TITLE: An optimized protocol for sequencing mammalian roadkill tissues with Oxford Nanopore Technology (ONT)
AUTHORS: Marie-Ka Tilak, Rémi Allio, Frédéric Delsuc

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. Tissue sampling  and preservationTissues 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).
?. Tissue preparationBefore 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.
?. DNA extraction3.1 - Tissue lysis1/ 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 precipitation1/ 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 controlQuantify the DNA with Qubit® 2.0 Fluorometer (Thermo Fischer Scientific) and NanoDrop™ Spectrophotometer (Thermo Fisher Scientific).1/ NanoDrop ratios must be: 1.8 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-tubes1/ 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 repair1/ 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 concentration5/ Visualize DNA quality by migration on a 1% agarose gel (Figure 3).
?. 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 DNA2/ 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 sequencingMinIONQC resultsLanfear 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.