Custom Illumina adapter synthesis

[kmcarr]

We are looking a making a set of barcoded adapters for Illumina sequencing. We know the sequences already but I wanted to ask people who have done it if they incorporated any modifications in the oligos. Specifically the use of phosphorothioate linkages. I'm asking because Roche/454 recently released their protocols and adapter sequences for doing MID tag sequencing on Titanium and they call for phosphorothioate links at the first and last 4 bases of each oligo.

I would appreciate hearing from people who have made their own adapters for Illumina sequencing. Did you use modified linkages? What were your experiences with homemade or custom adapters? Any lessons learned would be greatly appreciated. Thanks in advance.

[elaney_k]

Hi kmcarr,

I routinely use indexed adapters in place of the Illumina adapters and modify them as the Illumina adapters; with a 5' Phosphate on one and a phosphorothioate bond on the other. Both are HPLC purified.

Elaine

[seqer]

Could anyone explain why phosphorothiorate is needed?

[kmcarr]

Hi kmcarr,

I routinely use indexed adapters in place of the Illumina adapters and modify them as the Illumina adapters; with a 5' Phosphate on one and a phosphorothioate bond on the other. Both are HPLC purified.

Elaine

Elaine,

Just to make sure I understand you, do you mean that you use the phosphorothioate only for the overhanging 'T'?

[greigite]

According to supplementary info in the source paper for Illumina technology (Nature 2008; doi:10.1038/nature07517) the phosphorothioate bond protects the terminal T from excision by 3'-5' exonucleases. My guess is that this is used to make sure the adapters can ligate correctly to the A overhangs on the template. You might get less efficient ligation without the phosphorothioate bond, but I'm not sure it is completely necessary, since the ligase is supposed to be free of contaminating exonuclease activity.

Could anyone explain why phosphorothiorate is needed?

[seqer]

Thanks greigite! Although I do have experience on phosphorothioate, it is new to me to add it in the T-A ligation adapters.

[elaney_k]

Elaine,

Just to make sure I understand you, do you mean that you use the phosphorothioate only for the overhanging 'T'?

Hi kmcarr,

Yes only one phosphorothioate bond is used, see attached for a better explanation,

all the best,

Elaine

[kmcarr]

Thanks Elaine, I owe you a pint.

[elaney_k]

Thanks Elaine, I owe you a pint.

hmmm pints are always a good idea

[greigite]

BTW some people have told me that desalting is sufficient for purification. I did HPLC purification for the phosphorothioate adapters and desalt for the phosphate ones. I haven't tried them yet so we'll see! HPLC is quite a bit more expensive.

Hi kmcarr,

I routinely use indexed adapters in place of the Illumina adapters and modify them as the Illumina adapters; with a 5' Phosphate on one and a phosphorothioate bond on the other. Both are HPLC purified.

Elaine

[advanT]

custom adaptor synthesis

I've been also interested in custom adapters, but couldn't find anywhere that would make them. Recently I found Biooscientific.com has a custom adapter sequence service that you can order. Now, I send them all the adaptor sequences I need and have been very satisfied. They also make those tricky adenylated oligos with hplc purification or standard desalting (3' adapters for the illumina platform). This is the link to their page:

http://biooscientific.com/DetailProd...A&Name=Cloning

[charliewestin]

RNA adapters

Is it possible to use custom RNA adapters for barcoding, and if so does anyone have experience or advice on the subject? Thanks

[advanT]

Is it possible to use custom RNA adapters for barcoding, and if so does anyone have experience or advice on the subject? Thanks

Add your barcode (3-4 bases) to the 3' adenylated adapter, works great.

[charliewestin]

Thanks for the advice. Is the method you are suggesting the same as the G. Church 'Efficient microRNA capture and bar coding via enzymatic oligonucleotide adenylation' paper method? Is there a simpler method you know of?

[advanT]

Thanks for the advice. Is the method you are suggesting the same as the G. Church 'Efficient microRNA capture and bar coding via enzymatic oligonucleotide adenylation' paper method? Is there a simpler method you know of?

I haven't read that paper. Are they using a complicated method? We just purchase the adenylated barcodes and they work great for us...very simple.