Hi All,
I want to generate RRBS libraries where we can track each unique molecule with a UMI. Thus I have generated new TruSeq adaptors that we normally use.
The regular Truseq primers look like this:
A1_P5: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T
A1_P7 (AR005): ℗-GATCGGAAGAGCACACGTCTGAACTCCAGTCACACAGTGATCTCGTATGCCGTCTTCTGCTTG
What we did was to add 8 random nucleotides to the P5 so that it looks like this:
A1_P5 UMI8: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNN*T
From what i heard I should anneal the two adaptors. The annealing effeciency can be seen here: https://imgur.com/a/70LZU
What I did now was to try our regular protocol with old adaptors vs new adaptors.
The libraries look like this: https://imgur.com/a/mHZeF
Stupidly I did not generate another P5 without the 8 UMI's.
It seems like the adaptors form adaptor dimers, but for the library it seems like it does not bind.
Suggestions of other designs or ways to get it to work would be highly appreciated.
I have looked into the dual indexes and then exchanging one index with UMI's instead, but could not find the sequences, do any of you have them?
Best regards
Emil
I want to generate RRBS libraries where we can track each unique molecule with a UMI. Thus I have generated new TruSeq adaptors that we normally use.
The regular Truseq primers look like this:
A1_P5: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T
A1_P7 (AR005): ℗-GATCGGAAGAGCACACGTCTGAACTCCAGTCACACAGTGATCTCGTATGCCGTCTTCTGCTTG
What we did was to add 8 random nucleotides to the P5 so that it looks like this:
A1_P5 UMI8: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNN*T
From what i heard I should anneal the two adaptors. The annealing effeciency can be seen here: https://imgur.com/a/70LZU
What I did now was to try our regular protocol with old adaptors vs new adaptors.
The libraries look like this: https://imgur.com/a/mHZeF
Stupidly I did not generate another P5 without the 8 UMI's.
It seems like the adaptors form adaptor dimers, but for the library it seems like it does not bind.
Suggestions of other designs or ways to get it to work would be highly appreciated.
I have looked into the dual indexes and then exchanging one index with UMI's instead, but could not find the sequences, do any of you have them?
Best regards
Emil
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