EasySeq NGS Reverse Complement PCR

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Simultaneous Target Amplification and Barcoding

A single PCR reaction for Multiplex NGS library prep including flexible barcoding? It’s now possible. RC-PCR, the most straight forward, secure and robust method for NGS Library generation. 

Introduction 
Current PCR based methods for Amplicon library preparation for Next Generation Sequencing (NGS) typically require a two-step PCR reaction: A primary PCR is performed to amplify the ROI and add 5’ universal tags while the secondary PCR step, using the primary PCR as the template, add the required functional NGS sequences and indexes. This approach allows complex combinations of indexes to be added to PCR amplicons using a relatively small set of primers, however it requires multiple steps adding time and cost, particularly if large number of samples need to be analyzed.

In addition, as the primary and secondary PCR steps are performed separately, this requires opening tubes and/or tube transfers. Accordingly, there is a high risk of cross-­contamination or carry-over. Since PCR involves exponential amplification, even low levels of contamination can have very significant impact that is difficult to detect, particularly when the process is used as a preparation step for sensitive analysis like NGS. RC-PCR from NimaGen is the ideal solution to these problems, providing a new, patent protected method for generating amplicon constructs, with a single closed tube amplification and indexing reaction. Furthermore, the method is fast, simple to use, increases targeting specificity and quantification, has the potential for multiplexing and significantly reduces the chances of amplifying incorrect sequences.

Currently NimaGen is developing RC-PCR solutions in the field of:

  • Sample tracking kit / Human Exome (released April 2019)
  • Human Identification Kit
  • Custom Crop Genotyping assays
  • (Targeted GBS)
  • GMO detection kit
  • Meat detection kit (including speciate)
  • Pharmacogenetics (PGX)
  • Circulating Tumour DNA kit
  • Detection of aneuploidies


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