Amplicon Library Preparation Guidelines

The following guidelines apply to those submitting DNA (gDNA, amplified) for library preparation via PCR. These guidelines are appropriate for libraries requiring preparation through PCR with target-specific primer sets (16s, 18s, ITS, custom) or amplified products requiring additional PCR to add Illumina indexes and remaining adapter (Index PCR). Listed below are guidelines specific to Illumina, Oxford Nanopore, and PacBio instruments.

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Illumina Amplicon Library Preparation (16s, 18s, ITS, custom)

DNA Sequencing facility will prepared amplified Illumina libraries for 16s rRNA, 18s rRNA, and ITS using

  1. Follow Guidelines for All Submissions.
  2. Volume should be 15-25 µl at a concentration of 2-50 ng/µl, as measured by fluorometric assay (e.g., Qubit). Measurements by spectrophotometer (e.g., NanoDrop) are not acceptable measures of DNA concentration. All DNA is quantified prior to library preparation and DNA Sequencing facility staff will contact you if concentration is <1.67 ng/µl (12.5 ng). Library preparation can still be successful with low starting mass (hundreds of picograms), provided the DNA is enriched for the target of interest and genome copies in that starting mass are sufficient to match the complexity of the sample.
  3. A significant portion of the DNA, or the predominant DNA in the sample provided, should contain the target of interest. DNA consisting of a majority of untargeted DNA (e.g., host DNA in microbiome study) may lead to inefficient or failed library preparation.
  4. Custom amplicon projects must be discussed with DNA Sequencing facility prior to submission.

Illumina Index PCR

  1. Follow Guidelines for All Submissions.
  2. Follow the Fusion Primer Design to ensure the primers you use for target-specific amplification contain the correct sequence for subsequent PCR by the DNA Sequencing facility. If this is your first time, it is recommended you discuss with the facility staff prior to submission.
  3. Volume should be 20-50 µl of cleaned PCR product. Purification by column-based or SPRI beads is acceptable. You must provide advanced notice and receive approval to submit unpurified products. DNA Sequencing facility can provide purification of PCR products at an additional cost.
  4. There are no explicit concentration requirements.
  5. The DNA Sequencing facility doesn’t perform any upfront QC of the submitted amplified DNA. Ensure the following before you submit.
    • Optimize your reaction if you see primer dimer, spurious products, over-amplification/heteroduplex formation, or weak enrichment. The DNA Sequencing facility performs 8 additional cycles of PCR using 2.5 µl of the provided amplified DNA, which is sufficient for producing good libraries, provided successful initial amplification.
    • Check for effective target amplification. Verify the presence of the intended product and absence of primer dimers or spurious products by visualization on an agarose gel. Any and all products with the overhang adapter sequence present will be subsequently amplified. Smaller products are preferentially amplified.
    • If you are having trouble with your initial amplification, contact the DNA Sequencing facility before you submit.

Full-length 16s Amplicon Library Preparation (Oxford Nanopore and PacBio)

  1. Follow Guidelines for All Submissions.
  2. Requires double stranded DNA (RNase-treatment preferred).
  3. Volume should be 10-100 µl with a concentration ≥1 ng/µl.
  4. Elution buffers TE, Tris HCl, and water are acceptable. EDTA in elution buffers may inhibit enzymes used in library preparation. Provide an aliquot of elution buffer for blank measurement for our NanoDrop measurement.
  5. Verifying purity, size, and mass prior to submission is encouraged but not required. UWBC DNA Sequencing facility performs QC on all input DNA using NanoDrop, Femto Pulse, and Qubit (High Molecular Weight DNA QC) to check for:
    • A260/A280 of ~1.8 and A260/A230 of 2.0-2.2 as measured by spectrophotometer (e.g., NanoDrop).
    • Average fragment size >30 kb, as measured by pulsed-field gel electrophoresis (e.g., Femto Pulse)
    • Input mass of 10 ng to 5 µg (see application specifics below), as measured by fluorometric assay (e.g., Qubit)