[charliewestin]

Yes, the method is quite complex, and time consuming as well. I am thinking of just purchasing custom 5´ RNA adapters for simplicity's sake. However, attaching a barcode to the illumina supplied 3´ adapter sounds much cheaper, could you point me in the direction of a protocol?

Also, as I understand it, you are adding the bar code to the adapters that come with the illumina kit? Is this correct?

Thanks so much for you help, I am brand new to multiplexing and nextgen sequencing.

[advanT]

Yes, the method is quite complex, and time consuming as well. I am thinking of just purchasing custom 5´ RNA adapters for simplicity's sake. However, attaching a barcode to the illumina supplied 3´ adapter sounds much cheaper, could you point me in the direction of a protocol?

Also, as I understand it, you are adding the bar code to the adapters that come with the illumina kit? Is this correct?

Thanks so much for you help, I am brand new to multiplexing and nextgen sequencing.

I have been purchasing the 3'adapter barcodes. http://biooscientific.com/DetailProd...tGenSequencing
I purchased the set of 30 for miRNA multiplexing and they work well for me.

[charliewestin]

Do you need to unblock the 5´ end of the bar code before ligating illumina's 3´adapter sequence? As I understand it, the final construct would look like this.

--(Illumina)5´Adapter---(Experimental)SmallRNA---(AIR)Barcode---(Illumina)3´Adapter--

[advanT]

Do you need to unblock the 5´ end of the bar code before ligating illumina's 3´adapter sequence? As I understand it, the final construct would look like this.

--(Illumina)5´Adapter---(Experimental)SmallRNA---(AIR)Barcode---(Illumina)3´Adapter--

no, the 3' adapter barcodes are adenylated. the block is on the 3' end of the adapter, not the 5' end. its the 5' end that ligates to your small rna. you have to use RNA ligase 2 truncated for that. so simply take the adapter, mix it with your small RNA, add ligase and thats it.

[charliewestin]

Great, it is all coming together now. Thanks for all your help!

[marcaill]

Hi advanT,

About this 3' adenylated adapter strategy, do you have an idea about reproducibility between barcodes in a miRNA DGE context ?
I've tested a 5' adapter barcodes strategy. The bias between barcodes (for the same sample) is too important even though we add a 5 bases common linker at the 3' end.
[---(Illumina)5´Adapter-2b Barcode-5b Common linker---(Experimental)SmallRNA---(Illumina)3´Adapter v1.5---]

[advanT]

hi, we ran a 30 barcode RNA experiment and didn't see bias when the barcode was on the 3' adapter, we compared these results to individual runs and did not see anything greater than 8% variability.

I've heard from others that if you put the barcode on the 5' adapter there are issues (you are using a different type of ligase for this that is known to prefer some sequences over others), so l recommend using barcodes that are on the 3'adapter.

[marcaill]

hi, we ran a 30 barcode RNA experiment and didn't see bias when the barcode was on the 3' adapter, we compared these results to individual runs and did not see anything greater than 8% variability.

AdvanT, Could you please specify this 8% variability ?
I would like to know whether we can extrapolate this in our situation.

In our case, we test the reproducibility by measuring the correlation (Pearson) between 2 libraries with the same sample and 2 different barcodes.
After analysis, we obtain a relative count of miRNA for each condition and look at the scatter plot.
We consider a good correlation when the difference is not significant from a situation without barcode (Pearson coefficient near 0.99).
The best correlation for us with 5'adapter-indexing strategy was in average about 0.93 that is not enough...