TITLE: COI Metabarcoding Library Prep: Dual-PCR Method
AUTHORS: Colleen Kellogg, Andreas Novotny

[DESCRIPTION]
Draft!


[BEFORE_START]
Read background information, MIOP and BePOP-OBON information under the "Guidelines" tab.

[GUIDELINES]
MIOP: Minimum Information about an Omics Protocol
 
 
AUTHORS
 
 
 
RELATED PROTOCOLS
 
 
 
ACRONYMS AND ABBREVIATIONS

 

GLOSSARY

 

BACKGROUND

This document describes the required protocol to conduct insert name of the method/protocol.

Summary

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Please provide a brief summary of your method including, as appropriate, a brief description of what techniques your best practice is about, which ocean environments or regions it targets, the primary sensors covered, what type of data/measurements/observing platform it covers, limits to its applicability.

Method description and rationale

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Spatial coverage and environment(s) of relevance

If applicable, please specify the region where the protocol is applied. For regional term guidance see here. If applicable, please indicate here the environment(s) of relevance for the protocol, e.g. Abyssal plain. Select from the ENVO terminology.

Personnel Required

Insert the number of technicians, data managers, and scientists required for the good execution of the procedure

Safety

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Training requirements

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Time needed to execute the procedure

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[STEPS]
SECTION: PREPARATIONS
1. Ensure that the laboratory is appropriately configured and that staff has appropriate training. See "Guidelines" for more information. Pay attention to the separation of pre and post-PCR spaces and equipment.

SECTION: Triplicate PCR Amplification (1st PCR)
6. Preparations

 

 Reagents:
  (Or equal)
  
  
  
  

 
UV for 30 minutes the following:  
96-well PCR plates (or 8-strip tubes)  
 Sharpie 
 Pipette tips 
 Multichannel pipettes  
 Pipettes  
 Sterile Nuclease-Free Water

Thaw Taq, BSA, Primers, and nuclease-free water. Keep them in a cooling microcentrifuge tube rack. 

SECTION: Purification of first PCR product using SPRI beads
10. Preparations
 

Materials  
Magnetic beads working solution. If not already prepared:   
 Magnetic 96-well plate stand 
Anhydrous Ethanol to make a fresh 80% ethanol solution 
   

UV for 30 minutes the following:  
96-well PCR plates (or 8-strip tubes)  
Sharpie 
Pipette tips 
Multichannel pipettes  
Pipettes  
Sterile Nuclease-Free Water

Remove the magnetic beads from the fridge (allow 30 min to reach room temperature).
SECTION: BASIC TROUBLESHOOTING GUIDE
33. Identify known issues associated with the procedure, if any.

Provide troubleshooting guidelines when available.
SECTION: Triplicate PCR Amplification (1st PCR)
8. Seal the 96-well plates and transfer them to thermocyclers.

 
 
 

SECTION: Triplicate PCR Amplification (1st PCR)
9. Run a subset of the PCR product (5μl) on a 1.5% agarose gel to check the size of the amplicons and the success of the amplification.

 


SECTION: Triplicate PCR Amplification (1st PCR)
7. PCR reactions are carried out in triplicate 25μl reactions:
 


SECTION: Purification of first PCR product using SPRI beads
11. Vortex the beads before use.
Add 16 μl beads to 20 μl of PCR product to obtain a ratio of 0.8.
Pipette up and down ten times (or until the solution is well mixed – you will see that the color changes).
Spin tubes down to remove drops from the walls.
SECTION: Purification of first PCR product using SPRI beads
12. Incubate at room temperature without shaking for 5 min.
Then, place the plate on the magnetic stand until the supernatant has cleared (~ 3 min). 
SECTION: Purification of first PCR product using SPRI beads
13. Remove the supernatant with a multichannel pipette, ensuring to not disturb the beads. 
SECTION: Purification of first PCR product using SPRI beads
14. With the samples on the magnetic rack, wash the beads by adding 180 μl of freshly prepared 80% ethanol and incubate for 30s. Carefully remove the supernatant without disturbing the beads. 
SECTION: Purification of first PCR product using SPRI beads
15. Repeat the washing step   

SECTION: Purification of first PCR product using SPRI beads
16. Remove all residual ethanol using a pipette and air dry, leaving the samples on the magnetic stand (~ 5 min*). 

 

SECTION: Purification of first PCR product using SPRI beads
17.  Remove the plate from the magnetic stand and add 40 μl of nuclease-free water for elution. Gently pipet up and down ten times to resuspend the beads.  Incubate the plate at room temperature for 5 min. 
SECTION: Purification of first PCR product using SPRI beads
18. Place the plate back on the magnetic rack for at least 5 min or until the supernatant is cleared.  
SECTION: Purification of first PCR product using SPRI beads
19. Carefully transfer 30 μl of the clear supernatant to a new plate. Seal the plate.


SECTION: Purification of first PCR product using SPRI beads
20. Name the plate: Project, [Gene_name], PCR 1, Post-Purification Plate #, Date,  Initials.
Samples can be stored at -20°C for up to 7 days.

(IF this is the cleanup of the second PCR product   )
SECTION: Indexing PCR amplification (2nd PCR)
21. Preparations

Reagents:
  
  
  
  
i5 and i7 index plates (10 μM) – If not already prepared:   

UV for 30 minutes the following:  
- 96-well PCR plates (or 8-strip tubes)  
- Sharpie 
- Pipette tips 
- Multichannel pipettes  
- Pipettes  
- Sterile Nuclease-Free Water 

Thaw Taq, i5 and i7 indexes, and nuclease-free water. Keep them in the  IsoFreeze microcentrifuge tube rack. 
SECTION: Indexing PCR amplification (2nd PCR)
22. Dilute the cleaned-up PCR (1:10) with sterile nuclease-free water
SECTION: Indexing PCR amplification (2nd PCR)
23.  Prepare PCR reaction in 25μl reactions:
 

SECTION: Indexing PCR amplification (2nd PCR)
24.  Seal the 
 
 
SECTION: Purification of indexed libraries (Second bead cleanup)
26. Repeat the Ampure XP bead cleanup for all the indexed libraries.
  

SECTION: Quantification and pooling, and quality control
27. Use a fluorometric quantification method that uses dsDNA dyes to measure the concentration of your libraries (Qubit or plate reader). If using Qubit, give preference to theBroad Range kit (BR) if you visualize a strong band in the gel.

 

SECTION: Indexing PCR amplification (2nd PCR)
25. Run a subset of the PCR product (5μl) on a 1.5% agarose gel to check the size of the amplicons and the success of the amplification.

 


SECTION: Quantification and pooling, and quality control
28. Calculate sample volume to have a final amount of 10-40 ng. This amount may vary depending on the overall quantification. For example, if on average the concentration of your samples is about 3 ng/μl and you have 20 μl of product,  you can calculate the volume to make up to 60 ng per sample.

 

SECTION: Quantification and pooling, and quality control
29. Measure the final library pool concentration on Qubit using the High Sensitivity (HS) kit. 
SECTION: Quantification and pooling, and quality control
30. Label tube: [Gene_name], [Project_Name], Pooled Amplicons. Date, Initials, pool concentration.
SECTION: Quantification and pooling, and quality control
31. 
SECTION: PREPARATIONS
2. Ensure that all reagents are aliquoted in appropriate amounts, and stored according to manufacturers' recommendations. Never pipet directly from reagent stocks.
SECTION: PREPARATIONS
3. Prepare the SPRI beads' working solution, and test their efficiency following this protocol: 
SECTION: PREPARATIONS
4. Prepare primer working stocks (10μM) for both the first and second PCR steps.
We advise preparing the indexing primers according to this configuration:
  

SECTION: PREPARATIONS
5. We advise adding aliquots of the extracted DNA to a 96-Well PCR plate to facilitate the setup of the PCR reaction. This metadata template will help keep track of the samples, and if indexes are configured as described above, also the identity of sample indexes.
SECTION: Quantification and pooling, and quality control
32.