TITLE: HiDEF-seq
AUTHORS: Mei Hong Liu, Benjamin M. Costa, Emilia C. Bianchini, Una Choi, Rachel C. Bandler, Emilie Lassen, Marta Grońska-Pęski, Adam Schwing, Zachary R. Murphy, Daniel Rosenkjær, Shany Picciotto, Vanessa Bianchi, Lucie Stengs, Melissa Edwards, Nuno Miguel Nunes, Caitlin A. Loh, Tina K. Truong, Randall E. Brand, Tomi Pastinen, J. Richard Wagner, Anne-Bine Skytte, Uri Tabori, Jonathan E. Shoag, Gilad D. Evrony

[DESCRIPTION]
This is the HiDEF-seq library preparation protocol for bulk, single-molecule fidelity, long-read sequencing. This version of the HiDEF-seq protocol is designed for high-quality DNA. See our paper "DNA mismatch and damage patterns revealed by single-molecule sequencing" (Liu & Costa et al.) for more information.

[STEPS]
SECTION: Reagent Preparation
3. If necessary, create 75% PB Ampure Bead dilution as follows:

 

Vortex mix
SECTION: Reagent Preparation
4. Create fresh 80% Ethanol as follows:

 

Vortex mix

SECTION: E. Coli Nick Ligation
17. Prepare E. Coli Nick Ligation reaction as follows:
 

Pipette mix and spin down

SECTION: A-Tailing
22. Prepare A-Tailing reaction as follows:
 

Pipette mix and spin down

SECTION: Adaptor Ligation
27. Prepare Adaptor Ligation reaction as follows:
 

Pipette mix and spin down

SECTION: Nuclease Treatment
29. Prepare Nuclease Treatment Master Mix as follows:
 

Pipette mix and spin down

SECTION: Restriction Enzyme Digestion
13. Run Restriction Enzyme Digestion Thermocycler Protocol:
Lid: 105 °C 
37 °C 20 min ->4 °C Hold

SECTION: Restriction Enzyme Digestion
14. Dilute the reaction to a DNA concentration of 10ng/µL by adding 80µL of Nuclease Free Water
Note: If more or less than 1500ng of DNA was input into the library preparation, calculate the amount of water to add to obtain 10ng/µL of DNA concentration and then adjust the subsequent bead cleanup volume accordingly.

Vortex mix and spin down

SECTION: Restriction Enzyme Digestion
15. Perform a 0.8X Bead Clean:
Add 120µL of 75% PacBio Ampure Beads.
Note: If more or less than 1500ng of DNA was input into the library preparation, calculate the amount of 0.8X relative bead volume according to the prior step's post-dilution volume.
Continue with a standard bead clean up, with two 80% Ethanol washes.
Elute in 22µL of 10mM Tris pH8

SECTION: E. Coli Nick Ligation
20. Perform a 0.75X Bead Clean with 75% PacBio Ampure Beads
Calculate bead volume relative to the post-dilution volume of the sample after completing the prior step
Wash twice with 80% Ethanol
Elute in 22µL of 10mM Tris pH8

SECTION: E. Coli Nick Ligation
18. Run E. Coli Nick Ligation Thermocycler Protocol:
Heated lid off
16 °C 30 min ->4 °C Hold

SECTION: A-Tailing
24. Dilute the samples to a maximum of 10ng/µL based on the Qubit values after the cleanup that followed the E. Coli Nick Ligation, using the following equation:
Volume to add = (Qubit Concentration)(21µL)/(10ng/µL) - 30µL
Vortex mix and spin down

SECTION: A-Tailing
23. Run A-Tailing Thermocycler Protocol:
Lid: 105 °C
 37 °C 30 min ->4 °C Hold

SECTION: Adaptor Ligation
28. Run Adaptor Ligation Thermocycler Protocol:
Heated lid off
20 °C 60 min ->4 °C Hold

SECTION: Nuclease Treatment
32. Perform a 1.2X Bead Clean by adding 63µL 75% PacBio Ampure Beads
Wash twice with 80% Ethanol
Elute in 24µL of 10mM Tris pH8

SECTION: Nuclease Treatment
31. Run Nuclease Digestion Thermocycler Protocol:
Lid: 105 °C 
37 °C 60 min ->4 °C Hold

SECTION: QC
33. Measure concentration of library by inputting 1µL into Qubit.
Expected concentration: 2.5 - 7.5ng/µL

SECTION: Restriction Enzyme Digestion
12. Prepare Restriction Enzyme Digestion:

Input 1500ng of gDNA into a 70µL reaction as follows:
 

Pipette mix and spin down

SECTION: Reagent Preparation
9. Take out DNA from freezer
Thaw, vortex, and spin down

SECTION: Reagent Preparation
10. Measure concentration of DNA samples with Qubit.

SECTION: Reagent Preparation
11. Measure DNA size distribution and quality with Genomic DNA ScreenTape.

SECTION: Reagent Preparation
5. Create 10mM Tris pH8 as follows:

 

Vortex mix

SECTION: Restriction Enzyme Digestion
16. Measure concentration of DNA samples by inputting 1µL into Qubit.

SECTION: E. Coli Nick Ligation
21. Measure concentration of DNA samples by inputting 1µL into Qubit.

SECTION: E. Coli Nick Ligation
19. Dilute the samples to a maximum of 10ng/µL based on the post Restriction Enzyme Digest Clean Up Qubit values, using the following equation:
Volume to add = (Qubit Concentration)(21µL)/(10ng/µL) - 30µL
Vortex mix and spin down

SECTION: Reagent Preparation
7. If necessary, dilute 100mM stock dATP to 10mM dATP as follows:

 

Pipette mix and spin down

SECTION: Reagent Preparation
8. If necessary, make 1mM dATP/ddBTP Mix as follows: 
 

Pipette mix and spin down

SECTION: QC
34. Measure DNA size distribution with High Sensitivity D5000 ScreenTape.
Example size distribution:

 

SECTION: A-Tailing
25. Perform a 0.75X Bead Clean with 75% PacBio Ampure Beads
Calculate bead volume relative to the post-dilution volume of the sample after completing the prior step
Wash twice with 80% Ethanol
Elute in 22µL of 10mM Tris pH8

SECTION: A-Tailing
26. Add to the sample 3µL of 10X NEBuffer 4 and 5µL of Nuclease Free Water

SECTION: Nuclease Treatment
30. Prepare Nuclease Treatment reaction as follows::
 

Pipette mix and spin down

SECTION: Introduction
1. This is the HiDEF-seq v2 protocol with A-Tailing used for high-quality DNA as assessed by fragment size analysis (e.g., TapeStation). For low-quality DNA samples, refer to our paper for the relevant protocol: Liu & Costa et al. DNA mismatch and damage patterns revealed by single-molecule sequencing.

SECTION: Introduction
2. Reagent List:
 

SECTION: Reagent Preparation
6. If necessary, create 500µM aliquots of NAD+ as follows:

 
Pipette mix and spin down
Split into 10µL aliquots and store at -80C