This week I'm learning how to use a Flongle Flow Cell to sequence genes found in the cultures I have been keeping alive from Fairhaven Dock, Bellingham WA.
Based on microscopy, we think that we see crytophytes (see SEM image below).
However, the cells are very small. We attempted to identify them with SEM, but the cells are too fragile for the sample prep we are able to do in the lab.
Therefore, we are going to utilize Nanopore technology.
The above image is an SEM image of cryptophytes found on Wikipedia.
The image above is from Churko et al., 2013. The image depicts how the Nanopore technology uses pores inside a membrane to read nucleotides along a strand of DNA.
The Nanopore Ligation Sequencing kit is a method for preparing sequencing libraries from double stranded DNA (dsDNA). You start with 1ug of genomic DNA (gDNA) . The gDNA is the chromosomal DNA of an organism. It represents the bulk of an organism's genetic material. It is distinct from bacterial plasmid DNA, complementary DNA, or mitochondrial DNA.
With the Ligation Sequencing Kit from Nanopore technologies, DNA ends are repaired and dA-tailed using the NEBNext END Repair/dA-tailing module. And sequencing adapters are ligated.
Ligation is to join molecules together with a new chemical bond (see schematic where “A” and “D” are added to the ends of the double stranded DNA)
dA-tailed is tailed oligonucleotide probes
Prior to sequencing, DNA repair increases library yield. In our workflow, DNA repair is accomplished using NEBNext FFPE DNA Repair Mix.
This mix contains enzymes that repair deamination of main bases found in DNA and RNA. NEBNext Ultra II End Repair/dA-Tailing Module is also used at this step to repair the 3’ and 5’ ends of the DNA (see image below).
"A base pair refers to two bases which form a "rung of the DNA ladder." A DNA nucleotide is made of a molecule of sugar, a molecule of phosphoric acid, and a molecule called a base. The bases are the "letters" that spell out the genetic code. In DNA, the code letters are A, T, G, and C, which stand for the chemicals adenine, thymine, guanine, and cytosine, respectively. In base pairing, adenine always pairs with thymine, and guanine always pairs with cytosine."
The above quote is from: https://www.ncbi.nlm.nih.gov/Class/MLACourse/Modules/MolBioReview/basepair.html
This NEBNext Ultra II End Repair/dA-Tailing Module converts fragmented DNA to repaired DNA that has 5’ phosphorylated and 3’ dA-tailed ends.
The dA-tailing allows fragmented DNA strands that were not repaired by the FFPE mix to be sequenced.
Also, DNA CS is included in this reaction to act as a positive control. DNA CS consists of a standard DNA sequence that can be used to provide quality control for sequencing and alignment.
Purification is accomplished using magnetic AMPure XP beads.
Above image is from https://labplan.ie/product/ampure-xp/.
AMPure XP beads selectively bind RNA and DNA and a magnetic stand is used to separate bead-bound DNA from contaminants (excess nucleotides, oligonucleotides, salts and enzymes) left behind in the buffer. The DNA product is then eluted in nuclease-free water and can used in downstream steps.
These are reagents not included with the Ligation Kit that can be ordered from NEB:
NEBNext FFPE DNA Repair Mix.
NEBNext Ultra II End Repair/dA-Tailing Module
AMPure XP beads
T4 Ligase from NEBNext Quick Ligation Module
Instructions for DNA repair and end-prep:
Thaw DNA at room temperature, mix by pipetting, and place on ice
Prepare the following:
The NEBNext FFPE DNA Repair Mix is a cocktail of enzymes formulated to repair DNA, and specifically optimized and validated for repair of FFPE DNA samples.
NEBNext Ultra II End repari/dA-tailing reagents are optimized to convert 500 pg-1 μg of fragmented DNA to repaired DNA having 5′ phosphorylated, 3′ dA-tailed ends. The module is optimized for use with the NEBNext Ultra II Ligation Module (NEB #E7595), and is part of the Ultra II DNA workflow which enables high yield preparation of high quality libraries from 500 pg to 1 µg of input DNA.
3. Prepare the DNA in Nuclease-free water
4. Prepare DNA CS (yellow labeled tub, also see schematic below)
5. Resuspend AMPure XP beads by vortexing
6. Transfer the DNA sample to clean tube
7. Add 60uL of beads to the end-prep reaction
8. Incubate for 5 minutes
9. Spin down the sample and pellet on a magnet
10. Keep the tube on the magnet and wash the beads with 200uL of freshly prepared 70% ethanol
11. Resuspend the pellet in 61uL nuclease-free water
12. Quantify using a Qubit
The adapter ligation step:
The Adapters need to be ligated to help the DNA find and bind to the nanopores on the flow cell.
Adapter Mix F (AMX-F; green labeled tube; see schematic below), Ligation buffer (LNB; gray labeled tube; see schematic below), Elution buffer (EB), and S fragment buffer (SFB) are in the kit.
In a 1.5mL Eppendorf DNA LoBind tube mix DNA sample, LNB, NEBNext Quick T4 DNA Ligase, and AMX-F. Resuspend beads by vortexing. Add 40uL of resuspended AMPure XP beads to the reaction and mix. Spin down sample and pellet on a magnet.
Wash the beads:
Add 250uL Long -or- Short Fragment Buffer, resuspend, spin down, and pellet on magnet. Remove and retain 15uL of eluate containing the DNA library into a clean tube. Quantify using Qubit.
Using the library into a flow cell:
The flow cell is a consumable used with MinION devices. It contains the proprietary sensor array, Application-Specific Integrated Circuit (ASIC) and R9.4.1 nanopores suitable for all 1D experiments.
"A user simply adds their prepared library to the flow cell and starts their sequencing experiment. " (Nanopore)
The Sequencing Buffer II (SBII), Loading Beads II (LBII), Loading Solution (LS), Flush Tether (FLT), and Flush Buffer (FB) are in the kit.
Thaw SBII, LBII, LS, FLT, and FB at room temperature.
Mix SBII, FB, FLT by vortexing and spin down.
Above image is from Nanopore store website: https://store.nanoporetech.com/us/flowcells/spoton-flow-cell-mk-i-r9-4.html
References and Resources:
Churko et al., 2013, Overview of High Throughput Sequencing Technologies to Elucidate Molecular Pathways in Cardiovascular DiseasesCirc Res; 112(12): . doi:10.1161/CIRCRESAHA.113.300939.
http://www.science.smith.edu/cmbs/wp-content/uploads/sites/36/2018/12/MinION-Genomic-Sequencing-s.pdf