TITLE: 12S 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 mitochondrial 12S rRNA gene (Miya 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 give a species-level resolution of fish diversity.

[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 mitochondrial 12S rRNA gene (Miya et al 2015) using Pair-End Illumina MiseqSequencing.
 
Method description and rationale
Assuming extracted DNA as starting material, this protocol includes the following steps:

First PCR: Triplicate locus-specific amplification of the mitochondrial 12S rRNA gene (Miya et al 2015) .
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
Library concentration (using magnetic beads)
Gel purification with size selection
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 fish diversity.

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 will 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)
  
  (Or equal)
  (Or equal)
  (Or equal)
Custom-designed primers (Miya et al 2015) including:
 

SECTION: Purification of first PCR product using SPRI beads
12. 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)
11. Run sample pools on a 2% gel (90V, ~40-60 min) to check the size
of the amplicons and the success of the PCR (5μl/sample). 

 

SECTION: Triplicate PCR Amplification (1st PCR)
8. PCR reactions are carried out in triplicate 12.5 μl reactions:
 

SECTION: Purification of first PCR product using SPRI beads
14. Incubate a room temperature without shaking for 5 min. For samples that you know have a low DNA concentration, you can increase this incubation time to 30 min.
Place the plate on the magnetic stand until the supernatant has cleared (~ 3 min). 
SECTION: Purification of first PCR product using SPRI beads
15. Remove the supernatant with a multichannel pipette, ensuring to not disturb the beads. 
SECTION: Purification of first PCR product using SPRI beads
16. 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
17. Repeat the washing step   
SECTION: Purification of first PCR product using SPRI beads
18. 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
19.  Remove the plate from the magnetic stand and add 32 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
20. 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
21. Carefully transfer 30 μl of the clear supernatant to a new plate. Seal the plate.
SECTION: Purification of first PCR product using SPRI beads
22. 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.
SECTION: Indexing PCR amplification (2nd PCR)
23. 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)
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: Quantification and pooling, and quality control
38. 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 the product on a 2% agarose gel to check the size of the amplicons and success of the PCR (5μl).

 

SECTION: Quantification and pooling, and quality control
39. 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
40. Measure the final library pool concentration on Qubit using
  

SECTION: Quantification and pooling, and quality control
41. 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
75. Library fragment size (BP) is determined using   
SECTION: Sequencing parameters
77. 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 Platinum, Primers, and nuclease-free water. Keep them in a cooling microcentrifuge tube rack. 
SECTION: Triplicate PCR Amplification (1st PCR)
10. Pool the three replicates.
SECTION: Purification of indexed libraries (Second bead cleanup)
27. Preparations

Materials  
Prepared serapure SPRI beads.
 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: Purification of first PCR product using SPRI beads
13. Vortex the beads before use.
As you now have about 32.5μl reaction (pooled samples - 5μl used on a gel/Qiaxcel), add 26 μl beads to the product to obtain a ratio of 0.8. 
Pipette up and down about 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 indexed libraries (Second bead cleanup)
28. 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 indexed libraries (Second bead cleanup)
29. Incubate a room temperature without shaking for 5 min. For samples that you know have a low DNA concentration, you can increase this incubation time to 30 min.
Place the plate on the magnetic stand until the supernatant has cleared (~ 3 min). 
SECTION: Purification of indexed libraries (Second bead cleanup)
30. Remove the supernatant with a multichannel pipette, making sure not to disturb
the beads.
SECTION: Purification of indexed libraries (Second bead cleanup)
31. With the samples on the magnetic rack, wash the beads by adding 180 μl of freshly prepared 80% ethanol.
Incubate for 30 s.
Carefully remove the supernatant without disturbing the beads.
SECTION: Purification of indexed libraries (Second bead cleanup)
32. Repeat the washing step.   
SECTION: Purification of indexed libraries (Second bead cleanup)
33. Remove all residual ethanol using a pipette and air dry, leaving the samples on the magnetic stand (~ 5 min*). Keep an eye on the beads and do not over-dry, otherwise, you will not get an efficient DNA recovery.
 

SECTION: Purification of indexed libraries (Second bead cleanup)
34. Remove the plate from the magnetic stand and add 28 μl of nuclease-free water for elution.
Gently pipette up and down ten times to resuspend the beads.
Incubate the plate at room temperature for 5 min.
SECTION: Purification of indexed libraries (Second bead cleanup)
35. Place the plate back on the magnetic rack for at least 5 min or until the supernatant has cleared.
SECTION: Purification of indexed libraries (Second bead cleanup)
36. Carefully transfer 25 μl of the clear supernatant to a new plate. Seal the plate.
SECTION: Purification of indexed libraries (Second bead cleanup)
37. 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.
SECTION: Concentrating library pool with magnetic beads
42. 
 

Materials
Magnetic beads* (SPRI beads or AMpure XP, find aliquots in fridge)
Magnetic rack for 1.5-2 mL tubes
Anhydrous Ethanol to make a fresh 80% ethanol solution
Sterile Nuclease-Free water
UV for 30 minutes the following:
1.5 mL lo-Bind tubes
Sharpie
Pipette tips
Pipettes
Sterile Nuclease-Free Water

Remove the magnetic beads from the fridge (allow 30 min to reach room temperature).
SECTION: Concentrating library pool with magnetic beads
43. Aliquot your pool into 1.5 mL tubes (try to keep similar volumes between aliquots, a maximum of 350 μl per tube). If you have a 2 mL pool, you can aliquot 350 μl pool in 5 tubes, and have the 6th tube with 250 μl.
SECTION: Concentrating library pool with magnetic beads
44. Calculate the volume of beads to add into each tube (0.8x beads). For the tubes with 350 μl – add 280 μl beads, and for the tube with 250 μl, add 200 μl beads. You will 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. Incubate a room temperature without shaking for 5 min.
SECTION: Concentrating library pool with magnetic beads
45. Place the tubes on the magnetic rack until the supernatant has cleared (~ 3 min).
SECTION: Concentrating library pool with magnetic beads
46. Remove the supernatant with a P1000 pipette, making sure not to disturb the beads.
SECTION: Concentrating library pool with magnetic beads
47. With the samples on the magnetic rack, wash the beads by adding 500 μl of freshly prepared 80% ethanol and incubate for 30 s. Carefully remove the supernatant without disturbing the beads.
SECTION: Concentrating library pool with magnetic beads
48. Repeat washing step   

SECTION: Concentrating library pool with magnetic beads
49. Remove all residual ethanol using a pipette and air dry, leaving the samples on the magnetic rack (~ 5 min*). Keep an eye on the beads and do not over dry, otherwise you will not get an efficient DNA recovery.
 

SECTION: Concentrating library pool with magnetic beads
50. Remove the tubes from the magnetic rack and add 40 μl of nuclease-free water for elution. Gently pipette up and down ten times to resuspend the beads. Incubate the tubes at room temperature for 5 min.
SECTION: Concentrating library pool with magnetic beads
51. Place the tubes back on the magnetic rack at least 5 min or until the supernatant is cleared.
SECTION: Concentrating library pool with magnetic beads
52. Carefully transfer 30 μl of the clear supernatant to a single new tube (pooling the volumes of the 6 tubes).
SECTION: Concentrating library pool with magnetic beads
53. Repeat the bead cleanup to reduce the volume even more. You may now have 180 μl in your pool, so you must add 144 μl of beads to have a rate of 0.8.  
Make final elution in 50 μl of water, transferring 45 μl of the clear supernatant to a new tube.
SECTION: Concentrating library pool with magnetic beads
54. Name the tube: [Gene_name], Project, Concentrated Pool, Date, Initials.
SECTION: Gel purification with size selection
55. 
 


Materials: 
   (or similar)
  
  
  
  

SECTION: Gel purification with size selection
55.1. Prepare 1.5-2 L of 1x TBE buffer and autoclave it. Wait for it cool down to room
temperature. *safe stopping point.
SECTION: Gel purification with size selection
56. Prepare a 3% agarose gel. It may polymerize faster that the gels that you are used to prepare. You may need about 120 mL of buffer and 3.6 g of agarose if using the small electrophoresis system. Add 6 μl of RedSafe. Use a comb with large teeth – it may accommodate a higher volume of product.
SECTION: Gel purification with size selection
57. If you have 45 μl of product, add 8 μl of loading dye to the tube and mix well. Load the entire tube’s content in the well. You may need more than one well to load the entire content.
SECTION: Gel purification with size selection
58. Load the Ladder 100 bp in the first and last well (~3 μl).
SECTION: Gel purification with size selection
59. Run the gel for about 1h at 90V. In this mean time, weigh one 1.5 mL Eppendorf tube and record the weight. Place on your counter: sterile blade for scalpel, a scalpel, and the LED transilluminator.
SECTION: Gel purification with size selection
60. When the gel run is done, transfer the gel to the transilluminator, and excise the 350 bp band. Place it in the 1.5 mL tube that you have weighed. * Sometimes the stronger band is the non-target one. That’s why it is important to have the ladder running beside your samples to help you to find the right band. See Figure 1.
 
 

SECTION: Gel purification with size selection
61. Weigh the tube with the gel slice. Record the weight.
SECTION: Gel purification with size selection
62. Use the Promega Wizard SV gel and PCR clean-up to perform the gel purification (follow instructions in the manual -  https://www.promega.ca/products/nucleic-acid-extraction/clean-up-and- concentration/wizard-sv-gel-and-pcr-clean-up-system/?catNum=A9281#protocols).
SECTION: Gel purification with size selection
63. Add 10 μl Membrane Binding Solution per 10 mg of gel slice (for example, if you weight 102 mg of gel slice in the tube, add 102 μl of Membrane Binding Solution).
SECTION: Gel purification with size selection
64. Vortex and incubate at 50-65oC until gel slice is completely dissolved (~10 min). Vortex again to be sure that you do not have any gel in the tube. Spin down.
SECTION: Gel purification with size selection
65. Insert SV minicolumn into a collection tube.
SECTION: Gel purification with size selection
66. Transfer dissolved gel mixture to the minicolumn assembly. Incubate at room temperature for 1 min.
SECTION: Gel purification with size selection
67. Centrifuge at 16,000 x g for 1 min. Discard flowthrough, and reinsert the minicolumn into thecollection tube. *dry the collection tube edges using Kim wipes if necessary.
SECTION: Gel purification with size selection
68. Add 700 μl Membrane Wash Solution (ethanol added). Centrifuge at 16,000 x g for 1 min. Discard flowthrough, and reinsert the minicolumn into collection tube.
SECTION: Gel purification with size selection
69. Repeat the step 15 with 500 μl Membrane Wash Solution. Centrifuge at 16,000 x g for 5 min.
SECTION: Gel purification with size selection
70. Empty the collection tube and recentrifuged the column assembly for 1 min to allow evaporation of any residual ethanol.
SECTION: Gel purification with size selection
71. Carefully transfer minicolumn to a clean 1.5 ml tube.
SECTION: Gel purification with size selection
72. Add 35-50 μl of elution buffer (or Nuclease-Free water) to the minicolumn. Incubate at room temperature for 1 min. Centrifuge at 16,000 x g for 1 min. Perform a second elution if you consider it to be necessary.
SECTION: Gel purification with size selection
73. Quantify the purified product on Qubit using the ds DNA BR kit.
SECTION: Gel purification with size selection
74. Label tube: [Gene_name], [Project_Name], Final Library. Date, Initials, concentration.
SECTION: Sequencing parameters
76. Molarity of the final pool is assessed using