Welcome to the Methods for eDNA Researchers page! Here you’ll find links to up-to-date protocols for our standard CALeDNA DNA library preparation workflow. In the software link above, you will find our data analysis tools Anacapa: a toolkit to process multiple metabarcode eDNA libraries, and Ranacapa: a community ecology data exploration tool.

If you find anything from a typo to a phrase that is unclear or erroneous in any of the workflow, please let us know and we’ll fix it right away! Send your comment to uc.caledna@gmail.com.

Metabarcoding workflow:


1. Sample preparation.

  1. Soil or sediment: Prepare 3 50 mL Falcon tubes with 20% bleach, 70% ethanol, and 100% ethanol. Wash metal spatulas in that order and flame or glass bead sterilizers. Allow to cool. Thaw sample tubes on ice if frozen. There should be three biological replicate tubes per sample and you are making a pooled aliquot of them for DNA extraction. Vortex or shake the tubes. Using the sterilized spatulas, pool an aliquot of about 0.25-0.5mL of each together into a new epitube. Homogenize with the spatula or by vortexing. Label the tube with the kit (sample) ID. 

  2. Water: Archiving part of the filter? Do this: Use sterilized forceps and scalpel or razor blade to cut the filter on a sterile surface, and then reserve half of it as an archive, folding it in foil, and labeling the foil, placing the foil in a whirlpak bag that is labeled, and filing in the freezer for long term storage.

    1. Sterivex filter with Qiagen Blood and Tissue: Follow the Water Extraction Protocol for Sterivex filters with Blood and Tissue Kit. 

    2. Sterivex with Qiagen Powersoil Pro Kit. Use just the first part to excise the filter and buffer to go into the PowerSoil Pro Kit lysis tubes with glass beads. 

    3. Smith-Root filter with Qiagen Blood and Tissue: These are easier to excise. Cut the part of the filter you will process into small pieces before placing into lysis buffer for either Qiagen Blood and Tissue (as above) or Qiagen Powersoil Pro Kit.

2. DNA extraction

  1. Soil or sediment: Do not use more than 0.25g input from your pooled aliquot of sample in the DNA extraction. We typically use the Qiagen Powersoil Pro Kit. If the client wants the Qiagen DNEasy Plant Kit or the Qiagen Blood and Tissue Kit, carefully proceed with those protocols starting also with 0.25g input. Use at least a 12 hour incubation in buffer ATL and ProK prior to the Blood and Tissue Kit.

  2. Water extraction: Follow the Water Extraction Protocol.

3. First PCRs with three technical replicates

  1. Guide with detailed instructions for setting up 15uL reactions for each metabarcode in triplicate. Input DNA volume can vary; we typically use 2uL input per 15uL reaction. Do all PCR work in the PCR hood, with sterilized sleeves and gloves.

  2. Thermocycler settings for different primer sets.

  3. Primer sequences to order with Nextera Adapters. These are long and some will require the ultrapure specs when ordering.

  4. Check PCR products on a gel, repeat those that didn’t work. If no amplification, consider Qubiting the DNA and concentrating it or increasing the input DNA into your PCR. Rarely there are inhibitors. A qPCR inhibitor test should be done if 16S doesn’t amplify.

  5. Contamination in DNA extraction blanks and in PCR blanks happens from time to time. If it’s in 16S, 18S, or FITS, we proceed, as it’s usually a single microbe (usually someone’s face microbes like Malessia!) but if PITS (plants ITS2) CO1 or Vert12S are contaminated we repeat the PCR for the plate.

  6. Pool successful triple PCR technical replicates together into one well per sample/metabarcode and record the volume so you can do bean cleaning with precise ratios next.

4. Bead Cleaning PCR products

  1. You want to remove primer dimer and these can be up to 120 bp long. We use MagBio PCR Prep beads at 1.2:1 beads:DNA ratio. Here is the detailed Bead Cleaning Protocol.

5. Pooling and Indexing

  1. Qubit your pooled, bead cleaned PCR products. We use a Qubit protocol on a Varioskan Lux plate reader. We run a serial dilution of the standards for every batch of Qubit master mix solution we make (sometimes running many plates at a  time).

  2. Once you have your DNA concentrations for each marker’s PCR product per sample, you can calculate your quantities to pool together so all markers for a single sample are in a well ready to be vortexed and used in indexing. This is the Template for Amplicon Pooling to assist you with your calculations.

  3. With the Kapa HiFi Hotstart Ready Mix, you can index 10ng-100ng input DNA. You don’t need to make each PCR pooled product exactly the same input DNA going in to this reaction. You just need to make sure all values (say, you choose 5uL) are safely between 10ng and 100ng input DNA range.

  4. Prepare your indexing reaction


  1. Run a gel on some to confirm the index PCR worked.

6. Bead clean indexed PCR product

  1. Bead clean with a 1-1.2 beads:DNA ratio depending on the insert size. Usually 1:1 ratio is safe unless using a primer that only amplifies an 80bp or smaller insert.

7. Qubit, and pool final products together for sequencing

  1. Qubit on a plate reader again, usually using BR assay. 

  2. Pooling template for final library pools is here. Make sure there are no really low or high volumes to pipette. Adjust the ending volume and nM so everything is nicely pipettable.

8. SUBMIT FOR SEQUENCING

  1. We have been submitting libraries for sequencing to these fine facilities:

    UC Berkeley Vincent Coates Genome Sequencing Laboratory http://qb3.berkeley.edu/gsl/

    UCSC Paleogenomics Laboratory (best rate) https://pgl.soe.ucsc.edu/

    UCLA Technology Center for Genomics and Bioinformatics http://pathology.ucla.edu/tcgb

    UNH Hubbard Center for Genome Studies https://hcgs.unh.edu/