Sep 21, 2023

Public workspaceVirus and Prokaryote Enrichment in Coral DNA Metagenomes

DOI
dx.doi.org/10.17504/protocols.io.q26g7p1y3gwz/v1
  • Natascha Varona1,
  • Bailey Wallace1,
  • Cynthia Silveira1
  • 1University of Miami Department of Biology
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DOI: dx.doi.org/10.17504/protocols.io.q26g7p1y3gwz/v1
Protocol Citation: Natascha Varona, Bailey Wallace, Cynthia Silveira 2023. Virus and Prokaryote Enrichment in Coral DNA Metagenomes. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7p1y3gwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 29, 2023
Last Modified: September 21, 2023
Protocol Integer ID: 87107
Keywords: coral, bacteria, virus, bacteriophage, metagenome
Funders Acknowledgement:
National Science Foundation GRFP to BW
Grant ID: 2023353157
Maytag Fellowship to NV
Grant ID: PG015171
University of Miami Provost Research Award to CS
Grant ID: UM PRA 2022-2547
Abstract
Tissue preparation and DNA extraction protocol for enrichment of viruses and prokaryotes in DNA metagenomes of corals.
Image Attribution
Cynthia Silveira
Materials
10X DNase Buffer:
  • Concentration100 millimolar (mM) Tris-HCl (pH 7.5)
  • Concentration25 millimolar (mM) MgCl2
  • Concentration5 millimolar (mM) CaCl2

  • Fresh or thawed  ~1cm3 coral sample
  • Sterile mortar and pestle
  • Sterile forceps
  • Amount1.5 mL centrifuge epi tubes
  • Amount0.02 µm filtered and autoclaved artificial seawater (ASW)
  • Amount425-600 µm acid-washed glass beads (Sigma CAT#G9268)
  • Amount15 mL conical centrifuge tubes
  • Vortex
  • Centrifuge
  • Pipettes
  • DNaseConcentration10000 U/mL
  • 100X EDTA Concentration0.5 Molarity (M)
  • Sterile Amount10 mL Luer-LokTM syringes
  • Amount8 µm pore size, 25mm flat filters (Whatman, Nucleopore Track-Etched Polycarbonate (CAT#110614))
  • Sterile 25mm filter holders with Luer-LokTM connection (Swinnex CAT#SX0002500)
  • AmiconTM Ultra-15 Centrifugal Filter Units (MilleporeSigma‱ CAT#UFC910008)
  • NucleoSpinⓇ Tissue Kit (MACHEREY-NAGEL Inc. REF:740952.10/.50/.250) 

Before start
  • Glass beads must be washed with 0.1 N HCl
  • Incubator set to Temperature56 °C & Temperature70 °C
  • Check if Buffer B5 and Proteinase K were prepared according to section 3 of NucleoSpinⓇ Tissue Kit Manual.


Sample Preparation
Sample Preparation
3h
3h
Collect a coral sample of approximately 1cm3 containing coral tissue, mucus, and skeleton. Use a fresh sample if possible. If not, flash-freeze the sample and keep it at Temperature-80 °C until processing. Thaw the sample on ice before proceeding.

Weigh and record coral fragment weight.
Crush the coral sample using a sterile mortar and pestle until the skeleton and tissue fragments are the size of coarse sand. Do not overdo this step. Transfer the fragments, along with the mucus (coral homogenate), to a 2 mL microcentrifuge tube.
Suspend sample in Amount100 µL of Artificial Seawater (ASW) and Amount0.2 g of sterile glass beads.

Vortex at speed 3 for Duration00:05:00 min to dislodge tissue and mucus.
* To avoid lysis of bacterial cells, do not vortex the sample at higher speeds or longer durations than described.
5m
Pulse-centrifuge the sample (3-5 seconds at Centrifigation3.200 x g ).

Collect supernatant and transfer to a clean 15 mL microcentrifuge tube.

Bring sample up to Amount1 mL with sterile ASW.

Add Amount100 µL of 10X DNase Buffer and Amount2 µL of DNase 10,000 U/mL for a final concentration of 20 U/mL. Mix by gently inverting the tube.

Incubate at room temperature for Duration02:00:00 hours .

2h
Stop DNase activity by adding Amount10 µL of 100X EDTA.
Bring sample volume up to Amount5 mL with sterile ASW.

Transfer thesample to a 10 mL syringe and push through an 8.0 µm, 25 mm flat filter, collecting the flowthrough in a clean 15 mL centrifuge tube.
* If the sample clogs the filter, replacing the 8.0 µm filter may be necessary. In this case, pull back on the syringe plunger to recollect the sample in the syringe before transferring it to a new filter.
* In the case of samples with excessive mucus, you may further dilute the sample with sterile ASW prior to filtering.
Transfer flowthrough to an Amicon‱ Ultra-15 Centrifugal Filter Unit.
Centrifuge at room temperature for Duration00:30:00 min at ≥Centrifigation2600 x g .

30m
DNA Extraction
DNA Extraction
1h 12m
1h 12m
  1. Add Amount200 µL Buffer T1 and Amount20 µL of Proteinase K from the NucleoSpinⓇ Tissue Kit to the Amicon‱ filter unit.

Rinse one side of the filter with the lysis buffer 3-5 times using a pipette.
Securely close the Amicon‱ filter unit and incubate for Duration01:00:00 hour at Temperature56 °C , being sure to place the tube in a position where the rinsed side of the filter remains covered in the Buffer T1 & Proteinase K mixture.
* Some Amicon‱ filter unit lids may not seal properly. Check for leaks before placing in the incubator. Seal with parafilm if necessary.

1h
Repeat steps 17 and 18 on the other side of the filter.
Add Amount210 µL Buffer B3 to the Amicon‱ filter unit and vortex to mix.

Incubate for Duration00:10:00 min at Temperature70 °C .

10m
Add Amount210 µL ethanol (100%) to the Amicon‱ filter unit and vortex to mix.

Proceed with steps 5-7 of the NucleoSpinⓇ Tissue Kit Standard protocol for human or animal tissue and cultured cells.
On step 8 of the NucleoSpinⓇ Tissue Kit, perform the following:
a. Place the NucleoSpinⓇ Tissue Column into a 1.5 mL microcentrifuge tube (not provided in kit)
b. Add Amount50 µL PCR-grade water
c. Incubate at room temperature for Duration00:01:00
d. Centrifuge for Duration00:01:00 min at Centrifigation11000 x g
e. Repeat steps b, c, & d once, for a final volume of 100 uL

2m