Oct 07, 2024
BIT495 PGS Individual Project Protocol
- 1North Carolina State University
Protocol Citation: Joslene Morgan 2024. BIT495 PGS Individual Project Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxy65zl8j/v1
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: In development
I developed this protocol for my portable genome sequencing course at NC State.
Created: October 03, 2024
Last Modified: October 07, 2024
Protocol Integer ID: 109132
Keywords: Tissue, Sequencing, Preservation
Abstract
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.
Materials
DNA extraction:
- ZYMO Quick-DNA Tissue/Insect Microprep Kit
Library prep:
- Oxford Nanopore Technologies Ligation Sequencing Kit v14
Sequencing:
- MinION flow cell
Overview
Overview
The figure above shows an overview of the protocol. First, a sample from human organ tissue is obtained. This sample must be preserved until the rest of the protocol is ready to be done. Then, the DNA is extracted and the library is prepared. The sample is then sequenced and analyzed. The data obtained can be used for various clinical applications such as rare disease diagnosis, oncology, infectious diseases and microbiota, and transplantation (4) (5).
Obtain sample from the field and preserve it (1)
Obtain sample from the field and preserve it (1)
Note
The human organ tissue sample must be preserved so no degradation or contamination occurs during the transportation process, especially if the patient is not nearby to a lab that can sequence it. This protocol describes preservation with a DMSO-salt solution that will protect the sample and even prevent further fragmentation during the later extraction process.
Cut organ tissue into 4-6 mm diameter pieces
Prepare DMSO-salt solution
20% DMSO
0.25 M sodium–EDTA
NaCl to saturation
pH 7.5
Add 3X volume of DMSO-salt solution to the tissue
When ready for next steps, rinse tissue with distilled water
DNA extraction (2)
DNA extraction (2)
Note
This is a bead-bashing based protcol specialized for mammalian tissue. 18 minutes of bead-bashing (total time for protocol, 7 min original+6 min extension) yields 0.48ug DNA per mg of tissue. It utilizes Zymo's BashingBead technology followed by column purification. https://www.zymoresearch.com/products/quick-dna-tissue-insect-microprep-kit?srsltid=AfmBOopAxZU1gFOF_Liu6lnGJDXkYGfq7Gi0T_peogRr2-3Sfzim3JtX
Zymo BashingBead Lysis Tubes
For every 2 mg of sample, add 550 μl BashingBead Buffer and 10 μl RNase A 20 mg/ml (invitrogen)
Vortex max speed 7 min
Centrifuge at 12,000 rcf for 1 min
Retrieve 200 μl supernatant for column purification
Column purification
Mix supernatant with 3X volume of dna binding buffer
Load samples in Zymo IC Spin columns
Centrifuge 10,000 rcf 30 sec
Wash with 500uL DNA wash buffer and centrifuge 10,000 rcf 30 sec
Wash with 200 uL DNA wash buffer and centrifuge 10,000 rcf 30 sec
Add 40uL zymo dna elution buffer and incubate at 37C for 1-3 min
Centrifuge 10,000 rcf 30 sec
Library preparation (3)
Library preparation (3)
Note
This step describes library prep using Oxford Nanopore Technologies Ligation Sequencing Kit v14. This kit is good for long reads and helps repair any nicks in the DNA prior to sequencing. https://store.nanoporetech.com/us/ligation-sequencing-kit-v14.html
Ligation Sequencing Kit workflow (3)
Reagants included in Ligation Sequencing Kit (3)
DNA repair and end prep
Prep DNA in nuclease free water: 1 μg/100-200 fmol input DNA, adjust volume 47uL with nuclease free water
Mix the following:
(3)
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
Using a thermal cycler, incubate at 20°C for 5 minutes and 65°C for 5 minutes.
Resuspend the AMPure XP Beads (AXP) by vortexing.
Transfer the DNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
Add 60 µl of resuspended the AMPure XP Beads (AXP) to the end-prep reaction and mix by flicking the tube.
Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
Prepare 500 μl of fresh 80% ethanol in nuclease-free water.
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.
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.
Repeat the previous step.
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.
Remove the tube from the magnetic rack and resuspend the pellet in 61 µl nuclease-free water. Incubate for 2 minutes at room temperature.
Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
Remove and retain 61 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
Adaptor ligation and cleanup
Mix the following:
(3)
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
Incubate the reaction for 10 minutes at room temperature.
Resuspend the AMPure XP Beads (AXP) by vortexing.
Add 40 µl of resuspended AMPure XP Beads (AXP) to the reaction and mix by flicking the tube.
Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when clear and colourless.
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.
Repeat the previous step.
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.
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.
Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
Remove and retain 15 µl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.
Sequencing with MinION (3)
Sequencing with MinION (3)
Note
Next, the library is loaded onto a MinION flow cell and sequenced. Sequencing with MinION gives a good data output betweent that of the flongle and PromethION. Reagants for these steps were obtained from the Ligation Sequencing Kit used in the previous step. https://store.nanoporetech.com/us/ligation-sequencing-kit-v14.html
Prep flow cell priming mix according to the table below:
(3)
Draw 20-30uL from priming port to remove any air bubbles
Load 800uL of the priming mix into the priming port and wait 5 minutes
Load 200uL of priming mix into the priming port
Prep library for loading according to table below:
(3)
Add 75uL of prepped sample to SpotON port dropwise, hovering above the port
Analysis with EPI2ME
Analysis with EPI2ME
Note
The output data from the sequencing run is then analyzed on EPI2ME. EPI2ME can now be run locally or on the cloud.
Concatenate the data and upload the resulting file to EPI2ME
Run workflow(s). Some options are listed in the substeps below, but you can browse EPI2ME and choose others based on your experimental goals.
wf-human-variation
Small variant calling, structural variant calling, CNV calling, STR expansion genotyping
wf-somatic-variation
Analyze variation between tumor and normal DNA. Identifies potential SNVs, structural variants, and modified sites
Various specialized workflows for certain infectious diseases and viruses
Protocol references
(1) Noncryogenic Preservation of Mammalian Tissues for DNA Extraction: An Assessment of Storage Methods https://doi.org/10.1023/A:1014541222816
(2) A Rapid and Simple Bead-Bashing-Based Method for Genomic DNA Extraction from Mammalian Tissue https://doi.org/10.2144/btn-2019-0172
(3) Ligation sequencing DNA V14
https://nanoporetech.com/document/genomic-dna-by-ligation-sqk-lsk114
Additional sources for information on clinical applications:
(4) The application of long-read sequencing in clinical settings
https://doi.org/10.1186/s40246-023-00522-3
(5) Clinical Versus Research Sequencing
https://doi.org/10.1101/cshperspect.a025809