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Old 12-10-2014, 12:17 AM   #1
AcoBetalow
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Default Amplification free library preparation

Hello community,

Firstly I would like to greet everyone, for this is my first of hopefully many productive experiences on this forum.

My question involves amplification free library preparation for MiSeq. We have purified aptamers of 80 bp and would like to prepare them for sequencing with as little amplification as possible.

Is it possible to directly ligate indexes to aptamers are do we have to ligate adapters first?

Is there any other way?

Thank you in advance for any suggestions.
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Old 12-10-2014, 07:44 AM   #2
jlei_face
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The ligation of adapters (which contain your index) occurs right before amplification so as long as you have enough material/high enough library yield for clustering you don't necessarily need to amplify.
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Old 12-10-2014, 10:18 PM   #3
Asaf
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Alternatively, you can use UMI - unique molecule identifiers. Each adapter that you ligate contains abarcode (of the library) and a short sequence (4-6 nt) unique for that molecule. After amplification you count each UMI of each DNA species only once, even if you got multiple reads of it and overcome amplification bias.
Another amplification with less bias is IVT - in-vitro transcription which is linear rather than exponential as in PCR.
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Old 12-11-2014, 07:37 AM   #4
AcoBetalow
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Firstly, thank you for your help.

After wavering few possibilities, we have decided that probably easiest and cost effective way is to use cloning kit to repair ends of the aptamers, create single d-ATP overhangs on 3' end and ligate adapters to them. Afterwards we made compromise and we will use standard Illumina index PCR to label.

Another solution is KAPA hyper prep kit, that ligates adapters and indexes all together, but is a bit more expensive.
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