Mar 10, 2022

Public workspaceEnvironmental DNA (eDNA) metabarcoding protocol for fish species

DOI
dx.doi.org/10.17504/protocols.io.b4awqsfe
  • Omneya Ahmed1,
  • Tomas Larsson1,
  • Mats Töpel1,
  • Alexander Eiler1
  • 1eDNA solutions AB
Omneya Osman
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DOI: dx.doi.org/10.17504/protocols.io.b4awqsfe
Protocol Citation: Omneya Ahmed, Tomas Larsson, Mats Töpel, Alexander Eiler 2022. Environmental DNA (eDNA) metabarcoding protocol for fish species. protocols.io https://dx.doi.org/10.17504/protocols.io.b4awqsfe
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: January 25, 2022
Last Modified: March 10, 2022
Protocol Integer ID: 57398
Abstract

Environmental DNA metabarcoding universal primers targeting the hypervariable region of the 12S rRNA gene
Guidelines
Serial dilutions of mock community was prepared as a positive control
Materials
MATERIALS
ReagentAgencourt Ampure XPBeckman CoulterCatalog #A63AA0
ReagentUltraPure™ DNase/RNase-Free Distilled WaterThermo Fisher ScientificCatalog #10977015
Reagent10 mM dNTPsLife TechnologiesCatalog #10297-018
ReagentQ5 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.
DNA extraction can be performed using Qiagen DNeasy power water sterivex kit. The quality of the extracted DNA was 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.

MiFish primers
A modified version of the universal primers targeting the hypervariable region of the 12S rRNA gene (163-185 bp) (Miya et al., 2015) was used. The sequence of the primer set is
MiFish-UF: 5'ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT NNN NNN GTC GGT AAA ACT CGT GCC AGC
MiFish-UmR: 5'AGA CGT GTG CTC TTC CGA TCT NNN NNN CAT AGT GGG GTA TCT AAT CCC AGT TTG.


Mock community
DNA extract of 10 fish species were pooled and used as a positive control
-The fish species are Clupea harengus, Glypthocephalus cynoglossus, Scomber scombus, Thunnus alalunga, Pleuroneates platessa, Pollachius virens, Salmo salar, Gadus morhua, Reinhardtius hippoglossoides and Melanogrammus aeglefinus.


First PCR reaction
First PCR reaction

First PCR reaction for fish amplification
First PCR reaction for fish amplification

ComponentsWorking conc.Final conc.1 reaction (µl)
5xQ5 Reaction Buffer5X1X5
MiFish_F10 µM0,3 µM0,75
MiFish_R10 µM0,3 µM0,75
dNTPs 2 mM0,2 mM2,5
Q5 HF DNA polymerase2 U/µl0.02 U/µl0,25
Template DNA5
Nuclease-Free water 10,75
Total25
For environmental sample add 5 µl and for mock community add 1 µl as a template.
ABC
StepTempTime
Initial denaturation98 C30 sec
98 C20 sec
30 cycles60 C30 sec
72 C1 min
Final extension72 C7 min
Hold4 C
PCR visualization
PCR visualization
Check PCR products with Agarose gel electrophoresis (1%) - optional
Pool PCR duplicate samples together and perform purification with magnetic beads (Agencourt AMPure or similar)
1h 30m
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

STEPTEMP.TIME
Initial Denaturation98 C30 sec
98 C10 sec
15 cycles66 C30 sec
72 C30 sec
Final Extension72 C2 min
Hold6 C∞
Check second PCR products with Agarose gel electrophoresis (1%)
Quantification of the concentration of second PCR product before pooling using PicoGreen assay
http://tools.thermofisher.com/content/sfs/manuals/PicoGreen-dsDNA-protocol.pdf

Calculate PCR samples concentration and volume before pooling
Pool the PCR samples in equal DNA amount (ng) or for unequal length amplicons, in equal molecule amount (mol). You will get one tube including a mix of all the samples.

To calculate the volume of each sample to be pooled (DNA amount mixing):
- 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.




Gel purify the pool and requantify with PicoGreen before submitting to sequencing facility.
Sequencing
Sequencing
Sequencing was performed illumina paired end sequence strategy (150 bp).
Analysis was carried out by DADA2 pipeline https://benjjneb.github.io/dada2/tutorial.html