Amplicon sequencing

Planning your amplicon project

Clean, target-specific PCR products are extremely important for obtaining high-quality sequence data. Non-specific products can represent a substantial percentage of the sequencing reads if they are not removed. Please review the Best Practices section below for getting best results from PacBio sequencing.

Multiplexing of amplicons for sequencing on Sequel II instrument

Please note that we only recommend pooling amplicons of similar size, within +/- 15% of the mean size. 

Amplicons can be barcoded either during PCR (done by the customer) or by ligation of barcoded hair-pin adapters (done by NSC).

1. Options for multiplexing by PCR:

  • Barcoded target-specific primers are tailed with PacBio 16 bp barcodes to produce symmetrically or asymmetrically barcoded samples using a one-step PCR method.
  • Barcoded Universal primers are used for barcoding in a two-step PCR: first PCR is carried out using Universal Sequence-tagged target-specific primers. The second PCR uses barcoded Universal primers and adds 16 bp barcodes to both ends of the amplicon. Protocol can be found here.

After barcoding by PCR, amplicons should be cleaned and pooled equimolarly. NSC will then prepare a single PacBio library before sequencing. DNA amount needed for library prep depends size of the amplicon: 300 ng (<1.5 kb), 1 µg (1.5-3 kb) and 2 µg (3-10 kb) per 8M SMRT cell. However, please send us twice of the amount indicated above. 

2. Barcoding using barcoded hairpin adapters will be performed at NSC. Currently PacBio provides 96 barcoded adapters. Please deliver your amplicons in plate, normalized according to fragment length and DNA amount needed: 

Length DNA amount Delivery
250-1000 bp 1 µg 20 ng/µl in 50 µl
1-3 kb: 2 µg 40 ng/µl in 50 µl
3-10 kb: 4 µg 50 ng/µl in 80 µl
15 kb: 6 µg 75 ng/µl in 80 µl

Best practices for generating high-quality PCR products for PacBio sequencing

  • Use the highest-fidelity polymerase compatible with your PCR amplification system
  • Work with high-quality DNA in a clean environment
  • Use desalted or HPLC-purified oligo primers
  • Use long enough PCR extension time to generate full length PCR products
  • Use the lowest number of cycles needed for obtaining adequate yields (ng) of PCR product
  • If non-specific products are present, optimize PCR conditions or perform bead-based size selection using AMPure PB or Pronex beads. 
Published Nov. 16, 2021 1:18 PM - Last modified May 10, 2022 10:37 AM