How tos and FAQs

What is the Efficient amplicon multiplexing service?

This is the most economical way of amplicon sequencing and can be used also in ShareSeq (not necessarily).

For example, think of this project: you aim to sequence 50 different amplicons per sample (patients, environmental samples etc.) and you have 20 samples. This means you plan to generate 1000 amplicons. Employing Sanger sequencing for their sequence analysis would cost you a fortune.

The trick is to design PCR primers to contain our adapters. Each primer (100 in total for your 50 amplicons) bears at its 5’ end a universal adapter sequence (CS1 for forward and CS2 for reverse primer, respectively). You perform PCR as usual using these adapter-containing primers. At the end of the day you end up with 1000 PCR amplicons and you combine (pool) them into 20 tubes (1 sample/patient = 1 tube = 50 PCR amplicons). All have idential 5’ and 3’ ends.

Then you submit these 20 samples for our Efficient Amplicon Multiplexing service (available for ShareSeq also).

We perform a fusion multiplex PCR using primers whose 3’ ends match CS1 and CS2 and have specific barcodes (BC) at 5’ ends, unique for each sample (20 barcodes in total). Then all 20 barcoded-samples are combined together into one tube and only a single sequencing library is prepared! This way we generate and sequence a complex mixture of 1000 amplicons in a single tube but each of them can easily be identified after sequencing since it contains a sample-specific barcode and of course has its unique sequence making it easy to align it to a proper part of a reference sequence.

 

 

Sequences:

CS1: 5’-ACACTGACGACATGGTTCTACA-(amplicon-specific FW primer)-3‘

CS2: 5’-TACGGTAGCAGAGACTTGGTCT-(amplicon-specific RV primer)-3‘

 

Please contact us in case of interest.

Back to the list
© SEQme s.r.o., 2012 - 2019. All rights reserved. Disclaimer.
webdesign Beneš & Michl