Apr 02, 2020
Version 2
An optimized protocol for sequencing mammalian roadkill tissues with Oxford Nanopore Technology (ONT) V.2
- 1Institut des Sciences de l’Evolution de Montpellier (ISEM), CNRS, IRD, EPHE, Université de Montpellier, Montpellier, France
- High molecular weight DNA extraction from all kingdomsTech. support email: See@each.protocol
Protocol Citation: Marie-Ka Tilak, Rémi Allio, Frédéric Delsuc 2020. An optimized protocol for sequencing mammalian roadkill tissues with Oxford Nanopore Technology (ONT). protocols.io https://dx.doi.org/10.17504/protocols.io.beixjcfn
Manuscript citation:
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: April 02, 2020
Last Modified: April 02, 2020
Protocol Integer ID: 35127
Keywords: Roadkill, mammals, long reads, Oxford Nanopore Technology, MinION, DNA sequencing, genomics
Abstract
This protocol was developed to optimize DNA sequencing from mammalian roadkill tissue samples using Oxford Nanopore Technology (ONT). Roadkill tissues contain necrotized cells and impurities that result in DNA degradation. First, we observed that DNA preservation was generally better in RNAlater than in 95% EtOH preserved tissues. Second, we found that physically removing necrotized and epidermal cells before DNA extraction resulted in better DNA quality and purity. Finally, adjusting the ratio of AMPure beads to 0.4x at the DNA purification step permitted optimizing size selection for subsequent ONT library construction. These optimization steps allowed to significantly increase both read length and yield per flow cell for roadkill samples sequencing on the ONT MinION device.
Materials
MATERIALS
NanoDrop spectrophotometerThermo Fisher ScientificCatalog #ND-1000
Agencourt Ampure XPBeckman CoulterCatalog #A63880
RNAlaterThermo Fisher ScientificCatalog #AM7020
Phenol-chloroform-isoamyl alcohol 25:24:1 (PCI)Invitrogen - Thermo FisherCatalog #15593049
Proteinase K (2 ml)QiagenCatalog #19131
RNase A, DNase and protease-freeThermo Fisher ScientificCatalog #EN0531
Qubit Invitrogen - Thermo Fisher
DNA LoBind TubesEppendorfCatalog ##022431021
g-TUBECovarisCatalog #520079
Lysis buffer:
Stock solution (all solutions were sterilized, even SDS):
Tris Hcl 1M 1 mL
Nacl 5M 0.2 mL
EDTA 0.5M 0.5 mL
SDS 20% 10 mL
qsp sterilized water 100 mL
Final concentration:
Tris-Hcl 10 mM
Nacl 10 mM
EDTA 2.5 mM
SDS 2%
Lysis buffer will be preserved at 4°C.
SDS will be precipitated.
Keep the lysis buffer at least 1 hour at room temperature before using it.
Tissue sampling and preservation
Tissues were preserved in RNAlater Thermo Fisher® or 95% EtOH. We have observed that RNAlater preservation was often better to conserve DNA from internal organ tissues than 95% EtOH (Figure 1).
Figure 1: DNA extractions from spleen tissue of a fresh roadkill southern anteater (Tamandua tetradactyla) collected and preserved at the same time in either RNAlater or 95% EtOH.
Wells 1-2: Extraction from spleen tissue preserved in RNAlater.
Wells 4-5: Extraction from spleen tissue preserved in 95% EtOH.
Well 3: 200 bp - 10 Kb ladder.
Well 6: Lambda ladder (48.5 Kb).
Tissue preparation
Before the lysis step, put the tissue in a new fresh preservation liquid (RNAlater or 95% EtOH). Under a binocular magnifier, remove all necrotized or epidermal cells, hairs, or dust particles using a sterile scalpel blade. Try to keep the tissue with the best possible integrity in order to limit DNA degradation.
Figure 2: DNA extractions from an ear biopsy of a roadkill bat-eared fox (Otocyon megalotis) preserved in 95% EtOH.
Wells 1-2-3: DNA extractions from raw ear biopsies with hairs and epidermal cells.
Well 4: 200 bp - 10 Kb ladder.
Wells 5-6-7: Same tissue after removing hairs and epidermal cells using a sterile scalpel blade under a binocular magnifier.
DNA extraction
3.1 - Tissue lysis
1/ Use 25 mg of tissue for 225 μL of lysis buffer with 25 μL of proteinase K.
2/ Incubate at 56°C for at least 2 hours. Tissue must be fully digested.
3/ Add 4 μL of RNAse and incubate at Room Temperature (RT) for 30 min.
Optional: Check tissue lysis under the binocular magnifier. If there are still particles (hairs, dust…) in the solution, centrifuge 5 min at 14000 rpm and transfer the supernatant in new tube.
4/ Add an equal volume of UltraPure Phenol: chloroform:Isoamyl Alcohol (Invitrogen).
5/ Mix by inversion 30 times until an emulsion is formed.
6/ Separate the two phases by centrifugation at 12000 rpm for 5 min at RT.
7/ Gently transfer the upper phase in a new tube without dislocating the interphase.
8/ Add an equal volume of chloroform-isoamyl-alcohol (24:1).
9/ Mix by inversion 30 times until an emulsion is formed.
10/ Separate the two phases by centrifugation at 12000 rpm for 5 min at RT.
11/ Gently transfer upper phase in a new tube without dislocating the interphase.
3.2 - DNA precipitation
1/ Add 1/3 vol. of Amonium Acetate 7.5M (to improve protein precipitation: Crouse J. & Amorese D. 1987).
2/ Incubate at RT for 30 min.
3/ Centrifuge at 14500 rpm for 15 min. Sometimes a DNA pellet can appear.
4/ Under a binocular magnifier, gently transfer the supernatant in a new tube without dislocating the pellet.
5/ Check under the binocular magnifier if the supernatant is clear without suspension of slight particles.
At this stage the lysate must be clear to prevent the particles from condensing with DNA during the precipitation step. The presence of particles could block the nanopores on the flow cell during DNA sequencing.
6/ Add 2.5 vol. of RT isopropanol and mix by inversion a couple of times. You must see a mass of DNA (condensate DNA fragments).
7/ Fish the DNA pellet with a 200 μL tip cut at the end to avoid damaging DNA.
8/ Transfer the DNA in 1 mL of fresh 70% EtOH heated at 50°C.
9/ Repeat steps 8 and 9 three more times.
10/ Place the DNA pellet in a low binding 2 mL tube.
11/ Remove residual 70 % EtOH with a 10 μL tip.
12/ Air-dry for 5 min (to evaporate residual 70% EtOH).
13/ Add between 50 μL to 150 μL depending on the size of the condensed DNA.
14/ Place the DNA solution in the fridge at 4°C for 3-4 days so that it resuspends entirely before using it.
Crouse J. & Amorese D. (1987). Ethanol precipitation: ammonium acetate as an alternative to sodium acetate. Focus 9(2), 3-5.
DNA quality control
Quantify the DNA with Qubit® 2.0 Fluorometer (Thermo Fischer Scientific) and NanoDrop™ Spectrophotometer (Thermo Fisher Scientific).
1/ NanoDrop ratios must be: 1.8 < 260/280 < 2 and 2 < 260/230 < 2.2.
2/ Qubit/Nanodrop ratio must be between 0.6 and 1. Under 0.6, the DNA is not clean enough.
3/ Visualize DNA quality by migration on a 1% agarose gel.
Homogenization of DNA fragments size with Covaris G-tubes
1/ Load 4-5 μg (27 to 35 ng/μL) diluted in 150 μL of ultrapure water in a Covaris G-tube.
2/ Centrifuge the G-tube at 5000 rpm for 1 min.
3/ Reverse the G-tube and centrifuge at 5000 rpm for 1 min.
4/ Transfer the fragmented DNA in a new tube.
A-tailing and FFPE repair
1/ Transfer 3x48 μL of DNA in three 0.2 mL DNA LoBind tubes.
2/ Prepare mix for 3 tubes following 1D Genomic DNA by Ligation (SQK-LSK-109) protocol by ONT with slight modifications: Incubation time is increased up to 1 hour at 20°C followed by 5 min at 65°C before putting on ice.
3/ Pool 3 tubes in one and purify with 0.4x ratio (72 μL) of Agencourt AMPure XP beads following the manufacturer instructions. Elude in maximum 80 μL of ultrapure water. This ratio allows removing the small DNA fragments.
4/ Check Qubit concentration
5/ Visualize DNA quality by migration on a 1% agarose gel (Figure 3).
Figure 3. DNA size selection with different ratios of AMPure beads on extractions of a kidney tissue from a roadkill pygmy anteater (Cyclopes didactylus) preserved in 95% EtOH.
Well 1: Original DNA extract.
Wells 2-3-4: Size selection with 0.4x, 0.6x, and 1x of AMPure beads.
Well 5: 200 bp - 10 Kb ladder.
ONT Library preparation
Using this protocol for Covaris G-tubes (step 5), the average size of DNA fragments was between 3000 pb and 5000 pb.
1/ Use 0.2 pmole of DNA
2/ Follow 1D Genomic DNA by Ligation (SQK-LSK-109) protocol with increasing incubation time up to 1h at room temperature.
3/ Increase elution volume to 16 μL instead of 13 μL.
4/ Transfer 15.5 μL of the supernatant (library) in a new tube placed on the magnetic rack.
5/ Wait 5 min, very often a little pellet of residual magnetic beads appears.
6/ Gently transfer 13 μL of supernatant without beads in SQB+LB mix.
7/ Check Qubit concentration of library using 2 μL of residual supernatant.
ONT MinION sequencing
MinIONQC results
Figure 4: Read length distributions (log-scale) from MinION sequencing of a roadkill pygmy anteater (Cyclopes didactylus) obtained with MinIONQC (Lanfear et al. 2019). A. Results obtained from 10 flow cells with SQK-LSK108 ligation kit (total: 17.5 Gb) with basic Phenol/Chloroform extraction and following ONT library preparation protocol (FFPE+1x AMPure beads). B. Results obtained from 7 flow cells with SQK-LSK109 ligation kit (total: 22.2 Gb) following this optimized protocol.
Lanfear R., Schalamun M., Kainer D., Wang W. & Schwessinger B. (2019). MinIONQC: fast and simple quality control for MinION sequencing data. Bioinformatics 35(3):523-525.