TITLE: 18S rRNA-Gene Metabarcoding Library Prep: Dual-PCR Method
AUTHORS: rute.carvalho Carvalho, Colleen Kellogg, Matt Lemay

[DESCRIPTION]
This protocol is used for eDNA metabarcoding of the 18S SSU rRNA Gene (Balzano et al 2015) using Pair-End Illumina Miseq. Sequencing. As part of the Hakai Institute Ocean Observing Program, biomolecular samples have been collected weekly, from 0 m to near bottom (260 m), to genetically characterize plankton communities in the Northern Salish Sea since 2015. This protocol is developed to provide taxonomic annotations of Eukaryote Nuclear DNA.

[BEFORE_START]
Read Minimum Information about an Omics Protocol (MIOP) and other recommendations under the "Guidelines" tab.

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

 

GLOSSARY

 

BACKGROUND

This protocol is used for eDNA metabarcoding of the 18S SSU rRNA Gene (Balzano et al 2015) using Pair-End Illumina MiSeq Sequencing.
 

Method description and rationale
Assuming extracted DNA as starting material, this protocol includes the following steps:

First PCR: Triplicate locus-specific amplification of the 320 bp ling "Leray fragment" of the COI gene.
First PCR product purification (using magnetic beads)
Second PCR: Sample indexing using Nextera V2 indexing primers
Second PCR product purification (using magnetic beads)
Quantification and Pooling
Quality control
Pair End Sequencing on Illumina MiSeq V3 2*300 bp

Due to the risk of cross contamination, it is pivotal to separate work with amplified PCR products from pre-PCR steps. We preform pre-PCR steps (including DNA extractions) in separate clean rooms on surfaces steralized with hydrogen peroxide (PreEmpt) and UV.

Spatial coverage and environment(s) of relevance
As part of the Hakai Institute Ocean Observing Program, biomolecular samples have been collected weekly, from 0 to near bottom (260 m), to genetically characterize plankton communities in the Northern Salish Sea since 2015, developing a climatology from which we can begin uncover the physical, chemical and biological drivers of community and functional change in the dynamic coastal waters of coastal British Columbia. This protocol is developed to give a species-level resolution of marine eukaryotes.

Personnel Required
1 Technician
 
Safety
Identify hazards associated with the procedure and specify protective equipment and safety training required to safely execute the procedure!

Training requirements
Serile work technique, pipetting skills, PCR, gel electrophoresis.

Time needed to execute the procedure
This protocol may take several days to complete depending on sample size.

[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)
  
  
  
  
  
Custom-designed primers (Balzano et al 2015) including:
 

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

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

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: Triplicate PCR Amplification (1st PCR)
9. Seal the 96-well plates and transfer them to thermocyclers.

 
 
 

SECTION: Triplicate PCR Amplification (1st PCR)
10. 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)
8. PCR reactions are carried out in triplicate 25μl reactions:
 

SECTION: Purification of first PCR product using SPRI beads
12. 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
13. 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
14. Remove the supernatant with a multichannel pipette, ensuring to not disturb the beads. 
SECTION: Purification of first PCR product using SPRI beads
15. 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
16. Repeat the washing step   
SECTION: Purification of first PCR product using SPRI beads
17. 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
18.  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
19. 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
20. Carefully transfer 30 μl of the clear supernatant to a new plate. Seal the plate.
SECTION: Purification of first PCR product using SPRI beads
21. 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)
22. 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)
23. Dilute the cleaned-up PCR (1:10) with sterile nuclease-free water.
SECTION: Indexing PCR amplification (2nd PCR)
24.  Prepare PCR reaction in 25μl reactions:
 

SECTION: Indexing PCR amplification (2nd PCR)
25. Seal the 96-well plates and transfer them to thermocyclers.
 
 
SECTION: Purification of indexed libraries (Second bead cleanup)
27. Repeat the Ampure XP bead cleanup for all the indexed libraries.
  

SECTION: Quantification and pooling, and quality control
28. 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 the broad range kit if you visualize a strong band in the gel:   OR
  

 

SECTION: Indexing PCR amplification (2nd PCR)
26. 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
29. 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
30. Measure the final library pool concentration on Qubit using
  

SECTION: Quantification and pooling, and quality control
31. Label tube: [Gene_name], [Project_Name], Pooled Amplicons. Date, Initials, pool concentration.
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. Here we use Nextera V2 Kit Sets A, B, C, and D.  We advise preparing the indexing primers on 96-well plates 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: Sequencing parameters
32. Library fragment size (BP) is determined using   
Molarity of thefinal pool is assessed using   
SECTION: Sequencing parameters
33. COI libraries are sequenced an a MiSeq instrument using:
    with pair-end setup (2*300 bp),  spiked with 10%   .

SECTION: Triplicate PCR Amplification (1st PCR)
7. 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.