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:

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

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

A	B
Component	Volume (µL)
Stock PB Ampure Beads	2250
PB Elution Buffer	750
Total	=SUM(B2:B3)

• Vortex mix

Create fresh 80% Ethanol as follows:

A	B
Component	Volume (mL)
Ethanol	8
Nuclease Free Water	2
Total	=SUM(B2:B3)

• Vortex mix

Create 10mM Tris pH8 as follows:

A	B
Component	Volume (µL)
Nuclease Free Water	990
1M Tris pH8	10
Total	=SUM(B2:B3)

• Vortex mix

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

A	B
Component	Volume (µL)
Nuclease Free Water	198
50mM NAD+	2
Total	=SUM(B2:B3)

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

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

A	B
Component	Volume (µL)
Nuclease Free Water	9
100mM dATP	1
Total	=SUM(B2:B3)

• Pipette mix and spin down

If necessary, make 1mM dATP/ddBTP Mix as follows:

A	B
Component	Volume (µL)
Nuclease Free Water	60
10mM dATP	10
10mM ddCTP	10
10mM ddGTP	10
10mM ddTTP	10
Total	=SUM(B2:B6)

• 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.

Prepare Restriction Enzyme Digestion:

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

Calculation for gDNA Sample Input Volume (x) = 1500ng / gDNA Sample Concentration

• Pipette mix and spin down

Run Restriction Enzyme Digestion Thermocycler Protocol:

• Lid: 105°C
• 37°C 0h 20m 0s ->4°C Hold

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.

Prepare E. Coli Nick Ligation reaction as follows:

A	B	C	D
Component	Starting Concentration	Input (µL)	Final Amount
Eluted DNA		21
Nuclease Free Water		2.94
rCutSmart Buffer	10X	3	1X
NAD+	500µM	1.56	26µM
E. Coli DNA Ligase	10U/µL	1.5	15U
Total		30

• Pipette mix and spin down

Run E. Coli Nick Ligation Thermocycler Protocol:

• Heated lid off
• 16°C 0h 30m 0s ->4°C Hold

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.

Prepare A-Tailing reaction as follows:

A	B	C	D
Component	Starting Concentration	Input (µL)	Final Amount
Eluted DNA		21
Nuclease Free Water		1.5
NEBuffer 4	10X	3	1X
dATP/ddBTP Mix	1mM	3	0.1mM
Klenow Fragment (3'→5' exo-)	5U/µL	1.5	7.5U
Total		30

• Pipette mix and spin down

Run A-Tailing Thermocycler Protocol:

Lid: 105°C

37°C 0h 30m 0s ->4°C Hold

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

Prepare Adaptor Ligation reaction as follows:

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

• Pipette mix and spin down

Run Adaptor Ligation Thermocycler Protocol:

• Heated lid off
• 20°C 1h 0m 0s ->4°C Hold

Prepare Nuclease Treatment Master Mix as follows:

A	B
Component	Input (µL)
Enzyme A	2
Enzyme B	0.5
Enzyme C	0.5
Enzyme D	1
Total	4

• Pipette mix and spin down

Prepare Nuclease Treatment reaction as follows::

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

• Pipette mix and spin down

Run Nuclease Digestion Thermocycler Protocol:

• Lid: 105°C
• 37°C 1h 0m 0s ->4°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: