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Amplicon libraries (PCR)

Amplicon sequencing enables ultra-deep analysis of targeted PCR amplicons.  It is a cost-effective way to validate previous NGS findings and for large-scale screening of targeted DNA regions. This method allows mutations to be detected at extremely low levels and it is an excellent tool to identify low frequency somatic mutations. Powerful dual-indexing primer designs enable high multiplexing levels (currently over >500). Amplicon sequencing is also suited to metagenomics projects.

Primer design

Primers are designed for a region of interest manually or using automatic primer design pipeline. The length of amplification product depends on sequencing platform to be used.

Customer provides:

  • Genomic coordinates and genome assembly for the region of interest
  • Previous data if available

Sequencing unit delivers:

  • Primer design for the region of interest
  • Primer modifications prior to order if needed

Primer optimization

Service includes optimization of PCR amplification protocol for one primer pair.

Customer provides:

  • Primers (if primers come from customer)

Sequencing unit delivers:

  • PCR test(s) for one primer pair
  • Gel electrophoresis of PCR products
  • Choosing optimal PCR conditions
  • Capillary sequencing of a test amplification product

Targeted PCR amplification

The region of interest is amplified using primers (designed and) optimized by Sequencing unit. Also multiplex PCR reactions are possible but that needs to be taken into account when optimizing primers. DNA samples should be of good quality for optimal PCR results and the samples are expected to be in same concentration for equal yield and post-PCR processing.

Customer provides:

  • DNA sample(s), preferably in 10 ng/µl
  • If more than 16 samples, preferably in a 96-well plate
  • Sample information sheet with unique sample names and other required info.

Sequencing unit delivers:

  • PCR of the sample(s). If samples are sequenced by NGS sequencing, Illumina compatible tails are added to the PCR product
  • QC of PCR by gel electrophoresis (a batch of samples is checked)
  • Purification of the PCR products

Sequencing

Sequencing is performed using Miseq or alternatively on Hiseq 1500 or 2500. Also capillary sequencing is available. When considering the sequencing platform amount of amplicons, indexing and requested read depth are taken into consideration.

Last updated: 25.08.2016 - 13:17