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Old 07-27-2017, 07:27 AM   #8
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Location: UK

Join Date: Feb 2014
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Originally Posted by HESmith View Post
Ligation is always less efficient than incorporating the adapter ends directly into your amplicon primers. Is there a reason NOT to use the following type of primer?

adapter end - index - random k-mer - target sequence

If both primers have this structure, you can use dual indexing, alter the k-mer length to address phasing, and distinguish PCR duplicates, with only one round of PCR.
Thanks HESmith. This was the original idea I have been toying with.

The thought of ligating the adaptors on latterly was really just to keep costs down for the many samples we will need to run. I have heard that usual way this is done for metabarcoding is to PCR using a primer that includes the random k-mer + index + target sequence primer, and then ligate on the sequencing adaptors (avoiding further PCR). However, my understanding is that the latter part of this protocol is usually done by a commercial company and we will not have the resources to use their services in the future. Therefore I've been trying to develop a protocol that will be as cost effective (yet simple!) as possible to do inhouse.

I'm really grateful for your comments as they are allowing me to start ticking or scratching out some of my ideas I've had on how to tackle this. I have been going around in circles!

Thanks again.
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