TITLE: Workflow for generating HMW plant DNA for third generation sequencing with high N50 and high accuracy
AUTHORS: Patrick Driguez, Karen Carty, Alexander Putra, Luca Ermini

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We have developed a method based on the Qiagen Genomics-Tip kit for the extraction of HMW DNA from a wide range of plants suitable for third generation sequencing platforms such as PacBio and Oxford Nanopore. This HMW plant DNA was used to make sequencing libraries with high insert N50 (30-40 Kb) for GridION and PromethION Oxford Nanopore machines and Sequel I and Sequel II PacBio machines. This HMW DNA is also suitable for PacBio HiFi libraries. The resulting sequencing data is ideally suited for de novo assembly of plant genomes. </div></div>

[STEPS]
?. [Sample Preparation]
In a tray add excess liquid nitrogen to mortar and pestle. Allow most of the liquid nitrogen to evaporate and bring mortar and pestle down to cryogenic temperatures.
?. [Sample Preparation]
Grind > 1 g of frozen leaves to a very fine powder in mortar and pestle.Add small amounts of liquid nitrogen, as necessary, to maintain sample, and mortar and pestle at cryogenic temperatures.Resuspending powder in a small amount of liquid nitrogen is also very effective for pouring and transferring samples to storage tubes.
?. [Sample Preparation]
Process sample straight away using below protocol or return to -80°C for storage.
?. [Sample Preparation]
For best results use leaves that have been dark-treated for > 48 hours, freshly picked, weighed, flash frozen in liquid nitrogen and stored at -80°C
?. [DNA Extraction: Preparation]
Add the Lysis buffer to the ground powder depending on quantity and type of columns to be used for purification.Resuspend by inverting gently end-over-endDispense to multiple tubes if necessary.
?. [DNA Extraction: Preparation]
Add Proteinase K per 100/G column or  per 500/G column
500 µl
1000 µl
?. [DNA extraction: Lysis]
Incubate > 3.5 h at 50°C with slow rocking. Place the tube with a slight tilt so that the liquid moves very slightly during rotations. Note: The tube can easily leak, check the tube condition periodicallyWe use a platform rocker in a preheated incubator/shaker (with NO SHAKING)
50 °C
?. [DNA Extraction: Pre-Purification]
Preheat buffer QF in falcon tube in a  waterbath:  per 100/G column or  per 500/G column
50 °C
5 mL
15 mL
?. [DNA Extraction: Purification]
Take the lysed samples from the incubator
?. [DNA Extraction: Purification]
Centrifuge the 50ml tubes containing the samples for 15 minutes at room temp at maximum speed (3220g / 4000 rpm) with acceleration and deceleration at maximum (=9)After centrifugation minimal debris should be in the supernatantIf large amounts of debris still present repeat centrifugation up to 15 min
Centrifuge: 3220 34, 15 min
?. [DNA Extraction: Purification]
Pour the supernatant in the pre-calibrated Genomic tip column and allow the supernatant to pass through the column.
?. [DNA Extraction: Purification]
Add 1 ml of wash buffer QC by running along the walls of the tubes in order to precipitate any debris stuck on the walls. Remove the debris from the bottom by aspirating If debris still present wash with another of QC buffer
1 mL
?. [DNA Extraction: Purification]
Wash the column twice with buffer QC:2 x of buffer QC 100/G column.2 x of buffer QC 500/G column.
7.5 mL
15 mL
?. [DNA Extraction: Purification]
Transfer the column to a clean 50 ml Falcon tube.
?. [DNA Extraction: Purification]
Elute with elution buffer QF previously pre-heated at 50°Cof buffer QF per 100/G columnof buffer QF per 500/G column
5 mL
15 mL
?. [DNA Extraction: Purification]
Remove column and precipitate the DNA by adding 2-propanol (isopropanol) along the wall of the tube:per 100/G columnper 500/G column
3.5 mL
10.5 mL
?. [DNA Extraction: Purification]
Mix gently by slowly inverting the tube to form a visible mass of HMW DNA floating in solution (a “jellyfish”)You should observe the formation of the jellyfish during the process of inverting the tubes. Once the pellet HMW DNA is compacted slightly, stop mixing.Note: If the jellyfish is not formed, proceed to Precipitation of DNA with cold centrifugation (see below). Note: If the jellyfish is not formed, proceed to Precipitation of DNA with cold centrifugation (see below).
?. [DNA Extraction: Purification]
Capture the pellet HMW DNA with a disposable inoculation loop.This can be difficult and the pellet can easily be lost during transfers between tubesTry to trap the DNA pellet in the centre of the loop.
?. [DNA Extraction: Purification]
Transfer the DNA on the loop to a 1.5ml tube containing of fresh 80% ethanol.This step should remove starch and improve the quality of the DNA. Use separate tubes of ethanol for different samples
1 mL
?. [DNA Extraction: Purification]
Soak for 1 min
?. [DNA Extraction: Purification]
Air dry for 30 seconds approximately
?. [DNA Extraction: Purification]
Repeat twice more in two separate tubes of 80% ethanol
?. [DNA Extraction: Purification]
Air dry the DNA pellet for 30-60 seconds allowing most of the ethanol to evaporate
?. [DNA Extraction: Purification]
Dissolve DNA pellet in  to  of TE buffer in a new 1.5ml tube.Cut off the end of the loop with scissorsVolume of TE can be modified according to the amount of estimated DNA (size of the pellet)Several pellets can be combined
50 µl
100 µl
?. [DNA Extraction: Purification]
Leave overnight at room temperature to allow resuspension of the DNA.
?. [Day 2: Quantification]
Remove loop and carefully homogenize the DNA solution with wide bore tips. Use slow aspiration and discharge: 3 seconds up and 3 seconds down.DNA should be into solution; look for a clear solutionIf DNA is still not resuspended continue with resuspensionResuspension on a slow shaker or incubation at 4°CDon’t apply heat (even 37°C can degrade HMW DNA)
?. [Day 2: Quantification]
Quantify the DNA and check the qualityUse ds BR Qubit assay for DNA quantification Use Nanodrop to check the quality:A 260/280 ratio of 1.8-2.0 and 260/230 ratio of 2.0-2.2 is idealThis measures contamination with carbohydrates and other contaminants. The 260/230 ratio must be > 2 as sugars disrupt the polymerase during sequencing.Use Nanodrop to check the quality:A 260/280 ratio of 1.8-2.0 and 260/230 ratio of 2.0-2.2 is idealThis measures contamination with carbohydrates and other contaminants. The 260/230 ratio must be > 2 as sugars disrupt the polymerase during sequencing.- If 260/230 is low proceed with a gentle AMPure PB Bead clean and re-do the Nanodrop. Only very high molecular weight DNA is lost.
?. [Day 2: Quantification]
Run Pulse Field Gel Electrophoresis or FEMTO Pulse analysis of your DNA to check the size distribution of DNA.
?. [DNA Extraction: Preparation]
The following protocol is based on the "QIAGEN Genomic DNA Handbook" available at www.qiagen.com. We follow the recommendations for tissue with modifications.For 1 g of ground leaf tissue prepare enough Lysis Buffer for 4 x 100/G, or 1-2 x 500/G tip columns.
?. [DNA Extraction: Purification]
Calibrate the 100/G or 500/G columnsPut the column inside the placolumneeve on top of a 50ml Falcon tube (see figure in Supplementary section)Calibrate the column with the calibration buffer QBT:Add   of buffer QBT per 100/G columnAdd  of buffer QBT per 500/G columnAll QBT needs to go through the column. This step is quite quickAdd  of buffer QBT per 100/G columnAdd of buffer QBT per 500/G column
4 mL
10 mL
?. [Precipitation of DNA with cold centrifugation (Optional)]
Note: If a jellyfish of DNA was not present after addition of 2-propanol   this may indicate a poor quality extraction, dependent on species of plant, and a re-extraction may be appropriate.
?. [Precipitation of DNA with cold centrifugation (Optional)]
Prechill a high speed centrifuge to
4 °C
?. [Precipitation of DNA with cold centrifugation (Optional)]
Centrifuge the samples
Centrifuge: 5000 34, 15 min, 4 10
?. [Precipitation of DNA with cold centrifugation (Optional)]
Remove the supernatant and wash with 80% ethanol.
1 mL
?. [Precipitation of DNA with cold centrifugation (Optional)]
Carefully pour off supernatant, do not re-suspend. Drain at 45° on a paper towel to dry.
?. [Precipitation of DNA with cold centrifugation (Optional)]
Wash two more times.
?. [Precipitation of DNA with cold centrifugation (Optional)]
Centrifuge tha samples
Centrifuge: 5000 34, 10 min, 4 10
?. [Precipitation of DNA with cold centrifugation (Optional)]
Remove the supernatant and dry
?. [Precipitation of DNA with cold centrifugation (Optional)]
Drain on paper towel at 45°
?. [Precipitation of DNA with cold centrifugation (Optional)]
Resuspend in TE buffer Continue with resuspension and quality control of extracted DNA
?. [Day 2: Quantification]
Pulse Field Gel Electrophoresis1% agarose TBE gel over 24 hours (Initial switch 1 sec, finial switch 25 sec, 6 volt/cm, 120° included angle, Chef III, Bio-Rad, Hercules, CA, USA)Midrange PFG and Lambda PFG markers (N0342S, N0341S, NEB, Ipswich, MA, USA).
?. [Day 2: Quantification]
FEMTO Pulse analysis