Synthesis of i5 index

[stevenpei1]

Hi everyone,
i am new to the field and I have a problem of running out of the E505 and E506 index (of the Nextera DNA Exome (formerly TruSeq Rapid Exome Kit), while illumina said they dont offer individual purchase of these oligos. Therefore, i would like to order the sequence on my own.
I wonder if anyone can give me some advise how should i do it? i can only find the specific sequence of the index.

[N/S/E]505 GTAAGGAG CTCCTTAC
[N/S/E]506 ACTGCATA TATGCAGT

thanks a lot.

[Pauline dlp]

If you already have the sequence, I don't see what additional hurdles you have in ordering the oligos. I'm from the Philippines and we order custom-made oligos from IDT or Macrogen (South Korea) through local distributors.

[stevenpei1]

Hi Pauline,
Thanks a lot for your comments. I have read some threads here that it seems that some modification is required? Adding phosphate group to 5'' end etc.
So, i just see if anyone know any modification i might need after ordering the oligos.

[luc]

You have seen this info from Illumina:
https://support.illumina.com/content...0002694-06.pdf ?

Does this kit use transposase (no oligo modification required in this case) or adapter ligation?

[Pauline dlp]

It seems you are right. The adapters are modified to have a phosphate group on the 5' and phosphorothioate on the 3' end before the terminal T to protect it from 3' -> 5' exonuclease activity.

I am not sure about the Nextera Exome adapters but this article describes the structure of adapters for Truseq and Nextera:
http://bioinformatics.cvr.ac.uk/blog...mer-sequences/