May 16, 2024
Ultra-Long Sequencing of Yeast Cells (S. cerevisiae) on ONT Sequencers – A Modified FindingNemo Protocol
Forked from a private protocol
- 1University of Nottingham
Protocol Citation: Inswasti Cahyani 2024. Ultra-Long Sequencing of Yeast Cells (S. cerevisiae) on ONT Sequencers – A Modified FindingNemo Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzjbk5lx1/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: Working
We use this protocol and it's working
Created: January 19, 2023
Last Modified: May 16, 2024
Protocol Integer ID: 99850
Funders Acknowledgement:
Wellcome Trust
Grant ID: ANDREX project
Abstract
This protocol is a bespoke modification of the FindingNemo protocol (LongRead Club) to enable ultra-long (UL) sequencing of yeast on Nanopore sequencers.
We have tested and optimised this in Saccharomyces cerevisae using the obsolete SQK-ULK001 kit.
It's not tested yet but we think the new ULK114 can be used to replace ULK001, using exact volumes of the corresponding reagents.
Let us know if you have tried it with the new kit!
Our best read N50 so far was ~67 kb (look at profile pic). This is not (yet) ultra-long, but may also be due to the distribution of chromosome sizes which is generally smaller than more complex eukaryotes, e.g. mammals.
Materials
A. Chemicals/compounds
- Sorbitol
- Na2HPO4
- 5M Ammonium Acetate Sigma – Aldrich Catalog #A-7330
- Tris-HCl pH 8.0
- 10mM Tris-HCl pH 9.0
- Ethanol Absolute Honeywell Catalog #32221-2.5L
- Isopropanol Absolute Fisher Scientific Catalog #P/7500/15
- 1X Phosphate Buffer Saline Fisher Scientific Catalog #15453819
- Proteinase K, 2mL Qiagen Catalog #19131
- RNase A Qiagen Catalog #19101
- NaCl (5 M) RNase-free Thermo Fisher Scientific Catalog #AM9759
- EDTA (0.5 M, pH 8.0, nuclease-free) Thermo Fisher Scientific Catalog #AM9260G
- Cetyltrimethylammonium Bromide (CTAB) MP Biologicals Catalogue #02194004-CF
- Lyticase 5U/ul
- Hexamminecobalt(III) Chloride Alfa Aesar Catalog #A15470
- 40% Polyethylene Glycol MW 8000 Sigma – Aldrich Catalog #P1458
- ONT Ultra long kit #SQK-ULK001
B. Buffers
SpheroBuffer
- 1M Sorbitol
- 100 mM Na2HPO4
- 100 mM EDTA pH 8.0
Lysis Buffer
- 2M NaCl
- 20mM Tris-HCl pH 8.0
- 50mM EDTA pH 8.0
- 4% (w/v) CTAB
PEGW Buffer
- 10% PEG-8000
- 0.5M NaCl
C. Disposables
- DNA LoBind Tubes, 1.5 mL Eppendorf Catalog #0030108051
- DNA LoBind Tubes 2.0ml PCR Clean Eppendorf Tubes Eppendorf Catalog #0030 108.078
- Glass Beads 3 mm Scientific Laboratory Supplies Ltd Catalog #DD68501
- Wide-bore (or cut off) P1000 and P200 tips
- Monarch Bead Retainers New England Biolabs Catalog #T3004L (optional), or use 0.5 ml PCR tube cut 2-3 mm from the bottom to make a bead retainer
- Monarch Collection Tubes II - 100 tubes New England Biolabs Catalog #T2018L (optional), or use any 1.5 ml centrifuge tube as a collection tube
Spheroplasting
Spheroplasting
In a 2 ml DNA LoBind tube, pellet ~200 million cells (50 µL circa volume of pellet per reaction).
Wash cells in cold PBS and centrifuge at 10.000 x g, 00:01:00
1m
Remove supernatant.
Repeat step 2-3 once more.
Resuspend pellet in 480 µL SpheroBuffer.
Add 20 µL lyticase (5U/μl), mix well with a wide-bore pipette tip and incubate at 30 °C 00:30:00 . Check spheroplast formation from time to time (if this has not yet been optimised before).
30m
Cell Lysis
Cell Lysis
Add 480 µL lysis buffer (LB) and 20 µL Proteinase K, carefully mix with wide-bore pipette tip while avoiding any bubbles.
Incubate at 56 °C for 00:15:00 whilst shaking at 700 rpm . The solution should clear up when protein digestion takes place.
15m
Add 4 µL RNaseA and mix with a wide-bore pipette tip while avoiding any bubbles.
Incubate at 56 °C for 00:10:00 .
10m
Add 400 µL 5M Ammonium acetate and 100 µL 5M NaCl
Mix by vertical rotation (Hula mixer) 9 rpm, Room temperature , 00:02:00
Centrifuge at 16.000 x g, Room temperature, 00:10:00 .
10m
Transfer the supernatant to a new 2 ml tube, careful not to transfer protein layer/precipitate at the bottom. If lysate is too thick to have layers, move as much clear lysate as possible.
DNA Precipitation
DNA Precipitation
Add 3 glass beads (diameter = 3mm) to the cell lysate and 500 µL isopropanol.
Note
If the tube cannot accommodate 500 µl isopropanol, add as much isopropanol as possible (>400 ul). Then after DNA precipitation, take 200-300 µl from the upper part and replace with the same amount of isopropanol and rotate again to further precipitate DNA.
Mix on a Hula mixer at 9 rpm, 00:05:00 this can be extended to 10 minutes .
Check that the DNA has bound tightly around the beads and let sit for another minute.
Remove liquid by pipetting or carefully tipping it over while guarding the beads with the pipette tip.
Wash bound DNA with 1 mL of 70% ethanol, rotate the tube 2-3 times then discard the wash buffer.
Repeat wash once more.
Remove ethanol as much as possible, pour beads into a retainer tube on a collection column. Quickly spin (less than 1 second) on a table-top minicentrifuge.
Note
Alternatively, instead of spinning, just absorb excess ethanol with a wipe.
Quickly pour beads to a new 2 ml tube previously aliquoted with 100-120 µL 10mM Tris-HCl pH 9.0
Note
If handling multiple samples, do step 21-22 one sample at a time. Do not let the DNA on the beads to dry as it makes DNA elution and homogenization more difficult.
Incubate at 37 °C for at least 00:30:00 with regular pipetting every 10-15 min using a wide-bore tip.
30m
Pour the beads into a bead retainer on a new 1.5 ml tube and spin at maximum speed for 00:01:00 .
1m
Incubate at Room temperature for a few hours or Overnight (slow rotation may aid elution).
2h
HMW DNA QC
HMW DNA QC
An accurate measurement of DNA concentration is important as this will determine the
optimum ratio of transposase (FRA) to DNA molecules at the library prep step. Also, the
viscous nature of UHMW DNA requires that sample measurement represents all parts of the
DNA solution.
Two nucleic acid quantification methods, i.e., fluorometric (Qubit) and spectrophotometric
(Nanodrop), can be used in parallel to assess both the quantity and quality of the extracted
DNA.
Measure DNA concentration with Qubit.
Note
(Optional) measure RNA concentration to check that RNase treatment works efficiently. Reading should show only traces amount or <100% of [DNA]. If >100%, DNA concentration needs to be adjusted by this, e.g. halved.
For Nanodrop measurement, measure 3 positions from top, middle, and bottom part of the sample. Percent CV can then be calculated from these concentrations to gauge DNA homegeneity.
Note
If %CV is still >=100%, homogenise by more regular pipetting
and/or rotation at 9 rpm for few hours up to overnight as before.
Whenever possible, the quality of extracted DNA sample should be analysed by method(s) that
enable visual inspection of molecule length distribution such as:
Regular agarose gel electrophoresis
Pulsed-Field Gel Electrophoresis, e.g., using Pippin Pulse (Sage Science)
Agilent Bioanalyzer DNA
Agilent TapeStation DNA
Tagmentation and Adapter Ligation
Tagmentation and Adapter Ligation
Take 5 µg DNA and dilute with 10 mM Tris-HCl pH 9.0 to a total volume of 180 µL . Mix well or rotate and incubate for 10 min. Cool sample on ice.
Note
Final DNA concentration should be between 20-40 ng/µL. Scale-up (or down) final volume if necessary.
DNA sample has to be ice cold when mixing with FRA.
In another tube make up FRA dilution (1.2 µl FRA per microgram of yeast DNA):
| A | B | |
| Volume (µL) | ||
| FRA | 6 | |
| FDB (FRA Dilution Buffer) | 55 | |
| Total volume | 60 |
Mix the diluted FRA by vortexing. Cool on ice.
On ice, add 60 µL of the diluted FRA to the extracted DNA. Stir the reaction with the pipette tip whilst expelling the diluted FRA to ensure an even distribution.
Mix by gentle pipetting with a wide-bore pipette tip.
Note
Make sure sample is homogeneous and cold while mixing with FRA. The enzyme is fast acting and we want to make sure it difuses evenly through the DNA molecules before incubation.
Incubate the reaction as follows:
22 °C - 00:10:00
70 °C - 00:05:00
22 °C - 00:05:00
20m
Add 5 µL of RAP to the pooled sample with a regular pipette tip. Use a wide-bore tip to pipette mix. Visually check to ensure the reaction is thoroughly mixed.
Incubate for 00:30:00 at 22 °C .
30m
Nemo Clean-up
Nemo Clean-up
1h 34m
Add 3 glass beads into the tube with the adapted DNA.
Add 1:1 volume (240 µL ) of 10 mM CoHex.
Rotate tube at 9 rpm, 00:03:00 . Make sure there is no bead stuck at the bottom or at the cap, flick if this is the case.
Invert the tube again 2-3 times to ensure all DNA has precipitated and is tightly bound to the beads.
Discard supernatant. Take care not to disturb the DNA precipitated onto the beads.
Wash the glass beads by gently adding 750 µL PEGW buffer and gently invert 2-3 times.
Incubate for 00:03:00 at room temperature.
3m
Aspirate and discard the supernatant, taking care not to disturb the DNA precipitate.
Repeat wash once more.
Pipette 120 µL of EB into a clean DNA LoBind 2 ml tube.
Note
Can also use Tris-HCl pH 9.0
Discard the second wash buffer as much as possible.
Quick spin the tube and use a 10 µl tip to discard the rest of the buffer at the bottom of the tube (move the beads aside with the tip while doing it). There will be a few microliters buffer left as a dead volume. This will not affect sequencing.
Immediately pour the beads into the 2 ml tube with the elution buffer.
Incubate the library at 37 °C for 00:30:00 . Gently aspirate and dispense the eluate over the glass beads at regular intervals with a wide-bore pipette tip to aid elution.
30m
Continue with overnight incubation at room temperature.
The next day, insert a bead retainer into a clean 1.5-ml tube.
Pour the beads and spin at 10.000 x g, 00:01:00 .
1m
Incubate for at least 00:30:00 at Room temperature with regular pipette mixing.
30m
Quantify the library
Load 30-40 µL library with SQB in 1:1 volume ratio.
Note
For PromethION flow cell, DNA volume needs to be scaled-up into a total 150 µl. Use EB or Tris-HCl pH 9.0 for this.
Incubate for 00:30:00 at Room temperature
30m
Load the library on a primed MinION flow cell.