Dec 17, 2019
Isolation of supernumerary mini-chromosomes from fungi for enrichment sequencing
- Thorsten Langner1,
- Adeline Harant1,
- Sophien Kamoun1
- 1The Sainsbury Laboratory, University of East Anglia, Norwich, UK
External link: https://doi.org/10.1371/journal.pgen.1009386
Protocol Citation: Thorsten Langner, Adeline Harant, Sophien Kamoun 2019. Isolation of supernumerary mini-chromosomes from fungi for enrichment sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.9t7h6rn
Manuscript citation:
Langner T, Harant A, Gomez-Luciano LB, Shrestha RK, Malmgren A, Latorre SM, Burbano HA, Win J, Kamoun S (2021) Genomic rearrangements generate hypervariable mini-chromosomes in host-specific isolates of the blast fungus. PLoS Genet 17(2): e1009386. doi: 10.1371/journal.pgen.1009386
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: November 27, 2019
Last Modified: December 17, 2019
Protocol Integer ID: 30303
Keywords: Supernumerary chromosomes, Mini-chromosomes, DNA extraction, Genome sequencing
Abstract
Fungal genomes are highly dynamic and often contain supernumerary mini-chromosomes. However, our knowledge about the role of mini-chromosomes in dynamic genome rearrangements and evolution is scarce. Mini-chromosomes are usually smaller then core-chromosomes and are variable in size and numbers between individuals of a species. Mini-chromosome occurence in fungi is well documented since decades, but many genomic projects have neglected them, mainly because it was technically challenging to identify them in whole genome assemblies.
Here we present a protocol for isolation of mini-chromosomes for enrichment sequencing. This allows identification of mini-chromosomes in whole genome assemblies and thus comparative genomics analyses of core- and mini-chromosomes.
Guidelines
The protocol requires protoplast preparation of fungal cells and is therefore most suitable for culturable species.
Separation of chromosomes involves contour-clamped homogenous electric field (CHEF) gel electrophoresis.
This protocol has been developed for the blast fungus Magnaporthe oryzae. We tested this protocol by isolating mini-chromosomes from >10 individual isolates and identified corresponding contigs in whole genome assemblies.
The protocol is optimized for isolation of chromosome sized fragments between 400 kb and 3 Mb.
Materials
Required reagents:
YG medium:
5 g of yeast extract
20 g of glucose
Add ddH2O to 1l
Autoclave
Lysing enzyme solution:
10 mg/ml lysing enzymes (Trichoderma harzianum, Sigma #L1412)
15 ml 1M sorbitol
Filter-sterilize
1M Sorbitol:
182.17 g sorbitol
ddH2O to 1 l
Autoclave
1M Sorbitol/50 mM EDTA:
91.086 g sorbitol
50 ml EDTA 0.5 M
ddH2O to 500 ml
Autoclave
NDS buffer:
1 g laurylsarcosine
100 mM Tris-HCL pH9.5
500 mM EDTA
ddH2O to 100 ml
Filter-sterilize.
Add proteinase K freshly to a concentration of 200 µg/ml
Complete medium:
50 ml 20X Nitrate Salts (See Below)
1 ml Trace Elements (See Below)
10 g D-Glucose
2 g Peptone
1 g Yeast Extract
1 g Casamino Acids
1 ml Vitamin Solution (See Below)
15 g Agar
ddH2O to 1 l
Adjust pH to 6.5 with NaOH
Autoclave
20 X Nitrate Salts:
120 g NaNO3
10.4 g KCl
10.4 g MgSO4*7H2O (5.2 g if anhydrous)
30.4 g KH2PO4
ddHOH to 1 L
Autoclave
Store at 4°C.
1000 X Trace Elements:
Add the compounds in order!
80 ml ddH2O
2.2 g ZnSO4*7H2O
1.1 g H3BO3
0.5 g MnCl2*4H2O
0.5 g FeSO4*7H2O
0.17 g CoCl2*6H2O
0.16 g CuSO4*5H2O
0.15 g Na2MoO4*2H2O
5 g Na4EDTA
Boil briefly and let cool to 60°C
Adjust pH to 6.5 with KOH
Cool to room temperature
ddH2O to 100 ml
Store at 4°C
1000x Vitamin Solution:
0.01 g Biotin
0.01 g Pyridoxin
0.01 g Thiamine
0.01 g Riboflavin
0.01 g PABA (p-aminobenzoic acid)
0.01 g Nicotinic Acid
100 ml ddH2O
Store in a dark glass bottle at 4°C
Megabase agarose:
Certified Megabase Agarose (Biorad, #1613108)
TAE buffer 10x:
48.4 g Tris
3.72 Na2EDTA
ddH2O to 1 l
adjust pH to 8.0 with acetic acid
TBE buffer 10x
108 g Tris
55 g Boric acid
9.3 g Na2EDTA
ddH2O to 1 l
0.5 M EDTA
93.05 g Na2EDTA
Add 400 ml of ddH2O
Adjust pH to 8.0
ddH2O to 500 ml
Additional material required:
D-Tube Dialyzer Midi 3.5 kDa MWCO (Merck, #71506)
D-Tube™ Electroelution Accessory Kit (Merck, #71511)
Contour-clamped homogenous electric field (CHEF) gel electrophoresis system (e.g. Biorad, CHEF-DR II)
Preparation of the protoplasts
Preparation of the protoplasts
Prepare a fresh culture of M. oryzae on complete medium agar plates.
Incubate at 24°C for 7 days.
Inoculate 150 ml of YG medium in a 500 ml Erlenmeyer flask with 8 pieces (~0.5 x 0.5 cm) of 7 days old M. oryzae mycelium.
Shake at 120 rpm at 24°C for 3 days.
Prepare lysing enzymes solution and filter sterilise it.
Filter culture through 2 layers of miracloth and transfer mycelia to a 50 ml falcon tube or 100 ml beaker.
Note
Put the miracloth in a funnel on top of a bottle to collect the media.
Remove as much media residues as possible by squeezing gently.
Add 15mL of lysing enzyme solution to the mycelia.
Place the tube on a horizontal shaker at room temperature for 2 to 4 h. Shake at 30 rpm.
Note
Check the progress of protoplast formation under the microscope.
Filter protoplasts through 2 layers of miracloth and collect the flow through.
Note
Place the miracloth in a funnel on top of a 50mL falcon.
Centrifuge flow through at 1500 rpm at room temperature for 10 min. Discard the supernatant.
Note
Be careful not to dislodge the pellet. Use a pipette to remove supernatant.
Add 25 mL of Sorbitol 1 M and centrifuge again. Discard the Supernatant and repeat (2 wash steps in total).
After the washing steps, remove as much of the supernatant as possible using a pipette. Do not disrupt the pellet.
Add 100-200 µl of 1M Sorbitol/50mM EDTA and resuspend the protoplast with a cut 1mL tip.
Note
The protoplasts are very fragile! Pipette very slowly up and down or shake very gently to not damage the protoplasts.
Check quality and concentration of protoplast under the microscope using a hemocytometer.
Note
To visualize chromosome size fragments after CHEF gel electrophoresis and for subsequent DNA extraction, high protoplast concentration is required. Concentration of protoplasts should be ~ 3 x 109 protoplasts/ml.
Preparation of agarose gel plugs for contour-clamped homogenous electric field (CHEF) gel electrophoresis
Preparation of agarose gel plugs for contour-clamped homogenous electric field (CHEF) gel electrophoresis
Mix 1/3 volume of protoplasts with 2/3 volume of 1% megabase agarose in 1M Sorbitol/50mM EDTA at 45°C.
Note
Prepare the gel in advance and let it cool down in a water bath at 55°C.
Immediately after mixing, pipette protoplast mix into Bio-Rad plug mould (#1703713) and let solidify on ice for at least 30 min.
Note
Make sure to proceed quickly before the gel can solidify.
Tip: Pipette the liquid gel while keeping the tube in the water bath. Mix briefly by pipetting up and down twice and directly transfer the solution in the plug mould.
Alternatively, prepare protoplast/agarose gel mix in 1.5 ml Eppendorf tube and cut to size after the mix has solidified. For DNA extraction more than one plug might be required.
Remove the protoplast-agarose plug from the mould. Transfer to a 1.5 ml Eppendorf tube and add 1 ml NDS buffer containing 200 µg/ml proteinase K.
Incubate at 50°C for 48 h.
Remove the NDS buffer, add 1 ml of fresh 50mM EDTA and shake slowly at room temperature for 30 min to 1 h. Repeat 3 times.
Note
Washing steps can be carried out on a thermomixer set to 22°C and 200 rpm.
Carefully remove EDTA solution and store the plugs in NDS buffer (without proteinase K) at 4°C until separation by CHEF gel electrophoresis.
CHEF gel electrophoretic separation of mini-chromosomes
CHEF gel electrophoretic separation of mini-chromosomes
Prepare 1% megabase agarose gel in 0.5% buffer (TAE or TBE buffer) and let solidify.
Note
Use TBE buffer for better resolution during long runs and TAE buffer for extraction of DNA for library preparation. The borate in the TBE buffer can inhibit downstream enzymatic reactions.
Place the agarose plugs in the wells. Place the size markers.
Note
Saccharomyces cerevisiae and Schizosaccharomyces pombe chromosomes are commercially available and can be used as size markers (Biorad, #1703633, #1703605).
- Run the gel with following settings:
a.Initial switch interval 120s
b.End switch interval 3600s
c.Voltage 1.8 - 2V/cm
d.Run time 96 h
e.Temperature 14°C
Note
Prepare the system early and let the circulating buffer cool down to 14°C for at least 30 min. Do not move the system during the run, constant buffer flow and temperature are important for good resolution. We developed this protocol on a Biorad CHEF DRII system. The settings specified above are optimized to separate chromosomes between 400 kb and 3 MB.
To visualize chromosome sized fragments, place gel into ethidium bromide solution (1 µg/ml in 0.5% buffer) for 30 min and wash in 0.5% buffer (TAE or TBE) for 10 min and observe on a UV transilluminator.
Extraction of chromosome sized fragments from agarose gel by electroelution
Extraction of chromosome sized fragments from agarose gel by electroelution
Cut bands out of the gel using a scalpel and store gel slices in Eppendorf tubes at 4°C.
Note
Remove as much of the gel as possible. The gel slice should have clean edges to prevent contamination of the buffer with agarose in the next step.
Moisturize a D-Tube Dialyzer Midi 3.5 kDa MWCO (Merck, #71506) with 800 µl ddH2O for 1 min. Check membrane for leaks.
Remove water with a pipette and place agarose gel slice into the chamber.
Note
Do not touch the membrane with the pipette tip.
Fill the chamber of the D-Tube with 0.5% TAE buffer and close the lid tightly. Place tube in electrophoresis rack (D-Tube Electroelution Accessory Kit, Merck, #71511). Place in electrophoresis chamber.
Run electroelution in 0.5% TAE buffer at 90 V for 3 h at room temperature.
Note
Membranes should face in running direction. The chromosomal DNA will migrate out of the gel and gets collected in the buffer. Some DNA might stick to the membrane.
Carefully resuspend DNA by pipetting up and down close to the membrane.
Note
Do not touch the membrane with the pipette tip to prevent sample loss. Do not touch the gel slice with the pipette tip as this can lead to agarose contamination which can decrease efficiency during downstream enzymatic reactions or library preparation.
Transfer DNA solution to a 1.5 ml eppendorf tube and concentrate in a speedvac concentrator.
Note
Concentrate DNA solution to a volume of 20-50 µl. For library preparation adjust concentration following manufacturer's instructions. DNA concentration of 0.5 ng/µl is sufficient for low input library preparations. We recommend to use fluorometric methods to measure DNA concentration.
Proceed with Library preparation according to manufacturer’s instructions. We tested this protocol using a Nextera low input library preparation kit (Illumina) with custom barcoding primers and sequenced mini-chromosome derived libraries on a Nextseq 500 system.