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.
- 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.
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.
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 UIP400MTP – continuous 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
Sample 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 VialTweeter for Sample Praparation of up to 10 Vials
The 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
Available 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.
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.
Precise Process Control
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:
|Device||Power [W]||Type||Volume [mL]|
|UIP400MTP||400||for microplates||6 – 3456 wells||VialTweeter||200||for up to 10 vials plus clamp-on possibility||0.5 – 1.5|
|UP50H||50||probe-type||0.01 – 250|
|UP100H||100||probe-type||0.01 – 500|
|UP200Ht||200||probe-type||0.1 – 1000|
|UP200St||200||probe-type||0.1 – 1000|
|UP400St||400||probe-type||5.0 – 2000|
|CupHorn||200||CupHorn, sonoreactor||10 – 200|
|GDmini2||200||contamination-free flow cell|
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Literature / References
- Poptsova, M., Il’icheva, I., Nechipurenko, D. et al. (2014): Non-random DNA fragmentation in next-generation sequencing. Scientific Reports 4, 4532; 2014.
- Basselet P., Wegrzyn G., Enfors S.-O., Gabig-Ciminska M. (2008): Sample processing for DNA chip array-based analysis of enterohemorrhagic Escherichia coli (EHEC). Microbial Cell Factories 7:29. 2008.
- Doublier S., Riganti Ch., Voena C., Costamagna C., Aldieri E., Pescarmona G., Ghigo D., Bosia A. (008): RhoA Silencing Reverts the Resistance to Doxorubicin in Human Colon Cancer Cells. Molecular Cancer Research 6(10), 2008.
- Fredlund E., Gidlund A., Olsen M., Börjesson T., Spliid N.H.H., Simonsson M. (2008): Method evaluation of Fusarium DNA extraction from mycelia and wheat for down-stream real-time PCR quantification and correlation to mycotoxin levels. Journal of Microbiological Methods 2008.
- Fritsche C., Sitz M., Weiland N., Breitling R., Pohl H.-D. (2007): Characterization of the growth behavior of Leishmania tarentolae – a new expression system for recombinant proteins. Journal of Basic Microbiology 47, 2007. 384–393.
- Ristola M., Arpiainen S., Saleem M. A., Mathieson P. W., Welsh G. I., Lehtonen S., Holthöfer H. (2009): Regulation of Neph3 gene in podocytes – key roles of transcription factors NF-κB and Sp1. BMC Molecular Biology 10:83, 2009.
- Rodriguez J., Vives L., Jorda M., Morales C., Munoz M., Vendrell E., Peinado M. A. (2008): Genome-wide tracking of unmethylated DNA Alu repeats in normal and cancer cells. Nucleic Acids Research Vol. 36, No. 3, 2008. 770-784.
- Weiske J. Huber O. (2006): The Histidine Triad Protein Hint1 Triggers Apoptosis Independent of Its Enzymatic Activity. The Journal of Biological chemistry. Vol. 281, No. 37, 2006. 27356–27366.
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 must be performed. 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 DNA – known as nucleic acid sequence – is determined. The nucleic acid sequence is composed from four nucleotide bases – adenine, cytosine, guanine, thymine – which 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]