TITLE: BIT495 PGS Individual Project Protocol
AUTHORS: Joslene Morgan

[DESCRIPTION]
This protocol is designed for sequencing DNA from human organ tissue samples. The information obtained from sequencing can then be used for various clinical applications.

[STEPS]
SECTION: Obtain sample from the field and preserve it (1)
2.     
 

SECTION: Obtain sample from the field and preserve it (1)
3. Cut organ tissue into 4-6 mm diameter pieces
SECTION: Obtain sample from the field and preserve it (1)
4. Prepare DMSO-salt solution
SECTION: Obtain sample from the field and preserve it (1)
4.1. 20% DMSO
SECTION: Obtain sample from the field and preserve it (1)
4.2. 0.25 M sodium–EDTA
SECTION: Obtain sample from the field and preserve it (1)
4.3. NaCl to saturation
SECTION: Obtain sample from the field and preserve it (1)
4.4. pH 7.5
SECTION: Obtain sample from the field and preserve it (1)
5. Add 3X volume of DMSO-salt solution to the tissue
SECTION: DNA extraction (2)
8. Zymo BashingBead Lysis Tubes 
SECTION: DNA extraction (2)
8.1. For every 2 mg of sample, add 550 μl BashingBead Buffer and 10 μl RNase A 20 mg/ml (invitrogen)
SECTION: DNA extraction (2)
8.2. Vortex max speed 7 min

SECTION: DNA extraction (2)
8.3. Centrifuge at 12,000 rcf for 1 min

SECTION: DNA extraction (2)
8.4. Retrieve 200 μl supernatant for column purification
SECTION: DNA extraction (2)
9. Column purification
SECTION: DNA extraction (2)
9.1. Mix supernatant with 3X volume of dna binding buffer
SECTION: DNA extraction (2)
9.2. Load samples in Zymo IC Spin columns
SECTION: DNA extraction (2)
9.3. Centrifuge 10,000 rcf 30 sec
SECTION: DNA extraction (2)
9.4. Wash with 500uL DNA wash buffer and centrifuge 10,000 rcf 30 sec
SECTION: DNA extraction (2)
9.5. Wash with 200 uL DNA wash buffer and centrifuge 10,000 rcf 30 sec
SECTION: DNA extraction (2)
9.6. Add 40uL zymo dna elution buffer and incubate at 37C for 1-3 min
SECTION: DNA extraction (2)
9.7. Centrifuge 10,000 rcf 30 sec
SECTION: Library preparation (3)
11. DNA repair and end prep
 
SECTION: Library preparation (3)
11.1. Prep DNA in nuclease free water: 1 μg/100-200 fmol input DNA, adjust volume 47uL with nuclease free water
SECTION: Library preparation (3)
11.2. Mix the following:
 
 

SECTION: Library preparation (3)
11.3. Thoroughly mix the reaction by gently pipetting and briefly spinning down.
SECTION: Library preparation (3)
11.4. Using a thermal cycler, incubate at 20°C for 5 minutes and 65°C for 5 minutes.
SECTION: Library preparation (3)
11.5. Resuspend the AMPure XP Beads (AXP) by vortexing.
SECTION: Library preparation (3)
11.6. Transfer the DNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
SECTION: Library preparation (3)
11.7. Add 60 µl of resuspended the AMPure XP Beads (AXP) to the end-prep reaction and mix by flicking the tube.
SECTION: Library preparation (3)
11.8. Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
SECTION: Library preparation (3)
11.9. Prepare 500 μl of fresh 80% ethanol in nuclease-free water.
SECTION: Library preparation (3)
11.10. Spin down the sample and pellet on a magnet until supernatant is clear and colourless. Keep the tube on the magnet, and pipette off the supernatant.
SECTION: Library preparation (3)
11.11. Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
SECTION: Library preparation (3)
11.12. Repeat the previous step.
SECTION: Library preparation (3)
11.13. Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
SECTION: Library preparation (3)
11.14. Remove the tube from the magnetic rack and resuspend the pellet in 61 µl nuclease-free water. Incubate for 2 minutes at room temperature.
SECTION: Library preparation (3)
11.15. Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
SECTION: Library preparation (3)
11.16. Remove and retain 61 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
SECTION: Library preparation (3)
12. Adaptor ligation and cleanup
SECTION: Library preparation (3)
12.1. Mix the following:
 


SECTION: Library preparation (3)
12.2. Thoroughly mix the reaction by gently pipetting and briefly spinning down.
SECTION: Library preparation (3)
12.3. Incubate the reaction for 10 minutes at room temperature.
SECTION: Library preparation (3)
12.4. Resuspend the AMPure XP Beads (AXP) by vortexing.
SECTION: Library preparation (3)
12.5. Add 40 µl of resuspended AMPure XP Beads (AXP) to the reaction and mix by flicking the tube.
SECTION: Library preparation (3)
12.6. Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
SECTION: Library preparation (3)
12.7. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when clear and colourless.
SECTION: Library preparation (3)
12.8. Wash the beads by adding either 250 μl Long Fragment Buffer (LFB) or 250 μl Short Fragment Buffer (SFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.
SECTION: Library preparation (3)
12.9. Repeat the previous step.
SECTION: Library preparation (3)
12.10. Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
SECTION: Library preparation (3)
12.11. Remove the tube from the magnetic rack and resuspend the pellet in 15 µl Elution Buffer (EB). Spin down and incubate for 10 minutes at room temperature. For high molecular weight DNA, incubating at 37°C can improve the recovery of long fragments.
SECTION: Library preparation (3)
12.12. Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
SECTION: Library preparation (3)
12.13. Remove and retain 15 µl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.
SECTION: Sequencing with MinION (3)
14. Prep flow cell priming mix according to the table below:

 


SECTION: Sequencing with MinION (3)
15. Draw 20-30uL from priming port to remove any air bubbles
SECTION: Sequencing with MinION (3)
16. Load 800uL of the priming mix into the priming port and wait 5 minutes
SECTION: Sequencing with MinION (3)
17. Load 200uL of priming mix into the priming port
SECTION: Sequencing with MinION (3)
18. Prep library for loading according to table below:


 


SECTION: Sequencing with MinION (3)
19. Add 75uL of prepped sample to SpotON port dropwise, hovering above the port
SECTION: Analysis with EPI2ME
21. Concatenate the data and upload the resulting file to EPI2ME
SECTION: Analysis with EPI2ME
20. 
 

SECTION: DNA extraction (2)
7. 
 
 

SECTION: Library preparation (3)
10.     
 
 
 
  
 
 

SECTION: Sequencing with MinION (3)
13. 
 

SECTION: Overview
1. 
 

SECTION: Obtain sample from the field and preserve it (1)
6. When ready for next steps, rinse tissue with distilled water
SECTION: Analysis with EPI2ME
22. Run workflow(s). Some options are listed in the substeps below, but you can browse EPI2ME and choose others based on your experimental goals.
SECTION: Analysis with EPI2ME
22.1. wf-human-variation
Small variant calling, structural variant calling, CNV calling, STR expansion genotyping
SECTION: Analysis with EPI2ME
22.2. wf-somatic-variation
Analyze variation between tumor and normal DNA. Identifies potential SNVs, structural variants, and modified sites
SECTION: Analysis with EPI2ME
22.3. Various specialized workflows for certain infectious diseases and viruses