nanobind cbb big dna kit handbook hbk-cbb-001 vf

Nanobind CBB Big DNA Kit Handbook

Document ID: HBK-CBB-001

Release Date: 3/24/2021

For extraction of HMW (50 kb – 300+ kb) and UHMW (50 kb – 1+ Mb) genomic DNA from cultured cells, cultured bacteria, and blood

Page | 2 Nanobind CBB Big DNA Kit Handbook (HBK-CBB-001)

Table of Contents

Table of Contents .................................................................................................................................................................... 2 Kit Specifications ..................................................................................................................................................................... 4

Contents .............................................................................................................................................................................. 4 Prior to Starting ................................................................................................................................................................... 4 Kit Storage ........................................................................................................................................................................... 4 Safety Precautions .............................................................................................................................................................. 4 Product Use ......................................................................................................................................................................... 4

Equipment and Reagent List ................................................................................................................................................... 5 For All Protocols .................................................................................................................................................................. 6

Introduction .............................................................................................................................................................................. 7 Kit Overview ........................................................................................................................................................................ 7

Workflow .................................................................................................................................................................................. 9 Sample Information ............................................................................................................................................................... 10

DNA Size ........................................................................................................................................................................... 11 Preservation Methods ....................................................................................................................................................... 12

Fresh vs. Frozen Cells .................................................................................................................................................. 12 Fresh vs. Frozen Blood ................................................................................................................................................. 12 Blood Anti-Coagulant .................................................................................................................................................... 12 Ethanol-Preserved Nucleated Blood ............................................................................................................................. 12

PacBio Sequencing ........................................................................................................................................................... 13 PacBio Multiplex Microbial Sequencing ............................................................................................................................ 15 Oxford Nanopore Sequencing ........................................................................................................................................... 16 Nanobind Ultra Long Sequencing ..................................................................................................................................... 17 Short Read Eliminator Size Selection ............................................................................................................................... 18

Processing Tips ..................................................................................................................................................................... 19 Magnetic Rack Handling Procedure .................................................................................................................................. 19 Pipetting ............................................................................................................................................................................ 20 Heterogeneity and Viscosity (HMW DNA Only) ................................................................................................................ 21

HMW (50 kb – 300+ kb) DNA Extraction Protocols .............................................................................................................. 22 HMW DNA Extraction – Cultured Cells (EXT-CLH-001) ................................................................................................... 22 HMW DNA Extraction – Gram-Negative Bacteria (EXT-GNH-001) .................................................................................. 22 HMW DNA Extraction – Gram-Positive Bacteria (EXT-GPH-001) .................................................................................... 22 HMW DNA Extraction – 200 µL Mammalian Whole Blood (EXT-BLH-001) ..................................................................... 22 HMW DNA Extraction – 1 mL Mammalian Whole Blood (EXT-BLH-002) ........................................................................ 22 HMW DNA Extraction – Nucleated Blood (EXT-CLH-001) ............................................................................................... 22

UHMW (50 kb – 1+ Mb) DNA Extraction Protocols .............................................................................................................. 23 UHMW DNA Extraction – Cultured Cells (EXT-CLU-001) ................................................................................................ 23


UHMW DNA Extraction – Gram-Negative Bacteria (EXT-GNU-001) ............................................................................... 23 UHMW DNA Extraction – Gram-Positive Bacteria (EXT-GPU-001) ................................................................................. 23 UHMW DNA Extraction – Mammalian Whole Blood (EXT-BLU-001) ............................................................................... 23 UHMW DNA Extraction – Nucleated Blood (EXT-NBU-001) ............................................................................................ 23

QC Procedures ..................................................................................................................................................................... 24 Storage of DNA ..................................................................................................................................................................... 25 Troubleshooting FAQ ............................................................................................................................................................ 26 PacBio Sequel Sequencing Recommendations ................................................................................................................... 27 Oxford Nanopore Sequencing Recommendations ............................................................................................................... 28


Kit Specifications Contents

Nanobind CBB Big DNA Kit Part Number Number of Samples

v1 NB-900-001-01

20

Nanobind Disks 20

Proteinase K 0.45 mL

RNase A 0.45 mL

Buffer CLE3 0.45 mL

Buffer BL3 4.5 mL

Buffer CW1 Concentrate (Dilute to 60% final ethanol concentration as indicated on the bottle) 6.5 mL (16.5 mL after EtOH)

Buffer CW2 Concentrate (Dilute to 60% final ethanol concentration as indicated on the bottle) 10 mL (25 mL after EtOH)

Buffer EB 5 mL

Prior to Starting Buffer CW1 and CW2 are supplied as concentrates. This kit uses CW1 with a 60% final ethanol concentration. This kit uses CW2 with a 60% final ethanol concentration. Before using, add the appropriate amount of ethanol (96–100%) to Buffer CW1 and Buffer CW2 as indicated on the bottles.

Kit Storage RNase A should be stored at 4 °C upon arrival.

Nanobind Disks and all other buffers should be stored at room temperature (18–25 °C).

Safety Precautions Buffer BL3 and Buffer CW1 contain guanidine hydrochloride. Warning! Guanidine hydrochloride is harmful if swallowed or inhaled and causes skin and eye irritation. DO NOT mix with bleach or acidic solutions.

Product Use Nanobind CBB Big DNA Kits are intended for research use only.


Equipment and Reagent List

Equipment/Reagent Manufacturer (Part Number)

Magnetic Tube Rack Thermo Fisher DynaMag-2 (12321D)

Mini-Tube Rotator Fisher Scientific Mini-Tube Rotator (05-450-127)

Heat Block (or Water Bath) Fisher Scientific Isotemp Dry Bath Incubator (11-715-125DQ)

Mini-centrifuge Ohaus (FC5306)

Micro-centrifuge Eppendorf (5404000413)

ThermoMixer Eppendorf (5382000023)

1.5 mL Protein LoBind Microcentrifuge Tubes Eppendorf (022431081)

Ethanol (96–100%)

Isopropanol (100%)

1X PBS

UV/Vis Thermo Fisher Scientific NanoDrop 2000

Fluorescent DNA Quantification Thermo Qubit 3.0, dsDNA BR and RNA BR Assay Kits

Tris-HCl, 1 M, pH 8.0 Invitrogen (15568025)

Ethylenediaminetetraacetic Acid (EDTA), 0.5 M, pH 8.0 Thermo Fisher (15575020)

Sucrose Fisher Scientific (BP220)

Triton X-100 Sigma-Aldrich (X100)

Lysozyme MP Biomedicals (100831)

Lysostaphin Sigma-Aldrich (L7386)

26g Blunt End Needle SAI Infusion (B26150)

1 mL Syringe Fisher Scientific (14-823-30)


For All Protocols Eppendorf Protein LoBind tubes (Eppendorf #022431081) are highly recommended for all extractions to reduce protein contamination from tube carryover. Protein LoBind tubes are more effective in reducing carryover contamination than DNA LoBind tubes or other tubes and will result in improved UV purity.


Introduction Nanobind is a novel magnetic disk covered with a high density of micro- and nanostructured silica that can be used for rapid extraction and purification of high-quality DNA and RNA. The high surface area and unique binding mechanism give it an extraordinary binding capacity, allowing isolation of high purity, high molecular weight (HMW) and ultra high molecular weight (UHMW) DNA in a microcentrifuge tube format. It uses a standard lyse, bind, wash, and elute procedure that is common for silica DNA extraction technologies. A single disk is used in each tube. However, unlike magnetic beads and silica spin columns which shear large DNA, Nanobind disks bind and release DNA without fragmentation, to yield DNA up to megabases in length.

Kit Overview The Nanobind CBB Big DNA Extraction Kit is used for the extraction of HMW (50 kb – 300+ kb) and UHMW (50 kb – 1+ Mb) DNA from cultured cells, cultured bacteria, and blood. It has been used for both mammalian and non-mammalian cell culture samples, a diverse array of Gram-negative and Gram-positive bacteria, mammalian blood from a variety of species, and nucleated blood from birds, fish, and reptiles. The extracted DNA is suitable for analysis on long-range genomics platforms including PacBio RSII/Sequel/Sequel II, Oxford Nanopore MinION/GridION/PromethION, Bionano Genomics Irys/Saphyr, and 10X Genomics Chromium. Process time is approximately 45–90 minutes.

High-throughput automated extractions can also be performed using the Nanobind KF CBB Big DNA Kit.

The Sample Information section provides example extraction and sequencing results from a wide variety of sample types.

Protocols listed in the HMW (50 kb – 300+ kb) DNA Extraction Protocols and the UHMW (50 kb – 1+ Mb) DNA Extraction Protocols sections are updated frequently so please check the Nanobind Support page (http://www.circulomics.com/support-nanobind) for the most up-to-date list and for the current versions of the protocols.

The HMW (50 kb – 300+ kb) DNA Extraction Protocols are recommended for most long-read sequencing applications. This includes PacBio CLR and HiFi sequencing and nanopore sequencing using Oxford Nanopore Ligation and Rapid Sequencing Kits. For standard long-read sequencing applications these protocols will result in superior sequencing performance than the UHMW (50 kb – 1+ Mb) DNA Extraction Protocols.

The UHMW (50 kb – 1+ Mb) DNA Extraction Protocols generate large amounts of megabase sized DNA and are only recommend for Nanobind Ultra Long Sequencing on Oxford Nanopore instruments and for optical mapping. They are not recommended for use in the standard long-read sequencing applications. For standard long-read sequencing applications, superior results will be obtained using the HMW (50 kb – 300+ kb) DNA Extraction Protocols. Please contact us if you’d like to use the UHMW (50 kb – 1+ Mb) DNA Extraction Protocols in applications other than Nanobind Ultra Long Sequencing.

Differences between the HMW and UHMW protocols are illustrated in the table below.

SEM image of Nanobind's silica surface structure.


HMW DNA Extraction Protocols UHMW DNA Extraction Protocols

Extraction Processing Thermomixing Vortexing Pipette Mixing

Pipetting Standard Tips Wide Bore Tips

Typical DNA Size 50 kb – 300+ kb 50 kb – 1+ Mb

Sample Heterogeneity Low to Moderate (Conc CV = 5–25%)

High (Conc CV = 25–100%)

Recommended Applications PacBio HiFi Sequencing PacBio CLR Sequencing

Oxford Nanopore Ligation Sequencing Oxford Nanopore Rapid Sequencing

Oxford Nanopore Ultra Long Sequencing Bionano Genomics Optical Mapping


Workflow


Sample Information Yields of HMW genomic DNA will vary depending on the sample being processed. The following table provides suggested input ranges and expected yields for the validated sample types. Each sample has been validated by long read sequencing.

Expected Yields

Sample Suggested Input1 Example Input Example 260/280

Example 260/230

Example Yield (µg)

MCF-7 cells (tetraploid) 0.5x106 – 10x106 cells 1x106 cells 1.9 2.0 13.5

MDA-MB-231 (tetraploid) 0.5x106 – 10x106 cells 1x106 cells 1.9 2.0 14.9

MCF-10A cells (diploid) 0.5x106 – 10x106 cells 2x106 cells 1.9 2.0 12.3

GM12878 cells (diploid) 0.5x106 – 10x106 cells 5x106 cells 1.9 2.2 37.2

Escherichia coli2 5x108 – 5x109 cells 1 mL 1.8 1.4 18.3

Shigella sonnei2 5x108 – 5x109 cells 0.25 mL 1.8 1.4 27.7

Salmonella enterica2 5x108 – 5x109 cells 0.25 mL 1.8 1.5 23.4

Listeria monocytogenes2 5x108 – 5x109 cells 1 mL 1.8 1.9 21.7

Human Whole Blood 200 µL – 1 mL 200 µL 1.9 1.9 5.6 1Higher input levels can be used with appropriate optimization of buffer volumes and enzyme levels. 2Input based on cell pellet from stated volume of overnight culture.

Blood Anticoagulant Validation

Sample 260/280 260/230 DNA Yield (µg)

Whole Blood – K2 EDTA 1.9 2.0 5.5

Whole Blood – Sodium Citrate 1.9 2.0 5.0

Whole Blood – Heparin 1.9 1.9 5.1

Whole Blood – ACD 1.9 1.8 4.4

Nanobind was used to extract HMW DNA (up to 300 kb) from 200 µL of fresh human whole blood (n=3). All four samples showed similar yields and UV purity. Validation by long read sequencing likewise performed similarly, with EDTA stabilized blood displaying small advantages in read length.


DNA Size The HMW DNA Extraction Protocols typically yield DNA in the 50 kb – 300+ kb size range. The UHMW DNA Extraction Protocols typically yield DNA in the 50 kb – 1+ kb size range. The exact size will vary depending on sample type, the quality of the starting material, and processing parameters. For most long-read sequencing applications, superior sequencing performance will be obtained using the HMW DNA Extraction Protocols. This includes PacBio CLR and HiFi sequencing and nanopore sequencing using Oxford Nanopore Ligation and Rapid Sequencing Kits. The UHMW (50 kb – 1+ Mb) DNA Extraction Protocols generate large amounts of megabase sized DNA and are only recommend for Nanobind Ultra Long Sequencing on Oxford Nanopore instruments and for optical mapping. They are not recommended for use in the standard long-read sequencing applications. Please contact us if you’d like to use the UHMW (50 kb – 1+ Mb) DNA Extraction Protocols in applications other than Nanobind Ultra Long Sequencing.

Pulsed Field Gel Electrophoresis (PFGE) comparison of DNA extracted using the HMW DNA Extraction Protocol from various sample types (left). Pulsed Field Gel Electrophoresis (PFGE) comparison of HMW (center) and UHMW (center, right) DNA extracted from MCF-7 cells. The HMW protocols typically result in DNA from 50 – 300+ kb. The UHMW protocols are capable of obtaining megabase DNA surpassing 5.7 Mb in length.


Preservation Methods High quality samples are the key to obtaining high quality DNA. Either fresh or frozen samples can be used equivalently. However, care should be taken to minimize freeze-thaws and to minimize the time samples spend at ambient or at 4 °C.

Fresh vs. Frozen Cells

No systematic difference has been seen in either DNA QC or sequencing results between fresh and frozen cell pellets. Cell pellets should be frozen in minimal liquid after harvesting. No cryoprotectant is necessary. When using frozen cell pellets, it is important to fully resuspend the cell pellets before processing.

Fresh vs. Frozen Blood

No systematic difference has been seen in either DNA QC or sequencing results between fresh and frozen blood samples. Blood samples should be frozen as quickly as possible after being drawn. Storage at 4 °C should be limited to 2 days or less to prevent sample degradation. Blood samples should be aliquoted to avoid repeated freeze-thaws.

Blood Anti-Coagulant

K2 EDTA is the preferred anti-coagulant. However, heparin, citrate, ACD, and PAXGene tubes have also been tested and verified.

Ethanol-Preserved Nucleated Blood

Nucleated-blood preserved in ethanol (e.g.1:10 blood to ethanol) can be used by first centrifuging the blood at 10,000 x g for 2 minutes to pellet the cells and then following the nucleated blood protocols.


PacBio Sequencing HMW DNA was extracted from cultured cells, bacteria, and blood samples. The samples were then sequenced on PacBio RS II, Sequel I, or Sequel II using CLR or HiFi workflows. HMW DNA is recommended for standard CLR and HiFi sequencing.

PacBio Sequel I / II CLR Sequencing

Sample Library Prep Subread Length N50 (bp)

Max Subread Length (bp) Total Data (Gb)

GM12878 cells 5X NS, 30 kb BP Express Template 1.0, Sequel I 32,813 95,946 10.4

GM12878 cells 5X NS, SRE Express Template 2.0, Sequel II 26,789 N/A 142

E. coli 5X NS, 30 kb BP Express Template 1.0, Sequel I 29,611 80,713 6.8

S. sonnei 5X NS, 30 kb BP Express Template 1.0, Sequel I 35,251 83,705 9.0

S. enterica 5X NS, 30 kb BP Express Template 1.0, Sequel I 33,420 93,614 10.5

L. monocytogenes 5X NS, 30 kb BP Express Template 1.0, Sequel I 32,010 95,382 10.2

Human Whole Blood (200 µL)

5X NS, 30 kb BP Express Template 1.0, Sequel I 28,443 95,679 6.7

Human Whole Blood (1 mL)

5X NS, 30 kb BP Express Template 1.0, Sequel I 26,325 89,360 6.4

PacBio Sequel II HiFi Sequencing

Sample Library Prep Total Bases (Gb) ≥Q20 Mean Read Length (bp)

≥Q20 Read Yield (Gb)

≥Q20 Median Read Quality

GM12878 cells 17 kb shear, SRE Express Template 2.0 276 15,320 18.8 Q32



Data generated in collaboration with PacBio® NS Needle shear BP BluePippin size selection SRE Short Read Eliminator size selection


PacBio RS II CLR Sequencing – Blood Anticoagulant

Sample 260/280 260/230 DNA Yield (µg) Subread Length N50 (bp)

Whole Blood – K2 EDTA 1.88 2.04 5.5 ± 0.3 26,906

Whole Blood – Sodium Citrate 1.87 1.98 5.0 ± 0.1 25,610

Whole Blood – Heparin 1.87 1.93 5.1 ± 0.1 23,152

Whole Blood – ACD 1.85 1.81 4.4 ± 0.1 20,862


PacBio Multiplex Microbial Sequencing HMW DNA was extracted from a panel of Gram-negative and Gram-positive bacteria. The samples were then sequenced in multiplex on PacBio Sequel II using Procedure & Checklist – Preparing Multiplexed Microbial Libraries Using SMRTbell® Express Template Prep Kit 2.0 (101-696-100 Version 07). All bacteria assembled to a single chromosomal contig as expected. Detailed data is provided in the Microbial Multiplexing Workflow on the Sequel System using the SMRTbell Express Template Prep Kit 2.0 Application Note.

PacBio Sequel II Microbial Multiplexing

Barcode Sample Gram Status

Genome Size (bp)

Filtered Subread

Coverage Max Contig Length (bp)

Chromosomal Contigs (#)

Concordance w/ NCBI (QV)

BC1001 E. coli K12 – 4,653,240 64x 4,642,499 1 56

BC1002 E. coli K12 – 4,653,240 55x 4,642,500 1 57

BC1009 K. pneumonaie – 5,781,501 50x 5,435,746 1 48

BC1010 B. cereus + 5,427,083 59x 5,408,315 1 35

BC1012 L. monocytogenes + 3,032,269 66x 3,043,149 1 46

BC1015 S. sonnei – 5,062,953 53x 4,813,454 1 62

BC1016 N. meningitidis – 2,194,961 74x 2,213,947 1 50

BC1018 S. aureus + 2,806,345 92x 2,778,860 1 56

BC1019 E. coli Strain W – 5,005,347 68x 4,898,327 1 44

BC1022 S. aureus HPV107 + 2,901,406 82x 2,962,786 1 33

Data generated in collaboration with PacBio®. All samples other than BC1001 were extracted using the Nanobind CBB Big DNA Kit.


Oxford Nanopore Sequencing HMW DNA was extracted from cultured cells, bacteria, and blood samples. The samples were then sequenced on Oxford Nanopore MinION and GridION using the Ligation Sequencing Kits. HMW DNA is recommended for standard long-read sequencing using the Ligation Sequencing Kit (SQK-LSK108/109/110) and Rapid Sequencing Kit (SQK-RAD004). UHMW DNA is recommend for Nanobind Ultra Long Sequencing using the Ultra Long DNA Sequencing Kit (SQK-ULK001).

Oxford Nanopore MinION/GridION

Sample Library Prep Read Length N50 (bp) Longest Read (bp) Total Data (Gb)

GM12878 Cells 10 kb shear, No SS LSK109, FLO-MIN106D 9,026 128,601 17.8

GM12878 Cells 5X NS, No SS LSK109, FLO-MIN106D 38,243 287,388 8.1

GM12878 Cells 5X NS, SRE XL LSK109, FLO-MIN106D, 3X Load 45,517 302,612 13.0

E. coli1 5X NS, BP30 LSK108, FLO-MIN106C 46,156 191,836 6.8

L. monocytogenes1 5X NS, BP3 LSK108, FLO-MIN106C 48,391 254,985 8.0

Human Whole Blood 5X NS, No SS LSK109, FLO-MIN106D 38,985 280,103 7.7

Human Whole Blood 5X NS, SRE XL LSK109, FLO-MIN106D 53,159 346,216 7.9

NS – Needle shear, No SS – No Size Selection, BP30 – 30 kb Sage Science BluePippin, SRE XL – Circulomics Short Read Eliminator XL. 1Data generated in collaboration with Timp Lab at Johns Hopkins University.


Nanobind Ultra Long Sequencing Nanobind Ultra Long Sequencing can generate large numbers of ultra long (100+ kb) on Oxford Nanopore MinION/GridION/PromethION instruments. This method can obtain read length N50 of 100+ kb and reads surpassing 4 Mb in length. UHMW DNA is first extracted using the appropriate UHMW DNA Extraction Protocol and then prepared for sequencing using the Nanobind Library Prep – Ultra Long Sequencing Protocol. Please see the Nanobind UL Library Prep Kit Handbook for additional details.

Nanobind Ultra Long Sequencing on Oxford Nanopore MinION/GridION/PromethION

Sample Instrument Total Bases (Gb) N50 (kb) Gb

>100 kb Gb

>200 kb Number

Reads > 1 Mb Longest

Read (Mb)

HG02723 Cell Line (left graph) MinION 24.5 96.8 11.9 4.7 6 1.2

HG02723 Cell Line (right graph) PromethION 129.6 93.1 60.5 20.3 0 0.99

Human Blood MinION 18.6 104.6 9.9 4.2 11 1.5

Human Blood PromethION 67.6 84.4 28.4 8.1 6 1.3

E. coli MinION 25.9 66.4 8.6 2.7 1 1.1

E. coli1 PromethION 77.4 128 47.1 22.5 133 3.3

L. monocytogenes MinION 22 50 4.8 1.3 1 1.2

L. monocytogenes PromethION 58 53 13.2 3.6 3 1.0

Fish Blood MinION 16.8 98.6 8.3 3.4 1 1.0

Shingleback Lizard Blood2 PromethION 62.8 78.4 24.0 5.8 3 1.6

1Data generated in collaboration with Oxford Nanopore Technologies 2Data generated in collaboration with Garvan Institute

0100200300400500600700800900

0 100 200 300 400 500Th

roug

hput

(Mb)

Read Length (kb)

0

25

50

75

100

125

150

175

0 100 200 300 400 500

Thro

ughp

ut (M

b)

Read Length (kb)

HG02723 on MinION24 Gb, 97 kb N50, 1.2 Mb max

HG02723 on PromethION130 Gb, 93 kb N50, 1.0 Mb max

12 Gb 60 Gb

20 Gb4.7 Gb


Short Read Eliminator Size Selection Sequencing read lengths obtained using Nanobind extracted HMW DNA can be further enhanced by using the Circulomics Short Read Eliminator Kits to deplete short DNA. The Short Read Eliminator Kits are available in three versions with different size selection cutoffs (SRE XS: 10 kb, SRE: 25 kb, SRE XL: 40 kb). HMW DNA was extracted from human blood using the Nanobind CBB Big DNA Kit and then sequenced on Oxford Nanopore GridION (FLO-MIN106D) using the Ligation Sequencing Kit (SQK-LSK109). Size selection of the HMW DNA using the Short Read Eliminator Kit just before library preparation increased N50 from 30.9 kb up to 45.5 kb depending on which version was used.

Short-Read Eliminator Size Selection on Oxford Nanopore GridION

Sample Library Prep Size Selection Read Length N50 (bp)

Longest Read (bp) Total Data (Gb)

Human Blood No shear LSK109, FLO-MIN106D None 30,845 239,009 9.7

Human Blood No shear LSK109, FLO-MIN106D

Short Read Eliminator XS 32,767 325,081 7.4

Human Blood No shear LSK109, FLO-MIN106D Short Read Eliminator 39,456 247,234 7.2

Human Blood No shear LSK109, FLO-MIN106D

Short Read Eliminator XL 45,484 253,743 7.7

0

0.005

0.01

0.015

0.02

0.025

0.03

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

Nor

mal

ized

Dat

a Th

roug

hput

Read Length (kb)

No Size Selection, N50=31 kb

Short Read Eliminator XS, N50=33 kb

Short Read Eliminator, N50=40 kb

Short Read Eliminator XL, N50=46 kb


Processing Tips Magnetic Rack Handling Procedure To capture the Nanobind disk and enable simple processing, the microcentrifuge tubes are placed in a tube rack that is used with a magnetic base. Although DNA is bound quite robustly, proper pipetting and handling will ensure thorough washing and minimize disturbance of the bound DNA. For best results, the Nanobind disk should be captured near the top of the tube so that fluid can be easily removed from the bottom of the tube. The following procedure is recommended.

Recommended procedure for capturing Nanobind disk on a tube rack and magnetic base. This procedure ensures that the Nanobind disk is captured near the top of the liquid interface, minimizing disturbance of the bound DNA and facilitating processing.


Pipetting When removing liquid from the microcentrifuge tube, the Nanobind disk should not be disturbed. Carefully insert the pipette tip against the wall opposite the Nanobind disk and remove liquid by pipetting from the liquid surface. This will minimize the chances of accidentally pipetting bound DNA. Likewise, when adding liquid, dispense against the wall opposite the Nanobind disk.

Pipetting procedure for removal (left) and addition (right) of liquid during wash steps. Avoid disrupting the Nanobind disk and bound nucleic acids.


Heterogeneity and Viscosity (HMW DNA Only) The extracted HMW DNA can be highly viscous and heterogeneous. This is normal and is one of the challenges of working with HMW DNA. The heterogeneity and viscosity of the DNA eluate will vary depending on sample type, DNA size, sample input, and processing parameters. More gentle processing will yield larger DNA size but will also result in higher heterogeneity and larger amounts of highly viscous, unsolubilized “jellies.” Processing that is too gentle can dramatically reduce DNA purity and yield. To minimize the challenges of heterogeneity and viscosity, we recommend that new users err on the side of being overly aggressive. Listed below are tips for working with HMW DNA.

Following elution of the HMW DNA:

Pipette mix the extracted DNA 5-10X with a standard P200 pipette. Pipette mixing will help to loosen and coax the viscous DNA into solution. Moderate amounts of pipette mixing will not significantly impact DNA length. Pipette mixing is a standard part of our DNA elution process; we routinely use it for all long-read sequencing and optical mapping applications. For greater accuracy, the pipette mixed DNA should be left overnight at RT before quantifying the concentration.

In some cases, the extracted DNA will be very heterogeneous and contain large amounts of unsolubilized “jellies”:

The most common reason for high sample heterogeneity and low purity is insufficient mixing during lysis. More aggressive mixing will result in samples with improved purity due to more efficient lysis and digestion. Improved sample purity will lead to improved DNA homogeneity and reduced “jellies.” Aggressive mixing during lysis will not significantly impact DNA length. Mixing may be carefully scaled back by skilled users to achieve bigger DNA. For UHMW DNA, users should follow the appropriate UHMW DNA extraction protocol.

To accurately quantify the HMW DNA:

Pipette mix the DNA 5X with a standard P200 pipette again. Perform triplicate Nanodrop readings by sampling the top, middle, and bottom of the eluate. If the concentration %CV > 30, perform an additional 5X pipette mixing using a standard P200 pipette. Let the DNA rest for at least 1 hr and repeat the Nanodrop measurements.

To accurately determine the concentration of dsDNA, we recommend making triplicate measurements using the Qubit dsDNA BR Assay.

If the extracted DNA needs to be used immediately after extraction:

The extracted DNA can be sheared 5X using a 26g blunt stainless-steel needle and 1 mL syringe. The needle-sheared DNA can be used immediately for library preparation. Moderate amounts of needle shearing will not significantly impact DNA length. Nearly all samples we sequence have been 5X needle sheared.


HMW (50 kb – 300+ kb) DNA Extraction Protocols As of the document release date, the following protocols are available for HMW (50–300+ kb) DNA extraction. They are recommended for most long-read sequencing applications. This includes PacBio CLR and HiFi sequencing and nanopore sequencing using Oxford Nanopore Ligation and Rapid Sequencing Kits. For standard long-read sequencing applications these protocols will result in superior sequencing performance than the UHMW (50 kb – 1+ Mb) DNA Extraction Protocols.

Protocols are updated and added frequently. Please refer to the Circulomics Support Page (https://www.circulomics.com/support-nanobind) for the latest versions and the appropriate Nanobind Kit Handbook for additional data and guidance.

Contact us if you have any questions about which protocol should be used ([email protected]).

HMW DNA Extraction – Cultured Cells (EXT-CLH-001) This protocol describes the extraction of HMW DNA from cultured cells. This protocol has been validated on several cell types including GM12878, MCF-7, MCF-10A, and MDA-MB-231. This protocol requires the Nanobind CBB Big DNA Kit (NB-900-001-01) or Nanobind Tissue Big DNA Kit (NB-900-701-01).

HMW DNA Extraction – Gram-Negative Bacteria (EXT-GNH-001) This protocol describes the extraction of HMW DNA from Gram-negative bacteria. This protocol has been validated on several Gram-negative bacterial species including E. coli, S. sonnei, and S. enterica. This protocol requires he Nanobind CBB Big DNA Kit (NB-900-001-01) or Nanobind Tissue Big DNA Kit (NB-900-701-01).

HMW DNA Extraction – Gram-Positive Bacteria (EXT-GPH-001) This protocol describes the extraction of HMW DNA from Gram-positive bacteria. This protocol has been validated on several Gram-positive bacterial species including L. monocytogenes, S. aureus, and E. faecalis. This protocol requires the Nanobind CBB Big DNA Kit (NB-900-001-01) or Nanobind Tissue Big DNA Kit (NB-900-701-01).

HMW DNA Extraction – 200 µL Mammalian Whole Blood (EXT-BLH-001) This protocol describes the extraction of HMW DNA from 200 μL of mammalian whole blood. It has been validated using fresh and frozen whole blood. Other volumes can also be used with modification to the protocol. This protocol requires the Nanobind CBB Big DNA Kit (NB-900-001-01) or Nanobind Tissue Big DNA Kit (NB-900-701-01).

HMW DNA Extraction – 1 mL Mammalian Whole Blood (EXT-BLH-002) This protocol describes the extraction of HMW DNA from 1 mL of mammalian whole blood. It has been validated using fresh and frozen whole blood. Protocol available upon request. This protocol requires the Nanobind CBB Big DNA Kit (NB-900-001-01) or Nanobind Tissue Big DNA Kit (NB-900-701-01).

HMW DNA Extraction – Nucleated Blood (EXT-CLH-001) This protocol describes the extraction of HMW DNA from 5–30 μL of nucleated blood. It has been validated on fish, bird, and lizard blood including fresh, frozen, and ethanol preserved blood samples. This protocol requires the Nanobind CBB Big DNA Kit (NB-900-001-01) or Nanobind Tissue Big DNA Kit (NB-900-701-01).


UHMW (50 kb – 1+ Mb) DNA Extraction Protocols As of the document release date, the following protocols are available for UHMW (50 kb – 1+ Mb) DNA extraction. These protocols generate large amounts of megabase sized DNA and are only recommend for ultra long sequencing on Oxford Nanopore instruments and for optical mapping. They have been specifically optimized for Nanobind Ultra Long Sequencing and should take precedence over previous UHMW DNA extraction protocols. They are not recommended for use in the standard long-read sequencing applications. For standard long-read sequencing applications, superior results will be obtained using the HMW (50 kb – 300+ kb) DNA Extraction Protocols. Please contact us if you’d like to use the UHMW (50 kb – 1+ Mb) DNA Extraction Protocols in applications other than Nanobind Ultra Long Sequencing.

Protocols are updated and added frequently. Please refer to the Circulomics Support Page (https://www.circulomics.com/support-nanobind) for the latest versions and the appropriate Nanobind Kit Handbook for additional data and guidance.

Contact us if you have any questions about which protocol should be used ([email protected]).

UHMW DNA Extraction – Cultured Cells (EXT-CLU-001) This protocol describes the extraction of UHMW DNA from cultured cells. This protocol has been validated on several cell types including GM12878 and HG02723. This protocol requires the 1) Nanobind CBB Big DNA Kit (NB-900-001-01) or Nanobind Tissue Big DNA Kit (NB-900-701-01), 2) Nanobind UL Library Prep Kit (NB-900-601-01), and 3) UHMW DNA Aux Kit (NB-900-101-01).

UHMW DNA Extraction – Gram-Negative Bacteria (EXT-GNU-001) This protocol describes the extraction of UHMW DNA from Gram-negative bacteria. This protocol has been validated on E. coli. This protocol requires the 1) Nanobind CBB Big DNA Kit (NB-900-001-01) or Nanobind Tissue Big DNA Kit (NB-900-701-01), 2) Nanobind UL Library Prep Kit (NB-900-601-01), and 3) UHMW DNA Aux Kit (NB-900-101-01).

UHMW DNA Extraction – Gram-Positive Bacteria (EXT-GPU-001) This protocol describes the extraction of UHMW DNA from Gram-positive bacteria. This protocol has been validated on L. monocytogenes. This protocol requires the 1) Nanobind CBB Big DNA Kit (NB-900-001-01) or Nanobind Tissue Big DNA Kit (NB-900-701-01), 2) Nanobind UL Library Prep Kit (NB-900-601-01), and 3) UHMW DNA Aux Kit (NB-900-101-01).

UHMW DNA Extraction – Mammalian Whole Blood (EXT-BLU-001) This protocol describes the extraction of UHMW DNA from mammalian whole blood. The protocol is optimized on extraction from 1–3 mL of whole blood. It has been validated on human and bovine blood. This protocol requires the 1) Nanobind CBB Big DNA Kit (NB-900-001-01) or Nanobind Tissue Big DNA Kit (NB-900-701-01), 2) Nanobind UL Library Prep Kit (NB-900-601-01), and 3) UHMW DNA Aux Kit (NB-900-101-01).

UHMW DNA Extraction – Nucleated Blood (EXT-NBU-001) This protocol describes the extraction of UHMW DNA from nucleated blood. The protocol is optimized for extraction from 5–30 μL of nucleated blood. It has been validated on frozen fish and lizard blood. This protocol requires the 1) Nanobind CBB Big DNA Kit (NB-900-001-01) or Nanobind Tissue Big DNA Kit (NB-900-701-01), 2) Nanobind UL Library Prep Kit (NB-900-601-01), and 3) UHMW DNA Aux Kit (NB-900-101-01).


QC Procedures Accurate quantification of HMW and UHMW DNA can be challenging due to sample inhomogeneity, often leading to concentration measurements with high concentration CVs. We recommend performing replicate Nanodrop UV/Vis, replicate Qubit BR DNA Assay measurements, and a single, optional Qubit BR RNA Assay measurement.

See individual HMW and UHMW DNA extraction protocols for detailed guidance.


Storage of DNA DNA can be stored in Buffer EB at 4 °C for several months. Long term storage at -20 °C or -80 °C can be used if necessary. Avoid freeze/thaw cycles since this can degrade high molecular weight DNA.


Troubleshooting FAQ See individual DNA extraction protocols for details.


PacBio Sequel Sequencing Recommendations Below are our standard conditions and tips for sequencing on PacBio Sequel using the SMRTbell Express Template Prep Kit 2.0.

1. Isolate HMW DNA using Nanobind Big DNA Kit. 2. Perform a 5X needle shear using a 26g blunt end needle.

• Light needle shearing will improve sequencing throughput, DNA handling, and AMPure recovery during library preparation without noticeably affecting read length. The DNA concentration during needle shearing should be >100 ng/µL. Lower DNA concentration will result in increased rates of shearing.

• More aggressive shearing to 10–30 kb using Covaris G-tube or Diagenode Megaruptor can be used to obtain a fixed insert size.

3. Prepare DNA for sequencing using Procedure & Checklist - Preparing gDNA Libraries Using the SMRTbell Express Template Preparation Kit 2.0

• For BluePippin size selected CLR libraries, begin with 5 µg of HMW DNA input and use a >30 kb cutoff. • For SageELF or BluePippin size selected HiFi libraries, begin with 20 µg of sheared HMW DNA. • For Short Read Eliminator size selected CLR or HiFi libraries, begin with 5 µg of HMW DNA input.


Oxford Nanopore Sequencing Recommendations Below are our standard conditions and tips for sequencing on Oxford Nanopore MinION/GridION using the 1D Ligation Sequencing Kits (SQK-LSK109).

1. Isolate HMW DNA using Nanobind Big DNA Kit. 2. Perform a 5X needle shear using a 26g blunt end needle.

• Light needle shearing will improve sequencing throughput, DNA handling, and AMPure recovery during library preparation without noticeably affecting read length. The DNA concentration during needle shearing should be >100 ng/µL. Lower DNA concentration will result in increased rates of shearing.

• More aggressive shearing to 10–30 kb using Covaris G-tube or Diagenode Megaruptor can greatly increase throughput at the cost of read length.

3. Optional – Size select sample using Circulomics Short Read Eliminator. • Size selection can be used to improve read length distribution by depleting short DNA.

4. Prepare DNA for sequencing using the 1D Genomic DNA by Ligation (SQK-LSK109) Protocol. • For size selected samples, we generally begin library preparation with 2–4 µg of size selected HMW DNA (42–

83 ng/µL). After library preparation, we generally end up with 700–1500 ng of DNA ready for sequencing. • For non-size selected samples, we generally begin library preparations with 1–1.5 µg of HMW DNA (21–31

ng/µL). After library preparation, we generally end up with 200–500 ng of DNA ready for sequencing.

5. Library Preparation Notes • DNA precipitation may be visible during the adapter ligation step, especially when large amounts of HMW DNA

are input into the reaction. This does not appear to have any significant impact on sequencing throughput. • We typically elute the AMPure cleanups for 10 minutes and 20 minutes after the end-prep and ligation steps,

respectively.

6. Load a constant molar concentration of library onto the flow cell. • For a size selected DNA sample, we recommend loading 800–1000 ng of DNA onto the flow cell. • For a non-size selected DNA sample, we recommend loading 300–500 ng onto the flow cell. • For a moderately sheared DNA sample (i.e. 30 kb shear), we recommend loading 300 ng or less onto the flow

cell. • For consistent throughputs, more should be loaded for libraries with higher average size and less should be

loaded for libraries with shorter average size. Both overloading and underloading of the flow cell will decrease sequencing throughput as a constant molar concentration of DNA library is ideal.

nanobind cbb big dna kit handbook hbk-cbb-001 vf

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