Amplicon sequencing

PCR products (amplicons) can be sequenced on Illumina instruments after Illumina adapter sequences are appended to the primary PCR target sequences. This is typically achieved through two rounds of PCR:

• The first locus-specific PCR reaction uses ‘tailed’ primers containing partial Illumina adapter sequences at the 5’ ends. 
• The second indexing PCR reaction extends the adapter sequences and adds a sample barcode (index) to generate a complete Illumina library.

We support amplicon sequencing by providing services for the second ‘indexing’ PCR reaction, as well as Illumina sequencing runs. For primary PCR products ‘tailed’ with TruSeq or Nextera adapter sequences, we offer up to 192 UDIs (unique dual indices) or over 9,000 possible combinatorial dual barcodes. UDIs are highly recommended for patterned flowcells (NextSeq2000, NovaSeq6000, and NovaSeqX instruments) to minimize index hopping.

Indexing reactions

To be compatible with the indexing reaction, primary PCR products must contain Illumina adapter sequences at each end. PCR primers must be designed with the added “tails,” using either the TruSeq or Nextera adaptor sequences below. 

TruSeq

FOR: 5’ ACACTCTTTCCCTACACGACGCTCTTCCGATCT-[gene specific sequence] 3’
REV: 5’ GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-[gene specific sequence] 3’

Nextera

FOR: 5′ TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-[gene specific sequence] 3′
REV: 5′ GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-[gene specific sequence] 3′

The ‘tailed’ primers can also be phased to improve sequence complexity generated during Illumina sequencing. To use the phased approach, order a panel of tailed primers that incorporate additional bases between the adapter and gene specific sequence (at the ‘-‘ character in the primer sequences shown above) to offset the start of the Illumina read(s), as described here.

We will run the indexing PCR in a high throughput mode, which does not include checking each sample individually. This keeps the cost lower but can also lead to more variation in sequencing depth across samples. Customized pooling is also possible; additional fees will apply. 

Our indexing service includes the following steps:

• Quantify a subset of the submitted PCR products (from the first PCR reaction). Optionally, we can quantify each sample individually to confirm DNA is present (separate fee).
• If needed, dilute to average 2-5ng/ul (separate fee).
• Perform indexing PCR reaction, using UDIs or combinatorial dual barcoding.
• Pool an equal volume of each PCR product (1 pool per plate).
• Clean up and quantify the final pool.

Illumina Sequencing is required to be combined with the indexing service, and will include the size QC check on the final pool. Multiple plate-pools can be combined before sequencing (indexing barcodes must be unique). 

We are available to consult on your project! Please use the ‘Ask a question’ button at the top of the page to send us an email, or sign up for office hours with an expert.

UDI Primers

We also offer UDI primer stocks for your own use, compatible with both TruSeq and Nextera adapters - contact us for more information. 

Highly multiplexed amplicons

 We have experience with several approaches using multiplexed PCR to generate high complexity, targeted amplicon libraries. These custom projects require the development and testing of large sets of PCR primers and typically are used for projects sequencing the same panel of target loci for thousands of samples. Please contact us to inquire about how we can support custom projects using AmpSeq, rhAmpSeq, or rAmpSeq methods.