TITLE: High quality DNA from Fungi for long read sequencing e.g. PacBio
AUTHORS: Benjamin Schwessinger

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Extraction of high quality DNA for long read sequencing e.g. PacBio</div><div class = "text-block">Optimized for DNA extraction from wheat stripe rust spores and also tested on barley leaf rust.</div><div class = "text-block">Buffers are best when fresh and not older than 3-6 months. Buffered Phenol:Chloroform:Isoamylalcohol (25:24:1) should not be older than 3 months.</div><div class = "text-block">Critical steps to obtain high quality DNA:</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Do NOT heat samples during DNA extractions! Perform all steps at RT or 4oC as indicated.</li><li style = "counter-reset:ol0;list-style-type:disc;">Do NOT incubate samples with KAc for prolonged time periods</li><li style = "counter-reset:ol0;list-style-type:disc;">Perform two steps of buffered Phenol:Chloroform:Isoamylalcohol purification to reduce co-purifying metabolites.</li></ul></div><div class = "text-block">DNA fragments were well above the 40kb mark based on Pippin Pulse Gels. The sequencing center performed a second AMPure purification step before library construction. Summary statistics of sequencing runs to follow.</div></div>

[STEPS]
?. [Extraction I]
Make lysis buffer by mixing buffer A+B+C (2.5:2.5:1 + 0.1%PVP final) and briefly heat to 64 °C. Let cool to room temperature for use in 50mL Falcon tubes.All following steps are based on 17.5ml lysis buffer as starting volume.
?. [Extraction I]
add 10uL (10kU) RNAse T1 to lysis buffer
?. [Extraction I]
Grind tissue/spores with liquid nitrogen in a mortar with sand, use 1g of sand per 100mg of starting material. Grind for 2 mins in 4x 15 sec bursts adding liquid nitrogen after each 15 sec grinding burst.
?. [Extraction I]
Transfer powder to 50mL Falcon containing lysis buffer and RNAse, mix well by vortexing
?. [Extraction I]
Incubate at RT for 30 mins mixing by inversion every 5 mins
?. [Extraction I]
Add 200uL Proteinase K, incubate at RT for 30 mins mixing by inversion every 5 mins
?. [Extraction I]
Cool on ice for 5 mins
?. [Extraction I]
Add 3.5 mL (0.2 vol) of KAc 5M, mix by inversion, incubate on ice for max 5 mins
?. [Extraction I]
Spin at 4oC and 5000g for 12 mins
?. [Extraction I]
Transfer supernatant to fresh Falcon tube containing 17.5ml (1vol) (P/C/I) and mix by inversion for 2 mins
?. [Extraction I]
Spin at 4 °C and 4000g for 10 mins
?. [Extraction I]
Transfer supernatant (might be milky but do not worry) to fresh Falcon tube containing 17.5ml (1vol) P/C/I and mix by inversion for 2 mins
?. [Extraction I]
Spin at 4 °C and 4000g for 10 mins
?. [Extraction I]
Transfer supernatant (~17mL) to fresh Falcon tube and add 5uL RNAse T1
?. [Extraction I]
Incubate for 20-30mins at RT
?. [Extraction I]
Add 1.8mL (~0.1vol) NaAc and mix by inversion
?. [Extraction I]
Add 18mL (~1vol) RT isopropanol and mix by inversion
?. [Extraction I]
Incubate at RT for 5-10mins
?. [Extraction I]
Spin at 4 °C and 10000g for 30 mins
?. [Extraction I]
Carefully pipette off supernatant till about 1-2 mL left, DNA will form a mostly translucent to white film/pellet at the bottom of the tube
?. [Extraction I]
Use 1mL pipette tip to transfer pellet and remaining liquid into fresh 1.7mL eppendorf tube. If the pellet got loose during transfer add 1.5mL fresh 70% EtOH to the 50mL Faclon and spin for 5min at 4000g. Remove 1mL and transfer the remaining volume and DNA pellet to same 1.7mL eppendorf tube.
?. [Extraction I]
Spin in table top centrifuge for 5 mins at 13000g
?. [Extraction I]
Remove supernatant with pipette and wash with 1.5mL fresh 70% Ethanol, invert several times to dislodge pellet
?. [Extraction I]
Spin in table top centrifuge for 5 mins at 13000g
?. [Extraction I]
Remove supernatant with pipette and wash with 1.5mL fresh 70% Ethanol, invert several times to dislodge pellet
?. [Extraction I]
Spin in table top centrifuge for 5 mins at 13000g
?. [Extraction I]
Remove supernatant with pipette
?. [Extraction I]
Spin in table top centrifuge for 1 min at 13000g
?. [Extraction I]
Remove remaining ethanol with pipette
?. [Extraction I]
Air-dry pellet for 7 mins
?. [Extraction I]
Add 200uL of 10mM Tris pH9 leave at RT for 3 hours
?. [Extraction I]
Flick tube slightly for mixing and add 200uL of TE buffer. DO NOT! vortex as it shears DNA.
?. [Extraction I]
leave at RT over night
?. [Extraction I]
Next day add another 100uL TE buffer and incubate for 1h at 28 °C with 1400rpm shaking
?. [Extraction I]
Measure dsDNA concentration using BR Qubit and measure absorbance with Nanodrop. At this point Qubit to Nanodrop ratios were 1/10 -1/20.This might be also a good step to assess DNA quality by runing a 0.8% TBE agarose gel with 500ng dsDNA and a lamda-Hind-III ladder as control. If you have a Pulse Field Gel Electrophoresis around even better.
next step is 40. This has come out of order and will be fixed.
?. [Extraction II]
Elute in 10mM Tris pH8
?. [Extraction II]
Measure dsDNA concentration using BR Qubit and measure absorbance with Nanodrop
?. [Extraction II]
At this stage Qubit to Nanodrop ratios were 0.64, 260/280 1.87 and 260/230 1.37
?. [Results]
Samples were submitted to Ramaciotti (http://www.ramaciotti.unsw.edu.au/) sequencing centre in Sydney. Excellent personel performed quality control, prepared 15-20kb libraries and we ran 13 SMRT cells with P6 chemistry. Some summary statistics are shown below.
Summary statistics for all 12 SMRT cells combined ******************************************************************************** Total sequence amount: 1.263691e+10 Number of reads: 1.181543e+06 Median read length: 9632 Mean read length: 10695
Summary statistics for all 12 SMRT cells combined ******************************************************************************** Total sequence amount: 1.263691e+10 Number of reads: 1.181543e+06 Median read length: 9632 Mean read length: 10695
?. [Extraction II]
Use AMPure beads for secondary clean up at beads 0.45 (Vol/Vol) following the PacBio protocol.