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Cornell University

Indexing reactions and Amplicon sequencing

Amplicon sequencing

Also known as targeted sequencing, this set of technologies allows high-throughput, high multiplexing, targeted loci sequencing on Illumina NGS platforms.

AmpSeq and rhAmpSeq use a large set of primers with known sequences to create amplicons throughout the genome. rAmpSeq uses only a few pairs of primers that are anchored in repetitive DNA (i.e., transposable elements), and in thousands of copies within the genome.

These approaches are useful if you want cost-effective sequencing at a specific set of loci in the genome. The number of loci amplified (amplicons) typically ranges from hundreds to a few thousand. These loci are initially amplified by a first PCR with tailed, locus-specific primers, then barcoded by a second PCR with indexed primers for sequencing.


This library prep type is recommended when:

  • You have a limited set of primers (100-500)
  • A pilot project for to testing the approach on a small scale before moving to a larger number of primers or samples.

Indexing for Illumina library preps

We offer the following Nextera indices:

  • Unique Dual Indexes (UDI) for up to 96 samples.
  • Combinatory primers for up to 3072 samples.

We add adaptors on amplicons you already made during the first PCR amplification. Our service only includes the second PCR reaction to add the barcodes (indexing), and the sequencing of the amplicons for an additional fee.


This library prep type is recommended when:

  • You are looking for high multiplexing of primers and samples (over 500 primer pairs and over 50 samples)
  • You do not want primer-to-primer interactions (primer-dimers).

The technology relies on the RNA H2 enzyme to prevent primer-dimers by blocking the polymerase reaction when primers bind to another primer, and allowing it only when primers bind to template DNA. Our service is limited to processing the sample, and sequencing the sample for an additional fee. We ask you to order the primers though Integrated DNA Technologies (IDT) and purchase the master mix.


This library prep type enables identification of thousands of polymorphisms in the low copy intervening sequences of repetitive elements. The difference with AmpSeq or rhAmpSeq is that rAmpSeq does not amplify known sequences, but ALL the sequences in the genome that are flanking a known sequence. This technology is well suited for large genomic selection projects. The approach relies on creating a species or genus specific library.

Service pricing

Indexing reactions and Amplicon sequencing
DescriptionInternal price (Cornell and Cornell affiliates)External price
AmpSeq (users provide primers) Inquire Inquire
Indexing for Illumina library preps (UDI or Combinatory) $275 $451
Amplicon barcoding (AmpSeq) - Nextera indices. Full plate, 96 samples* $275 $451
rhAmpSeq (users provide primers + master mix) inquire inquire
rAmpSeq library preps (maize, sorghum, cassava). Full plate, 96 samples* $317 $520
* These numbers include one blank well