Metabarcoding Fecal Swabs or Stomach Contents for Fish and Crustaceans using 2-PCR protocol and Illumina MiSeq

Order metabarcoding primers with diversity spacers and Illumina overhang sequences (Illumina, 2013): (Miya et al., 2015):

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNGTCGGTAAAACTCGTGCCAGC

(Miya et al., 2015): GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNCATAGTGGGGTATCTAATCCCAGTTTG

Berry et al., 2017): TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNGGGACGATAAGACCCTATA

(Berry et al., 2017): GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNATTACGCTGTTATCCCTAAAG

We got ours from https://www.idtdna.com/ as custom oligos at 25nm scale, with standard desalting.

Reconstitute primers to 100micromolar (µM) stock solutions by adding 40µL of

Make 5micromolar (µM) working solutions of each primer by adding 95µL of and 5µL of primer stock solution for each 100µL of primer that you intend to use within the next week or so.

Determine which sample will go into each well. This should be the same for each primer set and each replicate. Include at least one extraction control (you can combine aliquots of the extraction controls from each round of DNA extraction into one tube, and use that as your single extraction control), and include a PCR negative control for each plate of PCR. See example below of 21 samples, a field negative sample, a combined extraction control, and a PCR negative.

Do not mix sample types between invasively sampled methods (fecal swabs, or stomach contents) and non-invasively sampled methods (eDNA from water or sediment) in the same PCR procedure. And don't plan to sequence both types in the same sequencing run with the combinatorial indexing scheme used here. The potential for contamination of the lower quantity eDNA samples by the higher quantity fDNA samples is too high.

Add 24µL of your MiFish metabarcoding mastermix to each well of

Add 1µL DNA extracted from stomach contents or fecal swabs.

Mix and stir together with pipette tip, swirling to make sure the liquid is in the bottom, and bringing any bubbles to the surface of each reaction.

Cap each row of reaction tightly before beginning any other PCR reaction in the same room.

95°C for 0h 1m 0s

35 cycles of:

95°C for 0h 0m 30s

66°C for0h 1m 0s

followed by:

68°C for0h 1m 0s

Hold at 4°C

Make your Crustacean_16S Mastermix:

For each PCR replicate of each sample you intend to process (+10% overage), mix:

2µL 5micromolar (µM)

2µL 5micromolar (µM)

20µL

For a full plate of 96 reactions, multiply 105.6*the per-sample volumes in the recipe to make the mastermix.

Add 24µL of your Crustacean_16S metabarcoding mastermix to each well of

Add 1µL DNA extract

Mix and stir together with pipette tip, swirling to make sure the liquid is in the bottom, and bringing any bubbles to the surface of each reaction.

Cap each row of reaction tightly before beginning any other PCR reaction in the same room.

95°C for 0h 1m 0s

35 cycles of:

95°C for 0h 0m 30s

50°C for 0h 1m 0s

68°C for 0h 0m 30s

followed by:

68°C for 0h 1m 0s

then hold at 4°C

Make a 1.7% to 2% agarose gel and run a representative sample of reactions on it to make sure the PCRs worked, producing bands in the 250-300bp range. Check some PCR negatives to see that they don't have bands. Be very careful opening the PCR plate wells at this point to avoid cross-contamination.

Get AmpureXP beads out of the refrigerator, and bring to room temp, swirl to mix occasionally, or use a rocking platform.

Make fresh 80% EtOH so that you will have at least 200µL of EtOH per well of the combined plate.

Get 2 sterile DNAase/RNAse free 96-well PCR plates out of their packaging and immediately cover with adhesive foil.

UV clean the plates for 0h 15m 0s

One plate will be for the bead-cleanup steps, and the other will be for the final, cleaned reactions.

in the bead-cleanup plate, do the following steps for one 8-sample row of the plate at a time, pulling back the foil cover for each row after the previous one has been completed.

pipette mix 10µL combined PCR product with 15µL .

Incubate 0h 5m 0s at room temperature.

After the 0h 5m 0s incubation, place 96-well plate on a

Equipment

Value	Label
96-well Magnetic Rack Separator	NAME
Magnetic Rack Separator	TYPE
Sergi Lab Supplies	BRAND
B08134P9RT	SKU

for0h 2m 0s or until liquid is clear.

remove and discard liquid from the row, being careful not to touch the beads with the pipette or to let the beads dry for more than 30 seconds.

Add 100µL of 80%EtOH to each well of beads. Incubate at Room temperature for 0h 0m 30s

Remove the EtOH, then immediately add another 100µL of 80% EtOH to the wells, incubate for 0h 0m 30s Room temperature .

Remove ALL EtOH, and let the row of beads dry just enough to lose some shine but not enough to start cracking. This should be approximately 0h 0m 30s to 0h 1m 0s.

Remove the plate with cleaned beads from the magnetic plate, and add 30µL of

to each well of beads, pipette mixing each well thoroughly. Incubate 0h 5m 0s at Room temperature

Place back on the magnetic rack for 0h 1m 0s until liquid is clear again.

Roll back the foil on the final cleaned reactions plate for the appropriate row. Remove the 30µL clear eluate from the bead-cleanup plate, and place in the appropriate wells of the final cleaned reactions plate. Immediately cover this cleaned PCR product with either 8-strip caps.

uncover the next row of samples for cleaning and

Create an indexing plate map and make sure your chosen indexes (iNext indexes) are color balanced if you aren't doing full 96-well plates at one time.

Indexing PCR Mastermix Recipe:

6µL

2.1µL

per sample.

Multiply by number of wells *10% as explained above, to create master mix.

In a new, clean 96-well plate (UV before use if possible and prepare in a pre-PCR space):

Add 8.1µL Indexing Mastermix to each well that will be used and add 0.7µL of the 5micromolar (µM) iNext forward indexed primer for each horizontal row of the plate (8 letters), and 0.7µL 5micromolar (µM) of the iNext reverse indexed primer for each vertical column of the plate (12 numbers) according to the indexing plate map.

Take the prepared indexing reactions to the post-PCR space to add the cleaned PCR product.

In the post-PCR area, add 2.5uL of cleaned PCR 1 product to their associated wells from the indexing plate map.

95°C 0h 3m 0s

8 cycles of:

98°C 0h 0m 20s

65°C 0h 0m 15s

72°C 0h 1m 0s

then hold 4°C

Optional: visualize PCR products in a 1.7-2% gel. Bands should be around 350-400bp.

Combine 10uL of up to 70 indexed samples (library) into a single 1.5mL tube. If there are more than 70 samples, you will need another tube.

Multiply the volume of the pooled libraries in each tube by 0.9 to get the volume of Ampure XP beads needed to clean up the reactions.

In the 1.5mL tube of pooled libraries, add 0.9x volume of Ampure XP beads and pipette mix well. incubate Room temperature for 0h 10m 0s

Make enough fresh 80% EtOH to have 2x the total volume of the beads+library pool plus a bit extra.

Place 1.5mL tube into a magnetic rack

Equipment

Value	Label
Magnetic Rack for for 1.5 mL Tubes	NAME
Magnetic Rack for DNA, RNA Purification; for 1.5 mL centrifuge Tubes	TYPE
Sergi Lab Supplies	BRAND
B0BZWXZMZ2	SKU

and incubate Room temperature for 0h 5m 0s

Discard liquid and add an equal or greater volume of 80% EtOH. Incubate Room temperature for 0h 1m 0s

Repeat the ethanol wash a second time , then after the second 80% EtOH wash, remove all EtOH and dry the beads slightly (just until no longer wet-looking but not cracking either).

Resuspend beads with 100µL by pipette mixing thoroughly. Incubate Room temperature``0h 10m 0s

Place 1.5 mL tube back on magnet rack and wait until liquid is clear, approximately 0h 2m 0s

remove 100uL of the clear eluate from the tube with beads while on the magnet and place in a new 1.5mL tube.

Quantify with Qubit Broad range and visualize in a gel, then send for sequencing on a lane of MiSeq.