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Cornell University

Indexing reactions and Amplicon sequencing

Indexing for Illumina library preps

We offer the following Nextera indices:

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

Amplicon sequencing

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

With AmpSeq and rhAmpSeq, a large set of primers with known sequences are used to create amplicons throughout the genome.

With rAmpSeq, the experiment uses only a few pairs of primers. These primers anchored in repetitive DNA (i.e., transposable elements), and therefore are in thousand copies in the genome.

These approaches are useful if you want a cost-effective sequencing at a specific set of loci in the genome. The number of loci amplified (amplicons) typically ranges between 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, and then sequenced.

Amplicon barcoding (AmpSeq)

This library prep type is the most cost-effective approach when:

  • you have a limited set of primers (typically in the 100s, and up to 500)
  • to test the approach on a small scale before moving the project to rhAmpSeq with a larger number of primers.

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.

rhAmpSeq

This library prep type allows high multiplexing without having to worry about primer-dimers. It allows for a high number of primers in the PCR mix (over 2000) and samples (over 50) that can be sequenced at the same time. 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.

rAmpSeq

This library prep type allows to identify thousands of polymorphisms in the low copy intervening sequences of repetitive elements. The difference with AmpSeq or rhAmpSeq is that rAmpSeq doesn't amplify known sequences, but ALL the sequences in the genome that are flanking a known sequence. Because up to 3,072 samples can be multiplexed in one sequencing lane, 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
Indexing for Illumina library preps (UDI or Combinatory) $275 $541
Amplicon barcoding (AmpSeq) - Nextera indices. Full plate, 96 samples* $275 $541
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