Feb 05, 2020
Version 1
Environmental DNA (eDNA) metabarcoding protocol for amphibians species V.1
- Alexander Eiler1,
- Mats Töpel2,
- Tomas Larsson1,
- Johan Andersson3
- 1eDNA solutions AB;
- 2eDNA solutions AB;
- 3Water Circle AB
- eDNAsolutionsTech. support phone: +0700264843 email: omneya@ednasolutions.se
Protocol Citation: Alexander Eiler, Mats Töpel, Tomas Larsson, Johan Andersson 2020. Environmental DNA (eDNA) metabarcoding protocol for amphibians species. protocols.io https://dx.doi.org/10.17504/protocols.io.973h9qn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 06, 2019
Last Modified: February 05, 2020
Protocol Integer ID: 30683
Abstract
Environmental DNA metabarcoding universal primers targeting the hypervariable region of the 12S rRNA gene
Attachments
Guidelines
Serial dilutions of mock community was prepared as a positive control
Materials
MATERIALS
Agencourt Ampure XPBeckman CoulterCatalog #A63AA0
UltraPure™ DNase/RNase-Free Distilled WaterThermo Fisher ScientificCatalog #10977015
10 mM dNTPsLife TechnologiesCatalog #10297-018
Q5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L
Safety warnings
The 1st part of the protocol is performed in the pre-PCR room.
The 2nd part in the post-PCR room.
Never bring back PCR products to the pre-PCR room.
Always add a negative control samples in each PCR run
Before start
Laboratory work space and equipment were sterilized by UV-light and DNase solution and 70% ethanol. Filter pipet tips were used in all steps of the laboratory work.
For DNA extraction use Qiagen DNeasy power water sterivex kit.
The quality of the extracted DNA can be estimated using Nanodrop.
Qiagen DNeasy power water sterivex kit: https://www.qiagen.com/se/resources/resourcedetail?id=c5fe7d5f-070a-4ebe-ac04-4bbf05a13e91&lang=en
Perform the first PCR (triplicates/duplicates of each sample) using Illumina adaptor attached primers that target the gene of your choice.
For Amphibians
Group specific mitochondrial 12S primers and human blocking primers (https://www.ncbi.nlm.nih.gov/pubmed/26479867) were used
batra_F: 5′-ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT NNN NNN ACA CCG CCC GTC ACC CT-3′ and
batra_R: 5′-AGA CGT GTG CTC TTC CGA TCT NNN NNN GTA YAC TTA CCA TGT TAC GAC TT-3′.
Human blocking primer: batra_blk: TCACCCTCCTCAAGTATACTTCAAAGGCA-SPC3I
which was used to bind to human DNA and prevent its amplification.
Amplification strategy
Amplification strategy
Paired end sequencing on the Miseq platfrom required two steps of PCR
First PCR step
First PCR step
| Components | Working conc. | Final conc. | 1 reaction (µl) | |
| 5x Q5 Reaction Buffer | 5X | 1X | 5 | |
| batra_F | 10 µM | 0,2 µM | 0,5 | |
| batra_R | 10 µM | 0,2 µM | 0,5 | |
| dNTPs | 2 mM | 0,2 mM | 2,5 | |
| batra_blk | 50uM | 4uM | 2 | |
| Q5 HF DNA polymerase | 2 U/µl | 0.02 U/µl | 0,25 | |
| Template DNA | 5 | |||
| Nuclease-Free water | 9,25 | |||
| ∑ | 25 |
For environmental samples add 5 µl of template DNA and for mock community samples add 1 µl DNA
| STEP | TEMP. | TIME | |
| Initial Denaturation | 98 C | 30 sec | |
| 98 C | 20 sec | ||
| 35 cycles | 57 C | 30 sec | |
| 72 C | 1 min | ||
| Final Extension | 72 C | 7 min | |
| Hold | 6 C | ∞ |
Check PCR products with Agarose gel electrophoresis (1%).
Pool PCR triplicated or duplicate samples together and perform purification with magnetic beads (Agencourt AMPure) https://research.fhcrc.org/content/dam/stripe/hahn/methods/mol_biol/Agencourt%20AMPure%20XP.pdf
Second PCR
Second PCR
A second PCR is conducted for attaching standard illumina handles and index primers
Multiplex_fwd
AATGATACGGCGACCACCGAGA{TCTACAC}-[i5 index] ACACTCTTTCCCTACACGACG
Multiplex_rev
CAAGCAGAAGACGGCATACGAGAT-[i7 index]-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
We have in total 20 different forward index/barcode primers and 20 different reverse index/barcode primers.
By combining both primers (20X20), it is possible to generate 400 tags in one final pool for sequencing.
| Components | Working conc. | Final conc. | 1 reaction (20 μl) | |
| 5xQ5 Reaction Buffer | 5X | 1X | 4 | |
| Forward index (i5, illu-N501-N508) | 5μM | 0.25 μM | 1 | |
| Reverse index (i7, illu-N701-N712) | 5μM | 0.25 μM | 1 | |
| dNTPs | 2mM | 200 μM | 2 | |
| Q5 HF DNA polymerase | 2 U/μl | 0.02 U/μl | 0.2 | |
| Template from 1st PCR | 2 | |||
| Nuclease-Free water | 9.8 | |||
| ∑ | 20 |
| STEP | TEMP. | TIME | |
| Initial Denaturation | 98 C | 30 sec | |
| 98 C | 10 sec | ||
| 15 cycles | 66 C | 30 sec | |
| 72 C | 30 sec | ||
| Final Extension | 72 C | 2 min | |
| Hold | 6 C | ∞ |
Check second PCR products with Agarose gel electrophoresis (1%)
Perform purification with magnetic beads (Agencourt AMPure).
Use a PicoGreen assay to quantify the concentration of the second PCR product before pooling.
Pool the PCR samples in equal DNA amount (ng) or for unequal length amplicons, in equal molecule amount (mol). This results in one tube including a mix of all the samples.
Calculate the volume of each sample to be pooled (DNA amount mixing) as follows:
- Use the lowest concentration sample to define the minimum amount of DNA (ng) that you have available from a single sample:
- DNA concentration (ng/μL) of the lowest concentration sample multiplied with its volume (μL). This will be your target DNA amount for each sample.
- Calculate how many μLs of each sample you need to achieve the target DNA amount: divide the target DNA amount with the concentration of each sample.
- Pipette into one tube the calculated volume of each sample.
- Aim to use the same pipette for all samples (dilute or pipette multiple times) to avoid pipette calibration errors.
Check the pooled samples by agarose gel electrophoresis (1%) to make sure only one band is displayed in the gel.
Gel purify the pool and re-quantify with PicoGreen before submitting to sequencing facility.
Sequencing
Sequencing
Software
Illumina MiDeq Next Generation sequencer
NAME
Sequencing was performed on the Illumina MiSeq platform that generated paired end sequence that where 150 bp in length.
Expected result