Nov 22, 2021
Version 1
Illumina Whole Genome Sequencing V.1
This protocol is a draft, published without a DOI.
- 1Lawrence Berkeley National Lab
- Agile BioFoundry
- LBNL
Protocol Citation: Rita Kuo 2021. Illumina Whole Genome Sequencing. protocols.io https://protocols.io/view/illumina-whole-genome-sequencing-bz9dp926
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
We are still developing and optimizing this protocol
Created: November 22, 2021
Last Modified: November 22, 2021
Protocol Integer ID: 55301
Keywords: Whole Genome Library Prep, Whole Genome Sequencing, Illumina
Abstract
This protocol is used to produce Ilumina libraries for whole genome sequencing. DNA input is 100 ng. User provide at least 300 ng of gDNA if it's possible to get enough materials for 2 attempts of library prep and sample QC. The enzymatic fragmentation reaction (for both Kapa and IDT kit) is very sensitive to the presence of EDTA, which must be removed or neutralized prior to fragmentation. EDTA in DNA preparations is usually introduced via elution buffers used in the final stages of the DNA extraction or purification process. The fragmentation condition of this protocol is for complex genome. Fragment size may reduce if the input DNA is from amplicon, plasmid or virus DNA.
Materials
Reagents
KAPA Hyperplus kit
Illumina Y adapter
KAPA DNA quantification kit
200 proof Ethanol
Nuclease free water
10 mM Tris-HCl (pH 8.0 – 8.5)
Ultra pure agarose
Disposables
PCR 8 stripe tubes
Tips
1.5 ml Tubes
Equipment
Thermocycler
Magnetic stand
Pipettes
Dano Drop
Qubit
Bioanalyzer
Sample QC
Sample QC
Use Dano Drop and Gel electrophoresis to quantify and qualify samples. If the sample is contaminated with RNA, inhibitors or degraded, do not proceed. A 260/280 ratio of ~1.8 is generally accepted as pure for DNA. If samples contain EDTA, proceed with bead clean up.
Bead clean up
Bead clean up
15m
15m
Bring the sample volume to 20 ul in PCR 8 stripe tubes
Add 18 ul of beads into the tube. Gently mix, incubate at room temperature for 5 minutes.
5m
Place the tube on a magnetic stand. Remove supernatant when it is clear.
Wash twice with freshly made 75 % Etoh and remove 75 % Etoh from the tube. Try to remove all residual ethanol without disturbing the beads.
Open the lid to let the bead dry at room temperature for 3 – 5 min, or until all of the ethanol has evaporated.
5m
Add 22 ul of 50 C nuclease free water. Quick vortex. Incubate samples in room temperature for 5 minutes.
5m
Repeat step 3-7 one more time.
Add 37 ul of 50 C nuclease free water. Quick vortex. Incubate samples in room temperature for 5 minutes.
Transfer 36 ul of eluted DNA to new 8 strip tubes.
Take 1 ul of the sample for Nanodrop measurement. If EDTA still presents in the sample, perform additional bead cleanup step using 1:1 sample bead ratio.
Enzymatic Fragmentation
Enzymatic Fragmentation
NOTE: If the DNA preparation does contain EDTA, dilute in the EDTA-containing buffer in which samples are currently suspended, in a total of 30 μL. To each reaction with 30 μL of EDTA-containing DNA, add 5 μL of diluted Conditioning Solution.
Assemble each fragmentation reaction on ice by adding the components in this order:
| A | B | C | |
| Component | Volume (ul) | ||
| Double-stranded DNA (with 5ul of conditioning solution if needed) | 35 | ||
| KAPA Frag Buffer (10X)* | 5 | ||
| KAPA Frag Enzyme* | 10 | ||
| Total volume | 50 |
Vortex gently by flicking the tubes and spin down briefly. Return the tube(s) to ice. Proceed immediately to the next step.
Incubate in a thermocycler at 37 °C for 3 mins and hold at 4 °C. Set the temperature of the heated lid to 50°C.
Transfer reactions to ice, and proceed immediately to End Repair and A-tailing.
End Repair and A-Tailing
End Repair and A-Tailing
IMPORTANT! The KAPA HyperPrep/Plus End Repair & A-tailing Buffer may contain white precipitates when thawed. Ensure the buffer is thoroughly vortexed until the precipitate has been resuspended. Heat at 37°C for 5 – 10 min, if indicated.
Pre-mix end repair & A-tailing buffer and enzyme based on the sample number. Add 10 ul of End Repair and A-tailing reaction premix to the fragmented DNA.
| Component | Volume(ul) | |
| Fragemented DNA | 50 | |
| End Repair & A-Tailing Buffer* | 7 | |
| HyperPrep/HyperPlus ERAT Enzyme Mix** | 3 | |
| Total volume | 60 |
Vortex gently and spin down briefly. Return the reaction tubes to ice. Proceed immediately to the next step.
Incubate in a thermocycler at 65 °C for 30 mins and hold at 4 °C. Set the temperature of the heated lid to 85°C.
Proceed immediately to Adapter Ligation. Keep 1 ul of the DNA for library QC.
Adaptor Ligation
Adaptor Ligation
IMPORTANT! The KAPA HyperPrep Ligation Buffer contains a high concentration of PEG 6000 and is very viscous. Small PEG 6000 droplets may be visible when thawed and require special attention during pipetting. Ensure the buffer is thoroughly vortexed until the droplets have been resuspended. Heat at 37°C for 5-10 min, if indicated.
Add 2.5 µl of 18 μM adapter to each reaction, quick vortex and spin. Document which index was used in which library.
| Sample ID | Index ID | Sample ID | Index ID | Sample ID | Index ID | |
| 1 | 7 | 13 | ||||
| 2 | 8 | 14 | ||||
| 3 | 9 | 15 | ||||
| 4 | 10 | 16 | ||||
| 5 | 11 | |||||
| 6 | 12 |
Pre-mix water, ligation buffer and DNA ligase based on the sample number. Add 47.5 ul of ligation premix to each reaction. quick vortex and spin.
| Component | Volume (ul) | |
| DNA with adaptor | 62.5 | |
| Water | 7.5 | |
| Ligation Buffer | 30 | |
| Ligase | 10 | |
| Total volume | 110 |
Incubate at 20 °C for 15- 60 minutes.
Library clean up
Library clean up
Add 88 ul of beads into the tube. Gently mix, incubate at room temperature for 5 minutes.
Place the tube on a magnetic stand. Remove supernatant when it is clear.
Wash twice with freshly made 75 % Etoh and remove 75 % Etoh from the tube. Try to remove all residual ethanol without disturbing the beads.
Open the lid to let the bead dry at room temperature for 3 – 5 min, or until all of the ethanol has evaporated.
Add 50 ul of nuclease free water. Quick vortex. Incubate samples in room temperature for 5 minutes.
Add 25ul of nuclease free water. Quick vortex. Incubate samples in room temperature for 5 minutes.
Transfer the 23 ul of the clear supernatant to a new tube.
Library QC
Library QC
Use 1 ul of the library and fragmented DNA to run on Bioanalyzer using DNA HS kit to determine the average size and concentration of the final library.
IMPORTANT: The final library size should be shifted up by ~150 bp compared to the insert size distribution. If there are no size shifts, it may indicate failure of adapter ligation. Consider failing the libraries with no size shifts.