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The Monarch® HMW DNA Extraction Kits provide a rapid and reliable process for extracting high molecular weight DNA (HMW DNA) from biological samples. These kits utilize an optimized process that combines gentle cell lysis with a tunable fragment length generation, followed by precipitation of the extracted DNA onto the surface of large glass beads. DNA size ranges from 50-250 kb for the standard protocol and into the Mb range for certain sample types when the lowest agitation speeds are used. Purified DNA is recovered in high yield with excellent purity, including nearly complete removal of RNA, and is ready for use in downstream applications including long-read sequencing. 

Reasons to Choose the Monarch HMW DNA Extraction Kits 



Extremely fast, user-friendly protocols utilizing a novel glass-bead-based approach

Comparison of HMW DNA Extraction Methods 
Extraction Method Approximate protocol/workflow time*  Notes** 
  Cells  Blood  Tissue   
NEB Monarch HMW DNA Extraction  30 minutes  60 minutes  90 minutes  Fast, convenient, High yield, pure, tunable DNA size 
Circulomics® Nanobind®  > 60 minutes  60 minutes  >2 hours  Several hands-on steps, difficult to process some samples due to high viscosity. 
Phenol/Chloroform Extraction  > 6 hours   > 6 hours   > 6 hours   Hazardous reagents, lengthy workflow, DNA is difficult to dissolve 
Qiagen® MagAttract® Not compatible  ~60 minutes  Overnight  
+ 40 minutes 
DNA is heavily sheared 
Qiagen® Genomic Tips  ~3.5 hours  3.5 hours  ~4 hours  Long workflow, sub-optimal yields, not very high molecular weight 
Revolugen® FireMonkey®  ~45 minutes  >60 minutes  Not compatible  Only second eluate is used (very low recovery); DNA heated to 80°C leading to denaturing
*workflow times are estimated based on protocol protocols published as of July 2020, and internal testing or usage 
**Notes are based on internal testing or usage 

Workflow for Processing Cell Samples 





Reproducibly purify high molecular weight genomic DNA (HMW DNA) from various sample types


 
Reproducible Extraction of HMW DNA from Cells and Blood with the Monarch HMW DNA Extraction Kit



DNA extracted with Monarch HMW DNA Extraction Kit for Cells & Blood. 1 x 106 fresh HEK293 cells and 500 μl fresh human blood were used as inputs and for preps performed according to the kit instructions using the agitation speed indicated above the gel lanes. 500 ng of DNA from the replicates was resolved by PFGE (1% agarose gel, 6 V/cm, 13°C for 20 hours, switch times ramped from 0.5–94 seconds on a BioRad® CHEF-DR® III System). Yield and purity ratios of the individual preps are shown in the accompanying tables. Lambda PFG Ladder (NEB #N0341) was used as molecular weight standard. 



The Monarch HMW DNA Extraction Kit for Tissue efficiently purifies high-quality, HMW DNA from a variety of sample types 



HMW genomic DNA extracted with the Monarch HMW DNA Extraction Kit for Tissue. 10 mg frozen rat kidney, 10 mg fresh mouse liver, 20 mg frozen mouse brain, 20 mg fresh mouse muscle, 2 x 109 frozen E. coli cells, 2 x 108 frozen B. cereus cells, 4 mg fresh X. laevis, 3.8 x 108 fresh S. cerevisiae cells and 15 mg frozen A. aegypti were used as inputs for preps. Preps were performed according to the kit instructions with sample agitation at 2000 rpm. A modified workflow was used to process S. cerevisiae preps. 500 ng of DNA from each sample prep was resolved by PFGE (1% agarose gel, 6 V/cm, 13°C for 20 hours, switch times ramped from 0.5-94 seconds on a BioRad CHEF-DR III System). Yield and purity ratios of the individual preps are shown in the accompanying tables. Lambda PFG Ladder (NEB #N0341) was used as a molecular weight standard. 



Tune fragment length by varying agitation speeds during lysis; achieve DNA in the Megabase size range with low speeds 


Use of varying agitation speeds during lysis produces tunable fragment length of extracted HMW genomic DNA from cells and blood



Preps were performed on duplicate aliquots of 1 x 106 HEK293 cells and 500 μl fresh human blood. Samples were agitated at the indicated speed during the lysis step to control the fragmentation of the DNA. Equal amounts of DNA from the replicates (cells: 500 ng; blood: 650 ng) were resolved by PFGE (1% agarose gel, 6 V/cm, 13°C for 20 hours, switch times ramped from 0.5–94 seconds on a BioRad CHEF-DR III System). Yield and purity ratios of the individual preps are shown in the accompanying tables. Lambda PFG Ladder and Lambda DNA-Hind III Digest (NEB #N0341 and #N3012) were used as molecular weight standards. Yield, purity ratios and DINs of the individual preps are shown in the accompanying tables. 



Use of varying agitation speeds during lysis produces tunable fragment length for HMW genomic DNA extracted from soft organ tissues and bacteria 



HMW genomic DNA from mouse kidney (10 mg, Figure 3A) and E. coli NEB10 beta (1 x 109 cells, Figure 3B) was purified using the Monarch HMW DNA Extraction Kit for Tissue. Samples were agitated at the indicated speed during the lysis step to control the fragmentation of the DNA. 300 ng (kidney) and 500 ng (E. coli) of DNA for each sample was resolved by PFGE (settings: switch time 0.5 to 94 sec; run time 20 hours at 6 V/cm, angle 120°, temperature 13°C on the BioRad CHEF-DR III System). Lambda PFG Ladder (NEB #N0341) was used as molecular weight standard. Yield, purity ratios and DIN values (kidney) of the individual preps are shown in the accompanying tables. 


Achieve outstanding results when compared to other commercially available solutions 


Monarch HMW DNA Extraction Kit produces high molecular weight DNA with excellent yields, purity, and fragment length as compared to other commercially available kits



HMW was isolated from 1x106 HEK293 cells and fresh human blood with the kits indicated in the figure legend. Blood input volumes were used as specified in manufacturers’ protocols (N: 500 μl, C: 200 μl, R: 500 μl, Q: 200 μl, P: 300 μl, Z: 200 μl).  

Monarch samples (lanes 1-2) were purified at maximum agitation speed during lysis (2000 rpm). Variation in fragment length of cellular DNA using the standard protocols for Monarch and Circulomics (lanes 1-2 and 3-4, respectively) results from agitation speeds during lysis (Monarch: 2000 rpm, Circulomics: 900 rpm). All other data presented are duplicate samples from each different kit and the standard protocols dedicated to blood or cells were followed. Qiagen does not provide a protocol for cultured cells; a modified version of the blood protocol was followed. Samples were eluted in 100 μl, except for Zymo which was eluted in 50 μl according to their recommendations.  

Yield and purity of the standard samples were analyzed on Trinean Dropsense 16 spectrophotometer (now Unchained Labs Lunatic. Reported blood sample yields were normalized per 100 μl. RNA content was determined by HPLC analysis of nucleoside content after digestion of 1 μg of eluted DNA with the Nucleoside Digestion Mix (NEB #M0649. The optional RNase treatment was performed with the Zymo prep. 



Excellent performance in long read sequencing

Single Run Sequencing Results of HEK293, Human Blood, and Mouse Kidney Samples on the Oxford Nanopore Technologies Platform

  HEK293 SAMPLE 1 HEK293 SAMPLE 2 HUMAN BLOOD SAMPLE 1 HUMAN BLOOD SAMPLE 2 MOUSE KIDNEY SAMPLE
Mean read length (bases) 21338.9 19249.9 21522.6 24677.7 27120.7
Mean read quality 12.8 13.2 13.4 13.3 13
Median read length 10388 9702 10130 12593 23150
Median read quality 13.2 13.7 13.9 13.8 13.5
Number of reads 377687 633636 538090 327314 164000
Read length N50 (bases) 45432 40415 46542 51394 44631
Total bases 8059414490
(8.1 Gb)
12197410796
(12.2 Gb)
11581090785
(11.6 Gb)

8077351338
(8.1 Gb)

4447789727
(4.4 Gb)


DNA used for the sequencing libraries was extracted by following the standard protocols for cultured cells and fresh mammalian blood samples, without further size selection. Mouse kidney DNA samples were extracted from fresh samples using a rotor-stator homogenizer for sample homogenization. Libraries were generated using the LSK109 ligation sequencing kit and loaded on a FLO-MIN106D flow cell. Sequencing was performed on a GridION® Mk1 for up to 48 hours, or shorter if no more data was generated by the flow cell. No additional treatment of the flow cell (e.g., flushing) was employed. In the samples that resulted in > 10 Gb of data, 800–1,000 ng of DNA library was loaded onto the flow cell for optimal sequencing performance and effective pore usage. Read lengths are indicated in bases unless otherwise noted.




Monarch can purify Mb sized DNA from blood and provides excellent nanopore sequencing results

Monarch_HMW_Blood_1020 

A. XL DNA was extracted from human blood samples following the Monarch protocol using an agitation speed during lysis of 300 rpm (lane 1). HMW DNA was isolated using the Circulomics Nanobind Kit following the protocol for ultra-high molecular weight (UHMW) DNA (lane 2). “Standard” HMW DNA samples were isolated using the Monarch kit using agitation speed during lysis of 2000 rpm (lanes 3 and 4) or the standard HMW protocol for blood from Circulomics (lanes 5 and 6). PFGE settings: 1% agarose gel, 6 V/cm, 13°C for 20 hours, switch times ramped from 0.5–94 seconds on a BioRad CHEF-DR III System.

B. Oxford Nanopore Technologies ligation-based sequencing from 12-plex run with 6 blood and 6 cell samples using the Ligation sequencing kit SQK- LSK109 kit, the Native barcode expansion EXP-NBD104. FLO-MIN106D R9.4.1 flow cells and the GridION Mk1 sequencer. Results of 3 samples are shown for NEB Monarch and Circulomics. Blood samples were extracted on the same day from the same fresh human blood sample following the standard HMW protocols for blood samples from the Monarch and Circulomics kit. DNA integrity of the isolated DNA was similar to that shown in lanes 3-6 in figure A. Monarch purified samples reproducibly provided significantly higher N50 read length values.

C. Spectrophotometric analysis of yield and purity using a Trinean Dropsense 16 of the same samples shown in B. Monarch provides for significantly higher yields with the standard input volume of 500 μl versus the Circulomics workflow which is limited to 200 μl. A260/230 ratios reveal higher purity on the Monarch samples.




PacBio® Sequel I sequencing data from Monarch purified HMW DNA from human blood sheared to 75 kb (size selected to >30 kb)

Monarch_HMW_PacBio_30to60_1020

HMW DNA was extracted from human blood with the Monarch HMW DNA Extraction Kit for Cells & Blood with agitation speed during lysis set to 2000 rpm. DNA was sheared to 75 kb with a Megaruptor, and SMRTbells were constructed with the SMRTbell Express Template Prep Kit 2.0 and size selected to a minimum of 30 kb with a Blue Pippin. CLR reads were obtained on a PacBio Sequel I using 12 pM on-plate loading concentration and a 20 hr movie with no pre-extension.




We’ve had great success with obtaining HMW DNA for long-read sequencing from a variety of cell types, using less input and obtaining a comparable yield…It is straightforward and easy to use.

– Inswasti Cahyani & Matt Loose, DeepSeq, University of Nottingham