HiDEF-seq

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

May 17, 2024

Version 1

HiDEF-seq V.1

DOI

dx.doi.org/10.17504/protocols.io.kxygxy9mwl8j/v1

Mei Hong Liu¹,
Benjamin M. Costa¹,
Emilia C. Bianchini¹,
Una Choi¹,
Rachel C. Bandler¹,
Emilie Lassen²,
Marta Grońska-Pęski¹,
Adam Schwing¹,
Zachary R. Murphy¹,
Daniel Rosenkjær²,
Shany Picciotto³,
Vanessa Bianchi⁴,
Lucie Stengs⁴,
Melissa Edwards⁴,
Nuno Miguel Nunes⁴,
Caitlin A. Loh¹,
Tina K. Truong¹,
Randall E. Brand⁵,
Tomi Pastinen⁶,
J. Richard Wagner⁷,
Anne-Bine Skytte²,
Uri Tabori⁴,
Jonathan E. Shoag³,
Gilad D. Evrony¹

¹New York University Grossman School of Medicine;
²Cryos International Sperm and Egg Bank;
³Case Western Reserve University School of Medicine;
⁴The Hospital for Sick Children;
⁵University of Pittsburgh School of Medicine;
⁶Children’s Mercy Kansas City;
⁷Université de Sherbrooke

Benjamin M. Costa

NYU Grossman School of Medicine

DOI: dx.doi.org/10.17504/protocols.io.kxygxy9mwl8j/v1

Protocol Citation: 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 2024. HiDEF-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxy9mwl8j/v1Version created by Benjamin M. Costa

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: May 13, 2024

Last Modified: May 17, 2024

Protocol Integer ID: 99842

Abstract

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.

Introduction

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.

Reagent List:
ABCDE
ReagentSupplierProduct #KitKit Product #
PB Elution BufferPacific Biosciences101-159-800SMRTbell express template prep kit 2.0100-938-900
Ligation MixPacific Biosciences101-654-100SMRTbell express template prep kit 2.0100-938-900
Ligation AdditivePacific Biosciences101-654-200SMRTbell express template prep kit 2.0100-938-900
Ligation EnhancerPacific Biosciences101-654-300SMRTbell express template prep kit 2.0100-938-900
Enzyme APacific Biosciences101-741-100SMRTbell enzyme cleanup kit 1.0101-746-400
Enzyme BPacific Biosciences101-741-700SMRTbell enzyme cleanup kit 1.0101-746-400
Enzyme CPacific Biosciences101-741-400SMRTbell enzyme cleanup kit 1.0101-746-400
Enzyme DPacific Biosciences101-741-500SMRTbell enzyme cleanup kit 1.0101-746-400
Stock PB Ampure BeadsPacific Biosciences100-265-900
Barcoded Overhang Adapter Kit 8APacific Biosciences101-628-400
Barcoded Overhang Adapter Kit 8BPacific Biosciences101-628-500
Qubit 1X dsDNA HS Assay KitThermo FisherQ33231
Genomic DNA ScreenTapeAgilent5067-5365
High Sensitividy D5000 ScreenTapeAgilent5097-5592
100mM dATPThermo FisherR0141
10mM ddNTP BundleJena BioscienceNU-1019
10X CutSmart BufferNEBB7204
Hpy166IINEBR0616S
10X rCutSmart Buffer NEBB6004S
β-Nicotinamide adenine dinucleotide (NAD+)NEBB9007S
E. Coli DNA LigaseNEBM0205S
10X NEBuffer 4NEBB7004S
Klenow Fragment (3'→5' exo-)NEBM0212S

Reagent Preparation

If necessary, create 75% PB Ampure Bead dilution as follows:

AB
ComponentVolume (µL)
Stock PB Ampure Beads2250
PB Elution Buffer750
Total3000

Vortex mix

Create fresh 80% Ethanol as follows:

AB
ComponentVolume (mL)
Ethanol8
Nuclease Free Water2
Total10

Vortex mix

Create 10mM Tris pH8 as follows:

AB
ComponentVolume (µL)
Nuclease Free Water990
1M Tris pH810
Total1000

Vortex mix

If necessary, create 500µM aliquots of NAD+ as follows:

AB
ComponentVolume (µL)
Nuclease Free Water198
50mM NAD+2
Total200
Pipette mix and spin down
Split into 10µL aliquots and store at -80C

If necessary, dilute 100mM stock dATP to 10mM dATP as follows:

AB
ComponentVolume (µL)
Nuclease Free Water9
100mM dATP1
Total10

Pipette mix and spin down

If necessary, make 1mM dATP/ddBTP Mix as follows: 
AB
ComponentVolume (µL)
Nuclease Free Water60
10mM dATP10
10mM ddCTP10
10mM ddGTP10
10mM ddTTP10
Total100

Pipette mix and spin down

Take out DNA from freezer
Thaw, vortex, and spin down

Measure concentration of DNA samples with Qubit.

Measure DNA size distribution and quality with Genomic DNA ScreenTape.

Restriction Enzyme Digestion

Prepare Restriction Enzyme Digestion:

Input 1500ng of gDNA into a 70µL reaction as follows:
ABCD
ComponentStarting ConcentrationInput (µL)Final Amount
Nuclease Free Water62 - X
10X Cutsmart Buffer10X71X
gDNA SampleX1500ng
Hpy166II10U/µL110U
Total70
Calculation for gDNA Sample Input Volume (x) = 1500ng / gDNA Sample Concentration

Pipette mix and spin down

Run Restriction Enzyme Digestion Thermocycler Protocol:
Lid: Temperature105 °C   
Temperature37 °C   Duration00:20:00   ->Temperature4 °C   Hold

20m

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

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

Measure concentration of DNA samples by inputting 1µL into Qubit.

E. Coli Nick Ligation

Prepare E. Coli Nick Ligation reaction as follows:
ABCD
ComponentStarting ConcentrationInput (µL)Final Amount
Eluted DNA21
Nuclease Free Water2.94
rCutSmart Buffer10X31X
NAD+500µM1.5626µM
E. Coli DNA Ligase10U/µL1.515U
Total30

Pipette mix and spin down

Run E. Coli Nick Ligation Thermocycler Protocol:
Heated lid off
Temperature16 °C   Duration00:30:00   ->Temperature4 °C   Hold

30m

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

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

Measure concentration of DNA samples by inputting 1µL into Qubit.

A-Tailing

Prepare A-Tailing reaction as follows:
ABCD
ComponentStarting ConcentrationInput (µL)Final Amount
Eluted DNA21
Nuclease Free Water1.5
NEBuffer 410X31X
dATP/ddBTP Mix1mM30.1mM
Klenow Fragment (3'→5' exo-)5U/µL1.57.5U
Total30

Pipette mix and spin down

Run A-Tailing Thermocycler Protocol:
Lid: Temperature105 °C  
 Temperature37 °C   Duration00:30:00   ->Temperature4 °C   Hold

30m

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

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

Add to the sample 3µL of 10X NEBuffer 4 and 5µL of Nuclease Free Water

Adaptor Ligation

Prepare Adaptor Ligation reaction as follows:
AB
ComponentInput (µL)
Eluted DNA with NEBuffer 430
PacBio Hairpin  Barcode Overhang Adapter2.5
Ligation Mix15
Ligation Additive0.5
Ligation Enhancer0.5
Total48.5

Pipette mix and spin down

Run Adaptor Ligation Thermocycler Protocol:
Heated lid off
Temperature20 °C   Duration01:00:00   ->Temperature4 °C   Hold

Nuclease Treatment

Prepare Nuclease Treatment Master Mix as follows:
AB
ComponentInput (µL)
Enzyme A2
Enzyme B0.5
Enzyme C0.5
Enzyme D1
Total4

Pipette mix and spin down

Prepare Nuclease Treatment reaction as follows::
AB
ComponentInput (µL)
Ligated DNA48.5
Nuclease Treatment Mastermix4
Total52.5

Pipette mix and spin down

Run Nuclease Digestion Thermocycler Protocol:
Lid: Temperature105 °C   
Temperature37 °C   Duration01:00:00   ->Temperature4 °C   Hold

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

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

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

A	B	C	D	E
Reagent	Supplier	Product #	Kit	Kit Product #
PB Elution Buffer	Pacific Biosciences	101-159-800	SMRTbell express template prep kit 2.0	100-938-900
Ligation Mix	Pacific Biosciences	101-654-100	SMRTbell express template prep kit 2.0	100-938-900
Ligation Additive	Pacific Biosciences	101-654-200	SMRTbell express template prep kit 2.0	100-938-900
Ligation Enhancer	Pacific Biosciences	101-654-300	SMRTbell express template prep kit 2.0	100-938-900
Enzyme A	Pacific Biosciences	101-741-100	SMRTbell enzyme cleanup kit 1.0	101-746-400
Enzyme B	Pacific Biosciences	101-741-700	SMRTbell enzyme cleanup kit 1.0	101-746-400
Enzyme C	Pacific Biosciences	101-741-400	SMRTbell enzyme cleanup kit 1.0	101-746-400
Enzyme D	Pacific Biosciences	101-741-500	SMRTbell enzyme cleanup kit 1.0	101-746-400
Stock PB Ampure Beads	Pacific Biosciences	100-265-900
Barcoded Overhang Adapter Kit 8A	Pacific Biosciences	101-628-400
Barcoded Overhang Adapter Kit 8B	Pacific Biosciences	101-628-500
Qubit 1X dsDNA HS Assay Kit	Thermo Fisher	Q33231
Genomic DNA ScreenTape	Agilent	5067-5365
High Sensitividy D5000 ScreenTape	Agilent	5097-5592
100mM dATP	Thermo Fisher	R0141
10mM ddNTP Bundle	Jena Bioscience	NU-1019
10X CutSmart Buffer	NEB	B7204
Hpy166II	NEB	R0616S
10X rCutSmart Buffer	NEB	B6004S
β-Nicotinamide adenine dinucleotide (NAD+)	NEB	B9007S
E. Coli DNA Ligase	NEB	M0205S
10X NEBuffer 4	NEB	B7004S
Klenow Fragment (3'→5' exo-)	NEB	M0212S

	A	B
	Component	Volume (µL)
	Stock PB Ampure Beads	2250
	PB Elution Buffer	750
	Total	3000

	A	B
	Component	Volume (µL)
	Nuclease Free Water	990
	1M Tris pH8	10
	Total	1000

A	B	C	D
Component	Starting Concentration	Input (µL)	Final Amount
Nuclease Free Water		62 - X
10X Cutsmart Buffer	10X	7	1X
gDNA Sample		X	1500ng
Hpy166II	10U/µL	1	10U
Total		70

	A	B
	Component	Input (µL)
	Eluted DNA with NEBuffer 4	30
	PacBio Hairpin Barcode Overhang Adapter	2.5
	Ligation Mix	15
	Ligation Additive	0.5
	Ligation Enhancer	0.5
	Total	48.5

	A	B
	Component	Input (µL)
	Ligated DNA	48.5
	Nuclease Treatment Mastermix	4
	Total	52.5

Public workspaceHiDEF-seq V.1

HiDEF-seq V.1