TITLE: Environmental DNA (eDNA) metabarcoding protocol for fish species
AUTHORS: Omneya Ahmed, Tomas Larsson, Mats Töpel, Alexander Eiler

[DESCRIPTION]

 Environmental DNA metabarcoding universal primers targeting the hypervariable region of the 12S rRNA gene 





[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.




[GUIDELINES]
Serial dilutions of mock community was  prepared as a positive control

[STEPS]
1. 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
2. 
Perform the first PCR (triplicates/duplicates of each sample) using Illumina adaptor attached primers that target the gene of your choice. 





2.1. 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.


SECTION: First PCR reaction
3. 
SECTION: First PCR reaction for fish amplification
3.1. 
        Components Working conc. Final conc. 1 reaction (µl)   5xQ5   Reaction Buffer 5X 1X 5   MiFish_F 10 µM 0,3 µM 0,75   MiFish_R 10 µM 0,3 µM 0,75   dNTPs  2 mM 0,2 mM 2,5   Q5 HF   DNA polymerase 2 U/µl 0.02 U/µl 0,25   Template   DNA     5   Nuclease-Free   water     10,75   Total     25    
  
        STEP TEMP. TIME   Initial   Denaturation 98oC 30 sec     98oC 20 sec   35 cycles 57oC 30 sec     72oC 1 min   Final   Extension 72oC 7 min   Hold 6oC ∞    
 
SECTION: PCR visualization
4. Check PCR products with Agarose gel electrophoresis (1%) - optional 




SECTION: PCR visualization
5. Pool PCR duplicate samples together and perform purification with magnetic beads (Agencourt AMPure or similar)




SECTION: Second PCR
6. 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. 
SECTION: Second PCR
6.1. 
 
 
 

 
 
          STEP TEMP. TIME   Initial   Denaturation 98oC 30 sec     98oC 10 sec   15   cycles 66oC 30 sec     72oC 30 sec   Final   Extension 72oC 2 min   Hold 6oC ∞    
 



SECTION: Second PCR
7. Check second PCR products with Agarose gel electrophoresis (1%) 





SECTION: Second PCR
8. Perform purification with magnetic beads (Agencourt AMPure)  
https://research.fhcrc.org/content/dam/stripe/hahn/methods/mol_biol/Agencourt%20AMPure%20XP.pdf




SECTION: Second PCR
9. 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 




SECTION: Second PCR
10. 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. 




SECTION: Second PCR
11. Gel purify the pool and requantify with PicoGreen before submitting to sequencing facility. 




SECTION: Sequencing
12. Sequencing was performed illumina paired end sequence strategy (150 bp). 
Analysis was carried out by DADA2 pipeline https://benjjneb.github.io/dada2/tutorial.html
2.2. 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.