Ultrasonic DNA Fragmentation for Next Gen Sequencing

Next Generation Sequencing (NGS) requires the reliable shearing and fragmentation of genomic DNA in order to sequence genomic DNA strands and to create genome libraries. The controlled fragmentation of DNA into DNA fragments is an essential sample preparation step required before the DNA is sequenced. Ultrasonication is proven as efficient and reliable technique for DNA fragmentation of certain length. Ultrasonic DNA fragmentation protocols achieve reproducible fragmentation results. Hielscher ultrasonicators are capable of producing a wide range of genomic DNA fragment size distributions, precisely controllable via the operating parameters. Since Hielscher ultrasonic DNA shearing systems are available for single and multiple vials as well as for microplates, sample preparation becomes highly efficient.

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UIP400MTP Plate Sonicator for high-throughput sample preparation: The UIP400MTP uniformly sonicates samples in multi-well, microtiter plates and 96-well plates disrupting cells, extracting proteins, fragmenting DNA, RNA and alpha-synuclein fibrils.

The UIP400MTP Plate Sonicator for high-throughput sample preparation uniformly sonicates samples in multi-well, microtiter plates and 96-well plates

Advantages of Ultrasonic DNA Fragmentation

  • repeatable / reproducible results
  • precisely adjustable to obtain certain fragment length
  • rapid processing
  • consistent DNA fragmentation results
  • devices for any sample volumes (e.g., multiple vials or microplates)
  • high throughput
  • precise temperature control
  • simple, user-friendly operation

Next-Gen Sequencing: Ultrasonic DNA Fragmentation for Library Preparation

In order to carry out a Next-generation sequencing, the three basic steps of (1) library preparation, (2) sequencing, and (3) data analysis must be performed. During library preparation, DNA is fragmented, then the fragment ends are repaired (polished) by adding a single adenine base and the target fragments are converted to double-stranded DNA. Finally so-called adapters are attached by ligation, PCR, or tagmentation so that the final library DNA product can be quantitated for sequencing.
DNA Fragmentation using Sonication: Especially when short-read sequencing technologies such as Illumina, which cannot read longer DNA fragments readily, the DNA stands must be fragmented to a certain size which can be achieved reliably by ultrasonication.
Ultrasonication can be reliably used for DNA, RNA and chromatin fragmentation.

Next Gen SequencingLibrary Preparation

Ultrasonic DNA fragmentation is commonly employed in the preparation of DNA sequencing libraries for next-generation sequencing (NGS) platforms. This technique is utilized to break DNA molecules into smaller fragments of a desired size range, which facilitates the subsequent steps in library preparation. Ultrasonic DNA fragmentation is typically required during the library preparation step of NGS workflows to fragment genomic DNA into smaller pieces suitable for downstream processing and sequencing. It plays a crucial role in ensuring the success of the sequencing experiment by generating DNA fragments of the appropriate size range for the specific sequencing platform being used.

Ultrasonic DNA fragmentation is frequently used as sample preparation step in Next Generation Sequencing (NGS)

Electrophoretic analyses of genomic DNA of E. coli EDL933 subjected to 0 – 15 min ultrasonication. L indicates the DNA Ladder. (Basselet et al. 2008)

Next Generation SequencingProcess Steps:

  • Ultrasonic DNA Fragmentation: Before library construction, the genomic DNA is fragmented into smaller, more manageable pieces. Ultrasonic fragmentation involves using high-frequency sound waves to shear the DNA molecules into fragments of the desired size range. This step is crucial because it helps ensure that the sequencing reads generated later will be of appropriate length for the sequencing platform being used. The size range of the fragments can be adjusted based on the specific requirements of the sequencing experiment.
  • Clonal Amplification by PCR: After ultrasonic fragmentation, the DNA fragments undergo end repair, adapter ligation, and PCR amplification to generate the final DNA sequencing libraries. These steps prepare the fragmented DNA molecules for the sequencing process by adding adapter sequences necessary for binding to the sequencing platform and providing priming sites for PCR amplification.
  • DNA Sequencing by Synthesis: Once the sequencing libraries are prepared, the DNA sequencing by synthesis (SBS) process begins. During SBS, the DNA sequence is determined by the sequential addition of nucleotides to the complementary strand. This step involves cyclic reactions of nucleotide incorporation, imaging, and cleavage, allowing the determination of the DNA sequence based on the fluorescent signals emitted by the incorporated nucleotides.
  • Massively Parallel Sequencing: In the final step, the spatially segregated, amplified DNA templates are sequenced simultaneously in a massively parallel fashion. This high-throughput sequencing approach enables the generation of millions to billions of sequencing reads in a single sequencing run, allowing for efficient and rapid determination of DNA sequences.

How Does Ultrasonic DNA Fragmentation Work?

Sonication, also known as acoustic sample processing, is a widely used method to fragment DNA. For ultrasonic DNA shearing, the samples are exposed to ultrasonic waves under controlled condition. The working principle of ultrasonic DNA fragmentation is based on the vibrations and cavitation generate by the ultrasound waves. The shear forces that result from ultrasonic (acoustic) cavitation break high molecular weight DNA molecules. The setting of sonication such as intensity (amplitude, duration), pulsation mode and temperature allow for precise DNA fragmentation to a certain desired length of DNA fragments. Whilst DNA often is reduced to 100 to 600 bp using ultrasonication, longer DNA fragments of up to 1300 bp can be obtained when milder ultrasonic conditions are applied.

Ultrasonic homogenizers are reliable for DNA shearing

Ultrasonic DNA shearing during ChIPchromatin Immunoprecipitation
Adapted from Jkwchui under CC-BY-SA.03

 

This tutorial explains what type of sonicator is best for your sample preparation tasks such as lysis, cell disruption, protein isolation, DNA and RNA fragmentation in laboratories, analysis, and research. Choose the ideal sonicator type for your application, sample volume, sample number and throughput. Hielscher Ultrasonics has the ideal ultrasonic homogenizer for you!

How to Find the Perfect Sonicator for Cell Disruption and Protein Extraction in Science and Analysis

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Temperature Control To Prevent DNA Degradation

The double-stranded molecular shape of DNA is highly sensitive to elevated temperatures so that exact control over temperature during sample preparation steps is a crucial factor for reliable analysis results.
Whether you are using Hielscher’s probe ultrasonicators, the VialTweeter or the UIP400MTPcontinuous temperature monitoring and control is ensured due to a pluggable temperature sensor and the smart device software. In order to maintain the temperature within a certain range, you can set an upper and lower temperature limit. Consequently, the ultrasonicator will pause as soon as this temperature limit is exceeded and automatically will continue to sonicate when the temperature has lowered by a set ∆T.
The sophisticated software of Hielscher ultrasonicators ensures the reliable maintenance of ideal sample treatment conditions.

Mass Sample DNA Fragmentation with the UIP400MTP Multi-Well Plate Ultrasonicator

Ultrasonic Multi-Sample Preparation Unit UIP400MTP for multi-well plate sonicationSample numbers in life science have increased significantly within the last decade. This means very high numbers of samples (e.g., 384, 1536, or 3456 wells per microplate) must be processed during sample prep and analysis under consistently equal conditions in order to obtain comparable and valid results. With the UIP400MTP, Hielscher Ultrasonics is following the trend of mass sample processing. The UIP400MTP is an ultrasonicator for sample preparation using microplates. The UIP400MTP can process plates with 6, 12, 24, 48, 96, 384, 1536, or 3456 wells. Depending on the microplate type, each well can typically hold sample volumes between tens of nanolitres to several millilitres. Widely used in life science research, the UIP400MTP is very commonly used for sample preparation before assays such as ELISA (enzyme-linked immunosorbent assay) or PCR, before protein analytics, as well as for chromatin preparation before CHiP and CHiP-seq, histone modification identification, and other analytical treatments (e.g., gel electrophoresis, mass spectrometry).

The UIP400MTP ultrasonic homogenizer can sonicate multi-well-plates and micro-titer-plates for cell lysis, DNA fragmentation, dispersing or homogenizing.

UIP400MTP for Multi-Well-Plate Sonication

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The VialTweeter for Sample Praparation of up to 10 Vials

Complete VialTweeter setup: VialTweeter sonotrode at ultrasonic processor UP200StThe VialTweeter is a widely used lab ultrasonicator VialTweeter that allows for the effective and comfortable sonication of up to 10 vials simultaneously. Since the vials and test tubes (e.g., Eppendorf vials, cryo vials, centrifuge tubes) are sonicated indirectly, any cross-contamination is avoided. As the same ultrasound intensity is delivered to each sample, all sonication results are homogeneous and reproducible. The VialTweeter offers all smart features like our other digital devices (e.g., smart menu, programmable settings, temperature control, remote control etc.) so that highest user-comfort is ensured.

Multi-Finger Probes for Microwell Plates

4 probe heads or 4 sonotrodes for simultaneous sonication of 4 samples at the same intensity with the Hielscher 200 watts ultrasonicator models UP200ST and UP200HTAvailable for the ultrasonic probe homogenizers UP200Ht and UP200St, multi-finger probes with 4 or 8 fingers are a comfortable option to sonicate multiple sample at the same time under same conditions. For instance, the sonotrode MTP-24-8-96 is an eight finger probe, which is ideal for the integration into automated systems or the efficient manual sample preparation of the wells of multi-well plates. The multi-finger sonotrode is ideal for automated for laboratories, where mostly beakers and test tubes using a standard ultrasonic sonotrode are processed. The multi-finger and standard probes can be rapidly inter-changed within a few minutes transforming the single-probe ultrasonicator into a multi-probe ultrasonic disruptor.

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Hielscher Ultrasonicators for DNA Fragmentation

Hielscher Ultrasonics offers various ultrasound-based platforms for DNA, RNA, and chromatin fragmentation. These different platforms include ultrasonic probes (sonotrodes), indirect sonication solutions for the simultaneous sample preparation of multiple tubes or multi-well plates (e.g., 96-well plates, microtiter plates), sonoreactors, and ultrasonic cuphorns. All platforms for DNA shearing are powered by frequency-tuned, high-performance ultrasonic processors, which are precisely controllable and deliver reproducible results.

Ultrasonic Processors for Any Sample Number and Size

With Hielscher’s multi-sample ultrasonicators VialTweeter (for up to 10 test tubes) and UIP400MTP (for microplates/ multiwell plates) it becomes easily possible to reduce sample processing time due to intense and precisely controllable ultrasonication whilst obtaining the desired DNA fragment size distribution and yield. Ultrasonic DNA fragmentation makes sample preparation efficient, reliable and scalable. Protocols can be linearly scaled from one to numerous samples by applying constantly controlled ultrasound.
Probe ultrasonicators with one to five fingers are ideal for the preparation of smaller sample numbers. Hielscher’s laboratory ultrasonicators are available at various sizes so that we can recommend you the ideal device for your application and requirements.

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Hielscher ultrasonicators can be remotely controlled via browser control. Sonication parameters can be monitored and adjusted precisely to the process requirements.Precisely controllable sonication settings are crucial since exhaustive sonification can destroy DNA, RNA and chromatin, but inadequate ultrasonic shearing results in too long DNA and chromatin fragments. Hielscher’s digital ultrasonicators can be easily set to precise sonication parameter. Specific sonication settings can be also saved as programmed setting for fast repetition of the same procedure.
All sonication are automatically protocoled and stored as CSV file on a built-in SD-card. This allows for accurate documentation of performed trials and makes it possible to revise sonication runs easily.
Via browser remote control, all digital ultrasonicators can be operated and monitored via any standard browser. Installation of additional software is not required, since a LAN connection allows a very simple plug-n-play setup.


Highest User-Friendliness in Ultrasonic Sample Preparation

All Hielscher ultrasonicators are designed to deliver high-performance ultrasound, whilst at the same time always being very user-friendly and easy-to-operate. All settings are well-structured in a clear menu, which can be easily accessed via coloured touch-display or browser remote control. The smart software with programmable settings and automatic data recording ensures optimal sonication settings for reliable and reproducible results. The clean and easy-to-use menu interface turn Hielscher ultrasonicators into user-friendly and efficient devices.
The table below gives you an indication of the approximate processing capacity of our lab ultrasonicators, which are ideal for sample preparation tasks such as DNA and RNA fragmentation, cell lysis as well as protein extraction:

DevicePower [W]TypeVolume [mL]
UIP400MTP400for microplates63456 wells
VialMagassugárzó200for up to 10 vials plus clamp-on possibility0.51.5
UP50H50probe-type0.01250
UP100H100probe-type0.01500
UP200Ht200probe-type0.1 – 1000
UP200St200probe-type0.1 – 1000
UP400St400probe-type5.02000
CupHorn200CupHorn, sonoreactor10200
GDmini2 200contamination-free flow cell

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The VialTweeter is a MultiSample Ultraonicator that allows for reliable sample preparation under precisely controlled temperature conditions.

The ultrasonic multi-sample preparation unit VialMagassugárzó allows for the simultaneous sonication of up to 10 vials. With the clamp-on device VialPress, up to 4 additional tubes can be pressed to the front for intense sonication.


The sonotrode MTP-24-8-96 has eight ultrasonic probes for the sonication of the wells of microtiter plates.

The sonotrode MTP-24-8-96 has eight ultrasonic probes for the sonication of the wells of microtiter plates.



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Facts Worth Knowing

What is Next Generation Sequencing?

Next-generation Sequencing, also Next Gen Sequencing (NGS), high-throughput sequencing or second-generation sequencing, refers to the approach of massive parallel sequencing, where very large (massive) amounts of DNA of millions of fragments are sequenced simultaneously in parallel per run.
In order to carry out a Next-generation sequencing, the three basic steps of

  1. library preparation,
  2. sequencing, and
  3. data analysis

are required.
During library preparation, DNA strands must be fragmented into DNA fragments of certain length. Sonication is one of the preferred technique to fragment DNA.
During the process of DNA sequencing, the order of nucleotides in DNAknown as nucleic acid sequence – is determined. The nucleic acid sequence is composed from four nucleotide bases – adenine, cytosine, guanine, thyminewhich code for information.
Next-generation sequencing is driving research in life science and personalized medicine since DNA and RNA sequencing are heavily used in genomic research, cancer research, the research of rare and complex diseases, microbial research, agrigenomics and many other research fields.

Next Generation Sequencing vs Sanger Sequencing

Whilst with Next Generation Sequencing (NGS) it is possible to sequence massive numbers of genomic samples, Sanger Sequencing (also known as chain termination method or First Generation Sequencing) has only the capability to sequence small sample numbers. Sanger sequencing only sequences a single DNA fragment at a time and can be accomplished in a single day. Due to its acccuracy, the Sanger sequencing is also considered the gold-standard technology, which is used to verify results obtained by Next-generation sequencing.
Sanger sequencing achieves read lengths of approximately 800bp (typically 500-600bp with non-enriched DNA). The longer read lengths in Sanger sequencing display significant advantages over other sequencing methods especially in terms of sequencing repetitive regions of the genome. A challenge of short-read sequence data is particularly an issue in sequencing new genomes (de novo) and in sequencing highly rearranged genome segments, typically those seen of cancer genomes or in regions of chromosomes that exhibit structural variation. [cp. Morozova and Marra, 2008]

DNADeoxyribonucleic AcidIts Forms and Functions

Each form of DNA has unique characteristics and applications, contributing to a broad spectrum of research fields, including oncology, genetics, forensic science, and evolutionary biology. Hielscher sonicators are a highly efficient and reliable solution to isolate and fragment DNA and RNA for your analysis purposes. In the list below, we explain you the specific forms of DNA and categorize them based on their biological context and functions:

  • Genomic DNA (gDNA)
    Genomic DNA (gDNA): The complete set of DNA in an organism, including both coding (genes) and non-coding regions.
  • Extracellular DNA
    Circulating Tumor DNA (ctDNA): Fragments of DNA released into the bloodstream by tumor cells.
    Cell-Free DNA (cfDNA): DNA fragments found freely circulating in the bloodstream, originating from various tissues.
  • Extrachromosomal Circular DNA (eccDNA): Circular DNA molecules found outside the chromosomes in eukaryotic cells.
    Viral DNA: DNA originating from viruses, either integrated into the host genome or as episomal DNA.
  • Mitochondrial DNA
    Mitochondrial DNA (mtDNA): DNA located in the mitochondria, inherited maternally, and involved in energy production.
  • Plasmid DNA
    Plasmid DNA: Small, circular DNA molecules that replicate independently of the chromosomal DNA, commonly found in bacteria and used in genetic engineering.
  • Nuclear DNA
    Nuclear DNA (nDNA): DNA contained within the nucleus of eukaryotic cells, comprising the majority of genetic material in an organism.
  • Single-Cell DNA
    Single-Cell DNA (scDNA): DNA extracted from a single cell, used for detailed genomic analysis at the individual cell level.
  • Recombinant DNA
    Recombinant DNA (rDNA): DNA molecules formed by laboratory methods of genetic recombination to bring together genetic material from multiple sources.
  • Specialized Forms
    Environmental DNA (eDNA): DNA collected from environmental samples (soil, water) without isolating the source organisms
    Ancient DNA (aDNA): DNA extracted from ancient specimens, providing insights into evolutionary biology and ancient populations.
  • Chromosomal DNA
    Chromosomal DNA: DNA that constitutes the chromosomes within the cell nucleus, encompassing both coding and non-coding regions.
  • Viral and Synthetic Forms
    Viral DNA: DNA derived from viruses, which can be either integrated into host genomes or exist as independent entities.
    Synthetic DNA: Artificially synthesized DNA sequences created through chemical processes, often used in research and biotechnology.

High performance ultrasonics! Hielscher's product range covers the full spectrum from the compact lab ultrasonicator over bench-top units to full-industrial ultrasonic systems.

Hielscher Ultrasonics manufactures high-performance ultrasonic homogenizers from lab to industrial size.


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