the Illumina's long PCR primer sequence extended to the 5'end so I don't have to do adaptor ligation again?

Check this article. Is anyone done something like this? (http://www.ncbi.nlm.nih.gov/pmc/arti...ukmss-3213.pdf).

Direct sequencing of short amplicons
To avoid unnecessary PCR amplification steps, which would potentially exacerbate biases, we can perform extremely deep sequencing of short amplicons using locus-specific primers that possess tails that are capable of hybridisation to the oligos tethered to the flowcell surface. The tailless forward and reverse oligos are then used as primers in the sequencing steps
(Supplementary Protocol 10 online).

[seqgirl123]

need some help

Hi,

I am a little confused with the various schematics I found on the Solexa adapter structure.

I followed Illumina's pdf (attached) and made the forked structure myself (jpeg). I also listed the variations I saw on the right taken from various schematics people have posted on this website which I think they took from other journal articles.

Can someone tell me if the sequences taken from Illumina's pdf are the correct forked structures as I've drawn them, meaning is that the way they're really supposed to look? My version has more bases ligating to each other versus the ones taken from this website which have fewer, why is that and does it make a difference? Thanks.

[anant]

Thanks kmcarr, it is very useful and information.