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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"> Environmental DNA metabarcoding universal primers targeting the hypervariable region of the 12S rRNA gene </div></div>

[STEPS]
?. 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.
?. For fish 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: ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT NNN NNN GTC GGT AAA ACT CGT GCC AGC MiFish-UmR: AGA CGT GTG CTC TTC CGA TCT NNN NNN CAT AGT GGG GTA TCT AAT CCC AGT TTG.
?. [First PCR reaction]
?. [First PCR reaction for fish amplification]
ABCD1ComponentsWorking conc.Final conc.1 reaction (µl)25xQ5
  Reaction Buffer5X1X53MiFish_F10 µM0,3 µM0,754MiFish_R10 µM0,3 µM0,755dNTPs&nbsp;2 mM0,2 mM2,56Q5 HF
  DNA polymerase2 U/µl0.02 U/µl0,257Template
  DNA58Nuclease-Free
  water  10,759Total25  ABC1STEPTEMP.TIME2Initial
  Denaturation98 C30 sec3 98 C20 sec430 cycles60 C30 sec5 72 C1 min6Final
  Extension72 C7 min7Hold6 C∞
ABCD1ComponentsWorking conc.Final conc.1 reaction (µl)25xQ5
  Reaction Buffer5X1X53MiFish_F10 µM0,3 µM0,754MiFish_R10 µM0,3 µM0,755dNTPs&nbsp;2 mM0,2 mM2,56Q5 HF
  DNA polymerase2 U/µl0.02 U/µl0,257Template
  DNA58Nuclease-Free
  water  10,759Total25
ABC1STEPTEMP.TIME2Initial
  Denaturation98 C30 sec3 98 C20 sec430 cycles60 C30 sec5 72 C1 min6Final
  Extension72 C7 min7Hold6 C∞
?. [First PCR reaction for amphibians amplification]
Check PCR products with Agarose gel electrophoresis (1%) - optional
?. [First PCR reaction for amphibians amplification]
Pool PCR duplicate samples together and perform purification with magnetic beads (Agencourt AMPure or similar)-
?. [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]-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTWe 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.
?. [Second PCR]
ABCD1								Components

													Working conc.

													Final conc.

													1 reaction (20
μl)

					2								5xQ5 Reaction
Buffer

													5X

													1X

					43								Forward index (i5,
illu-N501-N508)

													5μM

													0.25 μM

					14								Reverse index (i7,
illu-N701-N712)

													5μM

													0.25 μM

					15								dNTPs

													2mM

													200 μM

					26								Q5 HF DNA
polymerase

													2 U/μl

													0.02 U/μl

					0.27								Template from 1st
PCR

															28								Nuclease-Free
water

															9.89								∑

															20  ABC1STEPTEMP.TIME2Initial
  Denaturation98 C30 sec3 98 C10 sec415
  cycles66 C30 sec5 72 C30 sec6Final
  Extension72 C2 min7Hold6 C∞
ABCD1								Components

													Working conc.

													Final conc.

													1 reaction (20
μl)

					2								5xQ5 Reaction
Buffer

													5X

													1X

					43								Forward index (i5,
illu-N501-N508)

													5μM

													0.25 μM

					14								Reverse index (i7,
illu-N701-N712)

													5μM

													0.25 μM

					15								dNTPs

													2mM

													200 μM

					26								Q5 HF DNA
polymerase

													2 U/μl

													0.02 U/μl

					0.27								Template from 1st
PCR

															28								Nuclease-Free
water

															9.89								∑

															20
ABC1STEPTEMP.TIME2Initial
  Denaturation98 C30 sec3 98 C10 sec415
  cycles66 C30 sec5 72 C30 sec6Final
  Extension72 C2 min7Hold6 C∞
?. [Second PCR]
Check second PCR products with Agarose gel electrophoresis (1%)
?. [Second PCR]
Perform purification with magnetic beads (Agencourt AMPure)  https://research.fhcrc.org/content/dam/stripe/hahn/methods/mol_biol/Agencourt%20AMPure%20XP.pdf
?. [Second PCR]
Quantification of the concentration of second PCR product before pooling using PicoGreen assayhttp://tools.thermofisher.com/content/sfs/manuals/PicoGreen-dsDNA-protocol.pdfCalculate PCR samples concentration and volume before pooling
?. [Second PCR]
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.
?. [Second PCR]
Gel purify the pool and requantify with PicoGreen before submitting to sequencing facility.
?. [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