TITLE: Illumina Whole Genome Sequencing
AUTHORS: Rita   Kuo

[DESCRIPTION]
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.

[STEPS]
SECTION: Sample QC
1. 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.
SECTION: Bead clean up
2. Bring the sample volume to 20 ul in PCR 8 stripe tubes
SECTION: Bead clean up
3. Add 18 ul of beads into the tube. Gently mix, incubate at room temperature for 5 minutes.
SECTION: Bead clean up
4. Place the tube on a magnetic stand. Remove supernatant when it is clear.
SECTION: Bead clean up
5. Wash twice with freshly made 75 % Etoh and remove 75 % Etoh from the tube. Try to remove all residual ethanol without disturbing the beads.
SECTION: Bead clean up
6. Open the lid to let the bead dry at room temperature for 3 – 5 min, or until all of the ethanol has evaporated.



SECTION: Bead clean up
7. Add 22 ul of 50 C nuclease free water. Quick vortex. Incubate samples in room temperature for 5 minutes. 
SECTION: Bead clean up
8. Repeat step 3-7 one more time.
SECTION: Bead clean up
9. Add 37 ul of 50 C nuclease free water. Quick vortex. Incubate samples in room temperature for 5 minutes.
SECTION: Bead clean up
10. Transfer 36 ul of eluted DNA to new 8 strip tubes.
SECTION: Bead clean up
11. 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.
SECTION: Enzymatic Fragmentation
12. 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:
 
ComponentVolume (ul)Double-stranded DNA (with 5ul of conditioning solution if needed)35KAPA Frag Buffer (10X)*5KAPA Frag Enzyme* 10Total volume50
 
SECTION: Enzymatic Fragmentation
13. Vortex gently by flicking the tubes and spin down briefly. Return the tube(s) to ice. Proceed immediately to the next step.
SECTION: Enzymatic Fragmentation
14. 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.
SECTION: Enzymatic Fragmentation
15. Transfer reactions to ice, and proceed immediately to End Repair and A-tailing.
SECTION: End Repair and A-Tailing
16. 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. 

 
ComponentVolume(ul)Fragemented DNA50End Repair & A-Tailing Buffer*7HyperPrep/HyperPlus ERAT Enzyme Mix** 3Total volume60
 
SECTION: End Repair and A-Tailing
17. Vortex gently and spin down briefly. Return the reaction tubes to ice. Proceed immediately to the next step.
SECTION: End Repair and A-Tailing
18. 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.
SECTION: End Repair and A-Tailing
19. Proceed immediately to Adapter Ligation. Keep 1 ul of the DNA for library QC. 
SECTION: Adaptor Ligation
20. 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 IDIndex IDSample IDIndex IDSample IDIndex ID17132814391541016511612
 
SECTION: Adaptor Ligation
21. 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.
 
ComponentVolume (ul)DNA with adaptor62.5Water7.5Ligation Buffer30Ligase10Total volume110
 
SECTION: Adaptor Ligation
22. Incubate at 20 °C for 15- 60 minutes.
SECTION: Library clean up
23. Add 88 ul of beads into the tube. Gently mix, incubate at room temperature for 5 minutes.
SECTION: Library clean up
24. Place the tube on a magnetic stand. Remove supernatant when it is clear.
SECTION: Library clean up
25. Wash twice with freshly made 75 % Etoh and remove 75 % Etoh from the tube. Try to remove all residual ethanol without disturbing the beads.
SECTION: Library clean up
26. Open the lid to let the bead dry at room temperature for 3 – 5 min, or until all of the ethanol has evaporated.
SECTION: Library clean up
27. Add 50 ul of nuclease free water. Quick vortex. Incubate samples in room temperature for 5 minutes.
SECTION: Library clean up
28. Add 25ul of nuclease free water. Quick vortex. Incubate samples in room temperature for 5 minutes.
SECTION: Library clean up
29. Transfer the 23 ul of the clear supernatant to a new tube.
SECTION: Library QC
30. 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.