TITLE: Illumina metabarcoding protocol for the study of fungi in marine sediments
AUTHORS: Lluvia Vargas, Jennyfers Chong-Robles, Asunción Lago-Lestón, Meritxell Riquelme

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The study of fungi in marine sediments has received a growing attention in the recent years. Recent advances in metagenomics have allowed the discovery of a wide diversity of fungi in deep-sea environments, although standardized methods are needed to process the samples and to analyze the data. This protocol is a guidance to obtain good quality samples and subsequent ITS1 (Internal Transcribed Spacer 1) amplicons to be prepared for sequencing by Illumina platform. The collection of marine sediments is described, as well as the processing of the samples in laboratory; also, pertinent controls that should be included during all the processing steps to identify potential sources of contamination that could affect the sequencing and bias the interpretation of results are suggested.</div></div>

[STEPS]
?. [Preparation of syringes]
Grab a sterile syringe package and open it by the end, where the syringe tip is localized. Avoid moving the plunger.
Control #1: Keep a microcentrifuge tube containing 1 mL of sterile HPLC grade water open during the syringe preparation. The tube has to be close to the working area, where the syringes are manipulated.
This step should be done near a gas burner in order to preserve sterility.
?. [Preparation of syringes]
Attach a new scalpel blade to the handle and flame sterilize it by using a gas burner.
?. [Preparation of syringes]
Cut and remove the tip of the syringe carefully.
?. [Preparation of syringes]
Close a gain the syringe package and secure it with adhesive tape once the tip is cutoff.
?. [Preparation of syringes]
Sterilize again the scalpel before cutting a new tip.
?. [Sampling tubes preparation]
Open the tubes containing the sterile sucrose buffer near the gas burner.
Control #2: Keep an open microcentrifuge tube containing 1 mL of sterile HPLC grade water during the sucrose buffer preparation.Control #3: Once the sucrose buffer is prepared and sterilized, keep a microcentrifuge tube with 1 mL of sterile sucrose buffer.
?. [Sampling tubes preparation]
Pipette 5 mL of the sterile sucrose buffer (see recipe) into each of the 15 mL tubes.
Recipe - Sucrose buffer (100 mL)1. Mix the next reagents:  AB1  Reagent    Quantity  2  EDTA 0.5 M    4 mL  3  NaCl 5 M    8 mL  4  Tris-HCl 1 M    5 mL  5  Sucrose molecular grade    25.68 gr  2. Shake vigorously and add distilled water up to 80 mL.3. Adjust pH to 9.4. Fill to final volume (100 mL) with distilled water. 5. Near the gas burner, open the 0.2 µm pore filter, and place it in the syringe.6. Near the gas burner, filter the buffer through the 0.2 µm pore filter and collect the sterile solution in the 50 mL tubes.
?. [Sampling tubes preparation]
Cover the tube with a piece of parafilm, to prevent buffer leaks.
?. [Sampling tubes preparation]
All tubes should contain 25% w/v sucrose buffer (stored at room temperature) (Figure 1-next section).
?. [Sediment sampling]
Make a reference mark within the core to split it into two halves.
Note: Prior to sampling, each tube should be labeled with the station number, date, depth and sub-sampling number, and other information you need to identify the sample. Control #4: Keep a microcentrifuge tube containing 1 mL of sterile HPLC grade water open while the samples are prepared for storage.Control #5: Keep a microcentrifuge tube containing 1 mL of sterile sucrose buffer open while the samples are prepared for storage.
?. [Sediment sampling]
Open five pre-cut syringes (10 mL). Pull the plunger back.
?. [Sediment sampling]
Immerse syringes (cylinder) in one half of the core (Figure 1) to take the sample.
?. [Sediment sampling]
Remove the syringes and put the first 5 mL (corresponding to 10 cm depth) of sediment by pushing the plunger into 15 mL screw cap tubes, and the remaining 5 mL of sediment (corresponding to the first 5 cm) in another 15 mL screw cap tubes.
?. [Sediment sampling]
Shake the tubes to obtain a homogenous sample of buffer and sediment.
?. [Sediment sampling]
Seal the tubes with the screw cap and wrap them with parafilm.
?. [Sediment sampling]
Store tubes inside Ziploc® type bags at -20°C during the campaign and during the transport to the laboratory.
?. [Sample preparation]
Thaw the samples on ice.
?. [Sample preparation]
Centrifuge the samples at maximum velocity for 1 min at room temperature.
?. [Sample preparation]
Discard the supernatant. This step serves to discard the sucrose buffer used to preserve the samples. Store the samples at-80°C until processing.
Alternatively, the samples can be kept in the sucrose buffer at -20°C in the laboratory, until processing.
?. [ Lyophilization]
Place tubes containing the corresponding sample uncapped on ice and cover with parafilm the top of each tube.
?. [ Lyophilization]
Make a few holes onto the parafilm with a new (sterile) needle.
?. [ Lyophilization]
Put samples in the lyophilizer.
?. [ Lyophilization]
Check the samples with a sterile needle after 48 hours, before turning off the lyophilizer. The sample is ready if the sediment is totally dry. You can verify this by carefully shaking the sample to make sure the sediment is totally dry.
?. [ Lyophilization]
Cap the tubes and store the samples at -80°C until processing.
Samples can be processed without lyophilization
?. [DNA extraction]
Prepare the PCR chamber station: First, clean the entire chamber, including the glass walls. Lay all the material that is needed for the DNA extraction inside the chamber. Close the chamber and turn on the UV light for 15 min. Once the chamber is ready, without opening it, turn on the white light and the air circulator. Now the PCR chamber station is ready to be used.
Modified protocol from “Increasing DNA yield from PowerSoil kit - Info from rep Dr. Tetreault Carlson and from Tips and FAQ portion of MoBio website”.Important: We recommend not to process more than 5-6 samples per round of extraction; it is important to take care of the time that the sediment is in contact with the phenol chloroform reagent.Control #6: Keep a microcentrifuge tube containing 1 mL of sterile HPLC grade water open during the sample extraction.
?. [DNA extraction]
Weigh 0.15 grs of lyophilized sample and transfer it into a PowerBead tube. The weighing process must be done with care in order to prevent contamination and cross-contamination between samples.
If the samples are processed without prior lyophilization, take into account that the sediment is mixed with some water/sucrose buffer, and this would increase considerably the volume in the next steps of the DNA extraction procedure. To prevent an increase in volume, remove 100 µL of the PowerBead solution from the PowerBead tube.
?. [DNA extraction]
Return the remaining lyophilized samples to -80°C.
?. [DNA extraction]
Add 60 µL of C1 solution.
?. [DNA extraction]
Add 100 µL of phenol chloroform in the chemical fume hood.
?. [DNA extraction]
Incubate the samples 2 min at room temperature.
?. [DNA extraction]
Lay the samples horizontally in the vortex adapter and vortex for 30 secs (do notexceed this time).
?. [DNA extraction]
Centrifuge the samples for 1 min.
?. [DNA extraction]
Transfer the supernatant to a sterile 2 mL tube.
?. [DNA extraction]
Add 250 µL of C2 solution and vortex for 5 secs. Incubate the samples on ice for 5 min.
?. [DNA extraction]
Centrifuge the samples for 2 min.
?. [DNA extraction]
Transfer 600 µL of the supernatant to a sterile 2 mL tube.
?. [DNA extraction]
Add 200 µL of C3 solution and vortex for 5 secs. Incubate the samples on ice for 5 min.
?. [DNA extraction]
Centrifuge the samples for 3 min.
?. [DNA extraction]
Transfer up to 850 µL of the supernatant to a sterile 2 mL tube.
?. [DNA extraction]
Add 650 µL of C4 solution and 450 µL of absolute ethanol and vortex for 5 secs.
?. [DNA extraction]
Load up to 500 µL onto a column tube. Centrifuge for 30 secs and discard the flow through.
?. [DNA extraction]
Repeat step 18 until the complete sample has been processed.
?. [DNA extraction]
Centrifuge the samples for 2 min and carefully, transfer the column to a new sterile 2 mL tube.
?. [DNA extraction]
Add 600 µL of absolute ethanol and centrifuge for 30 secs. Discard the flow through.
?. [DNA extraction]
Centrifuge the samples for 3 min and discard the flow through.
?. [DNA extraction]
Add 650 µL of C5 solution and centrifuge for 30 secs. Discard the flow through.
?. [DNA extraction]
Centrifuge the samples for 4 min and carefully, transfer the column to a new sterile 2 mL tube.
?. [DNA extraction]
Add 50 µL of C6 solution and incubate at room temperature for 15 min.
?. [DNA extraction]
Centrifuge for 1.5 min.
?. [DNA extraction]
Add 50 µL of C6 solution and incubate at room temperature for 15 min.
?. [DNA extraction]
Centrifuge for 2 min.
?. [DNA extraction]
Discard the column.
?. [DNA extraction]
Visualize the extracted DNA by running an electrophoresis on a 1% agarose gel using 5 µL of DNA for each sample.
?. [Measuring the genomic DNA with a NanoDropTM Lite Spectrophotometer]
Once the NanoDrop is on, select the DNA option, and then the option dsDNA (double strain DNA).
?. [Measuring the genomic DNA with a NanoDropTM Lite Spectrophotometer]
Carefully lift the upper pedestal.
?. [Measuring the genomic DNA with a NanoDropTM Lite Spectrophotometer]
Clean the upper and lower pedestals by pipetting 2 µL of sterile HPLC grade water onto the bottom pedestal and, lower the upper pedestal and wait 2 min. Lift the pedestal and clean the upper and lower pedestal with Kimwipes.
?. [Measuring the genomic DNA with a NanoDropTM Lite Spectrophotometer]
Establish a blank by pipetting 1 µL of the blank solution onto the bottom pedestal, lower arm and press Blank.
?. [Measuring the genomic DNA with a NanoDropTM Lite Spectrophotometer]
When the measurement is complete, rinse the arm and wipe the blank solution from the upper and lower pedestal using Kimwipes.
?. [Measuring the genomic DNA with a NanoDropTM Lite Spectrophotometer]
Repeat step 5 to confirm the Blank. Now you are ready to measure the samples.
?. [Measuring the genomic DNA with a NanoDropTM Lite Spectrophotometer]
Measure the sample by pipetting 1 µL (mix the DNA with the pipette) of the sample in the lower pedestal, then, lower the upper pedestal and press Measure. Once ready, the equipment screen displays the results (Figure 2). Lift the pedestal and clean the upper and lower pedestal with Kimwipes and the instrument is ready to measure the next sample.  ABC1  <span style="font-size:10.0pt;font-family:
  &quot;Arial&quot;,sans-serif">Ratio    <span style="font-size:10.0pt;font-family:
  &quot;Arial&quot;,sans-serif">Value    <span style="font-size:10.0pt;font-family:
  &quot;Arial&quot;,sans-serif">Explanation  2<span style="font-size:10.0pt;font-family:
  &quot;Arial&quot;,sans-serif">A260/A280  1.8-2    Optimal purity DNA  3  1.6-1.8  DNA of acceptable purity4  &lt;1.6  Presence of aromatic  compounds5  &gt;2.1    RNA contamination
A total of two or three measures are necessary for each sample, in order to confirm the values of DNA concentration.
ABC1  <span style="font-size:10.0pt;font-family:
  &quot;Arial&quot;,sans-serif">Ratio    <span style="font-size:10.0pt;font-family:
  &quot;Arial&quot;,sans-serif">Value    <span style="font-size:10.0pt;font-family:
  &quot;Arial&quot;,sans-serif">Explanation  2<span style="font-size:10.0pt;font-family:
  &quot;Arial&quot;,sans-serif">A260/A280  1.8-2    Optimal purity DNA  3  1.6-1.8  DNA of acceptable purity4  &lt;1.6  Presence of aromatic  compounds5  &gt;2.1    RNA contamination
?. [Measuring the genomic DNA with a NanoDropTM Lite Spectrophotometer]
Once all samples are measured, repeat step 3.
?. [Measuring the genomic DNA with Qubit 3.0]
In a 2 mL microcentrifuge tube prepare the working solution by diluting the Qubit BR Reagent (Component A) 1:200 in the Qubit BR buffer (Component B). Prepare enough working solution for samples and standards. The final volume in each tube must be 200 µL. Each standard tube requires 190 µL of the working solution, and each sample requires anywhere from 180-199 µL. 3 µL of each sample is enough for the DNA measurement.
All steps are conducted at room temperature as recommended in the Qubit™ dsDNA HS Assay Kit user guide. Be careful to grab the tubes by the lid and try not to touch the rest of the tube, to avoid leaving marks on them.
?. [Measuring the genomic DNA with Qubit 3.0]
Prepare the two standards by adding 10 µL of the standard to 190 µL of working solution.Standard 1: 0 ng/µLStandard 2: 10 ng/µL
?. [Measuring the genomic DNA with Qubit 3.0]
Prepare the samples by adding 3 µL of DNA to 197 µL of working solution.
?. [Measuring the genomic DNA with Qubit 3.0]
Mix the samples and standards by vortexing (2-3 secs) and apply spin just for a few secs.
?. [Measuring the genomic DNA with Qubit 3.0]
Allow all tubes to incubate at room temperature for two min. Proceed with themeasurement of the DNA:- Connect the Qubit 3.0.- On the Home screen of the Qubit® 3.0 Fluorometer, press DNA, then select dsDNA High Sensitivity as the assay type. The “Read standards” screen is displayed. Press Read Standards to proceed.- Insert the tube containing Standard 1 into the sample chamber, close the lid, then press Read standard. When the reading is complete, remove Standard 1.- Insert the tube containing Standard 2 into the sample chamber, close the lid, then press Read standard. When the reading is complete, remove Standard 2.- The instrument displays the results on the Read standard screen.- Press Run samples.- On the assay screen, select the sample volume and units:Press the + or – buttons on the wheel to select the sample volume added to the assay tube (3 μL).From the dropdown menu, select the units for the output sample concentration (ng/µL).- Insert a sample tube into the sample chamber, close the lid, then press Read tube. When the reading is complete (~3 secs), remove the sample tube. The instrument displays the results on the assay screen. The top value (in large font) is the concentration of the original sample. The bottom value is the dilution concentration.
?. [Fungal PCR and library preparation]
The PCR is aimed to amplify the ITS1 region of rDNA (Figure 2-A). The primers used are ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′; Gardes and Bruns, 1993) and ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′; White et al. 1990).A two-PCR strategy is followed to obtain the amplicon libraries for Illumina sequencing. The first PCR includes the ITS1F and ITS2 primers attached to a Sequencing primer (Figure 3-B), and the second PCR is designed for the attachment of the Illumina adapter and barcode (identification sequence of each sample) (Figure 3-C).Controls 1 to 6 should be processed as the samples, and they should be included in all PCR runs. Besides these controls, the PCR must include an additional negative control, using as template sterile HPLC grade H2O, and a positive control; for instance, a mock community: a mix of genomic DNA extracted from different fungal strains.
?. [PCR1: Touchdown PCR for the ITS1 region amplification]
Prepare the PCR chamber station: First, clean the entire chamber, including the glass walls. Lay all the material that is needed to prepare the DNA and prepare the PCR reactions inside the chamber. Close the chamber and turn on the UV light for 15 min. Once the chamber is ready, without opening it, turn on the white light and the air circulator. Now the PCR chamber station is ready to be used.
Label all the tubes necessary for all the process.
?. [PCR1: Touchdown PCR for the ITS1 region amplification]
Keep the samples genomic DNA on ice until processing, and adjust the DNA concentration to 2 ng/µL.
?. [PCR1: Touchdown PCR for the ITS1 region amplification]
Prepare a master mix by multiplying the volume of each PCR reagent (Table 2) times the number of samples to be processed and add an extra 10% to account for pipetting errors. The Touchdown PCR program is indicated in Figure 3-B.
?. [PCR1: Touchdown PCR for the ITS1 region amplification]
Run 5µL of PCR product on a 2% agarose gel electrophoresis to check the correct amplification of the desired size fragment (>300 bp).  ABC1  Reagent    Concentration  50 µL final volume - 1 reaction (µL)2  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">HPLC grade water    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">----    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">17.7  3  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">BSA    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">10 mg/ µL    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">4  4  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">Buffer    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">5x    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">10  5  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">MgCl2    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">25 mM    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">6  6  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">dNTP mix    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">10 µM    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">4  7  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">Forward primer    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">10 µM    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">1.5  8  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">Reverse primer    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">10 µM    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">1.5  9  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">Polymerase    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">5 U    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">0.3  10  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">Genomic DNA    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">2 ng/ µL    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">5
ABC1  Reagent    Concentration  50 µL final volume - 1 reaction (µL)2  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">HPLC grade water    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">----    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">17.7  3  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">BSA    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">10 mg/ µL    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">4  4  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">Buffer    <span style="font-size:10.0pt;
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  font-family:&quot;Arial&quot;,sans-serif">10  5  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">MgCl2    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">25 mM    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">6  6  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">dNTP mix    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">10 µM    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">4  7  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">Forward primer    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">10 µM    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">1.5  8  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">Reverse primer    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">10 µM    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">1.5  9  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">Polymerase    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">5 U    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">0.3  10  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">Genomic DNA    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">2 ng/ µL    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">5
?. [Purification of PCR product]
Prepare the PCR chamber station. First, clean the PCR chamber, including the glass walls, put and prepare all the material that is needed for the PCR purification inside the PCR chamber. Close the chamber and turn on the UV light during 15 min. Instructions of how to use are on the side of the PCR chamber. Once the chamber is ready, without opening it, turn on the white light and the air circulator. Now the PCR chamber station is ready to use.
From PCR 1, a primer-dimer band is usually obtained (~120 bp) and should be removed, in order to avoid amplification of this undesirable product in the PCR 2 step. To do this, use the Just-a-Plate ™ 96 PCR Purification Kit.Solutions from the protocol are included in the kit.
?. [Purification of PCR product]
First time using the kit: Add 96 mL of 100% ethanol to Washing Buffer II (WB2) and mix well and mark bottle that ethanol has been added.
?. [Purification of PCR product]
Add 100 µL of Binding Buffer (KB5) to and 400 µL Isopropanol (IPP) to 1.5 mL microtube. Mix well by inverting tube 10 times after close the cap.
?. [Purification of PCR product]
Repeat 8 times, so you have total 8 wells with 45 µL PCR products. Then for each well, add 37 µl of working Binding Buffer (KB5) prepared in step (3) to each well, mix well by pipetting the solution up and down 10 times (use clean pipet tips each time).
?. [Purification of PCR product]
Cover the plate with a clean adhesive plate cover.
?. [Purification of PCR product]
Spin the plate for 10 min at 2250 x g  speed at room temperature (use another plate for balance).
?. [Purification of PCR product]
Remove the plate cover.
?. [Purification of PCR product]
Quickly flip over the plate over a waste container and shaking briskly to remove allthe solution in each well.
?. [Purification of PCR product]
Add 150 µL of washing buffer II to each well. Pipet up and down 2 times. Wait for 30 secs.
?. [Purification of PCR product]
Repeat steps 8 and 9.
?. [Purification of PCR product]
Repeat step 8.
?. [Purification of PCR product]
Blot dry the inverted plate on a piece of clean absorbent paper 3 – 4 times gently.
?. [Purification of PCR product]
Air dry for 10 min.
?. [Purification of PCR product]
Add 20 µL of elution buffer to each well, and cover the plate with an adhesive cover, and vortex it for 30 secs. After vortexing, spin the plate for 30 secs to collect all the liquid at the bottom of each well.
?. [Purification of PCR product]
Load 5 µl into a 1% agarose gel.
?. [PCR 2: Illumina adapter and barcode ligation to PCR product]
ABC1  Reagent    Concentration  30 µL final volume  - 1 reaction (µL)2  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">HPLC grade water    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">----    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">12.55  3  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">BSA    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">10 mg/ µL    <span style="font-size:10.0pt;
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  font-family:&quot;Arial&quot;,sans-serif">MgCl2    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">25 mM    <span style="font-size:10.0pt;
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  font-family:&quot;Arial&quot;,sans-serif">Polymerase    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">5 U    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">0.25  10  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">Purified PCR product    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">-----    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">1  Prepare a master mix by multiplying the volume of each PCR reagent (Table 3) times the number of samples to be processed.The PCR program is indicated in the Figure 2-C.Visualize the PCR products by electrophoresis in a 2% agarose gel. Load 5 µL from each sample. Since the ITS region is variable in size, differently sized PCR products, ranging from 300 bp to even 800-900 bp, should be expected.
We have a total of 20 indexes available (i7 index: 12; i5 index: 8). These indexes are sufficient for a total of 96 samples. A certain combination of indexes for each sample should be assigned.
ABC1  Reagent    Concentration  30 µL final volume  - 1 reaction (µL)2  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">HPLC grade water    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">----    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">12.55  3  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">BSA    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">10 mg/ µL    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">2.4  4  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">Buffer    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">5x    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">6  5  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">MgCl2    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">25 mM    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">3.6  6  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">dNTP mix    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">10 µM    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">2.4  7  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">Index    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">10 µM    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">0.9  8  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">Index    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">10 µM    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">0.9  9  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">Polymerase    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">5 U    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">0.25  10  <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">Purified PCR product    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">-----    <span style="font-size:10.0pt;
  font-family:&quot;Arial&quot;,sans-serif">1
?. [PCR purification and normalization]
For this step, follow the supplier instructions from the SequalPrep™ Normalization Plate Kit.
?. [Illumina sequencing]
For the sequencing, the MiSeq Reagent Kit v3 (600-cycle) is used in order to obtain 300 bp for each forward and reverse ends.