Next-Gen Sequencing has quickly become one of the most important tools in genomics research and nucleic acid fragmentation is a crucial first step in the sequencing workflow. While there are a variety of methods available to fragment nucleic acids, mechanical shearing remains the method of choice for achieving high sensitivity and unbiased results. Adaptive Focused Acoustics™ (AFA) technology is firmly established as the fragmentation method of choice for NGS, and Covaris continues to innovate the tools required to shear DNA and RNA without GC bias or thermal damage.
AFA Process for DNA and RNA Shearing
The Covaris AFA process is conducted under isothermal conditions, ensuring the integrity of the nucleic acid sample is maintained and providing high recovery of double-stranded DNA. Combined with the specifically engineered AFA Tubes it is possible to precisely and accurately fragment DNA and RNA to the 100 – 1500bp range (microTUBE), or 2 – 5kb range (miniTUBE). Focused-ultrasonicators range from the inexpensive M-Series to the single-tube full-power S-Series to higher throughput E and L-Series instruments.
g-TUBETM for Longer DNA Fragments
For sequencing applications requiring longer DNA fragments, Covaris developed the patented g-TUBE™. Generating shearing forces through centrifugation, the g-TUBE produces your selected fragment length in the 6 kb – 20 kb range. Apart from a benchtop centrifuge, no other equipment is needed.
Mechanical DNA and RNA shearing with AFA technology is the gold standard for nucleic acid fragmentation prior to NGS library preparation. All major sequencing instrument providers recommend the use of Covaris technology to obtain the highest quality data:
Unbiased results without GC or temperature bias
Consistent DNA/RNA shearing to a wide range of target sizes with low c.v. (quantify)
Concentration independent shearing
Optimization free protocols readily available
Highest quality data from amplified, non-amplified and whole genome sequencing experiments
AFA processing completely scalable from a single sample to 96 well plate
Wide range of consumables for different volumes and desired fragment size with easy selection
For customers requiring 1.5-5 Kb fragments, Covaris also offers the miniTUBE for use with the AFA™ Focused-ultrasonicators. miniTUBEs are an ideally suited for use in RainDance Technology workflows, as an example.
The Covaris AFA process is extensively cited in peer reviewed research articles for DNA fragmenting on all available sequencing platforms. Covaris technology is a mainstay for leading genome centers worldwide, including The Broad Institute, Wellcome Trust Sanger Institute, and Beijing Genome Institute.
Features & Benefits
Processing at controlled temperatures provides highest yields while preserving sample fidelity
Accurate and precise.
Highly reproducible process, day to day, user to user. Protocols can be transferred instrument to instrument without further optimization
Non-contact, closed vessel
No cross-contamination, clean-up, or sample loss
Generate tight fragment distribution centered from 100bp to 20 kbp
For DNA fragments distribution in the 150bp - 1,500kb range: microTUBE
For DNA fragments distribution in the 2 - 5kb range: miniTUBE
For DNA fragments distribution in the 6 - 20kb range: g-TUBE
A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries. Fisher et al. Genome Biology 2011, 12:R1
In this paper, scientist from the Broad Institute describe how the use of Covaris E210 system in conjunction with AFA microTUBEs is part of the process improvements that resulted in an dramatic increase in scale and throughput of sequence ready libraries produced. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3091298/?tool=pubmed
A large genome center’s improvements to the Illumina sequencing system. Quail et al. 2008. Nature Methods Vol 5 No 12.
In this paper, scientists from the Wellcome Trust Sanger Intitute discus how the use of AFA energy improves the Illumina protocol, making it more reliable in a high throughput environment. Main benefits from Covaris AFA technology are the tight fragment size distribution, allowing to skip the size selection step in some cases, and its compatibility with high throughout workflows. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2610436/?tool=pubmed
Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Aird et al. Genome Biology 2011, 12:R18
In this paper, scientists from the Broad Institute studied the origin of GC bias in Illumina sequencing libraries. They isolated each step of the library preparation process, and checked for GC bias. Their results show that DNA shearing with Covaris AFA technology doesn’t introduces any bias in the library preparation http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3188800/?tool=pubmed
Genome-wide copy number analysis of single cells. Baslan et al. Nature Protocols. 2012, 7:6 1024-1041
“Previously we reported sonication of WGA DNA using the Bioruptor ultrasonic disruptor24. However, we have switched to using the Covaris focus acoustics system, as it allows for higher throughput.” http://www.nature.com/nprot/journal/v7/n6/abs/nprot.2012.039.html
Solution-based targeted genomic enrichment for precious DNA samples. Shearer, AE et al. BMC Biotech 12:20 2012
“We found that after shearing with the Covaris, a tight size range is attained… negating the need for any size selection with gel electrophoresis, bead-based selection, or specialized equipment. The primary benefit of this method is that DNA loss is minimized during elution or tube transfers, as the sample is maintained in the same tube. “ http://www.biomedcentral.com/1472-6750/12/20/abstract
Impact of three Illumina library construction methods on GC bias and HLA genotype calling “Compared to standard TruSeq Nano, GC bias was more prominent in transposase-based protocols, particularly Nextera XT, likely through a combination of transposase insertion bias being coupled with a high number of PCR enrichment cycles. Importantly, our findings demonstrate non-uniform read depth can have a direct and negative impact on the robustness of HLA genotyping, which has clinical implications for users when choosing a library construction strategy that aims to balance cost and throughput with data quality.” http://www.ncbi.nlm.nih.gov/pubmed/25543015
Shearing protocols differ between instruments. The current DNA shearing protocols are published on our web site. Click here and follow the link “DNA Shearing” for a protocol that is related to your specific Covaris instrument type for the desired fragment size.
What are the DNA fragment sizes I can get using Covaris instruments?
Covaris instruments can provide tunable DNA fragment sizes from 150 bp to 5kbp. Random shearing with Adaptive Focused Acoustics™ (AFA) energy generates a log normal distributed DNA fragment profile. We provide protocols for the most commonly required DNA sizes. Click here and follow the link “DNA Shearing” to see information on instrument specific DNA fragment sizes. Typically, sizes between the defined protocols may be achieved by increasing the time of the treatment of the nearest size DNA fragment (upper side).
How can I shear genomic DNA into fragment sizes larger than 5 kb?
The single use g-TUBE can be used to shear DNA into large fragment sizes ranging from 6 kb to 20 kb using a bench top centrifuge. Click here and click on the link “g-TUBE User Manual” for the current settings required to obtain your desired fragment size.
We have 300bp DNA which we want to shear to 150-200bp. Can we use the recommended protocol?
Random DNA shearing generates a log normal distribution of fragments. The smaller the size of the input DNA, an exponentially higher amount of energy is required to fragment it to the desired smaller size range. The relationship between size and energy is demonstrated in the figure below. When starting with an input sample containing large fragments of DNA, less energy is required to shear those large fragments randomly to a desired average fragment size. When starting with a smaller input fragment size, more energy is required to shear randomly and generate a distribution of fragments around a desired average fragment size.
Our recommended protocols for shearing genomic DNA as starting material require a more than tenfold larger input starting material than the desired fragment size. When the difference between the size of DNA starting material and desired fragment size is small, increasing the AFA treatment time but maintaining all other AFA parameters is recommended. For example, on the M220 instrument using a microTUBE-130 to shear gDNA to 150 bp, treatment time is 330 sec; if using 300 bp DNA as starting material for the same size, you will need to increase shearing time beyond 330 sec. Please run a time course with 10% incremental steps, and check the results on the Bioanalyzer.
Relationship between DNA fragment sizes and total energy needed.
I do not see any settings for the fragment size I am interested in, but I see settings for size ranges a bit over and a bit below what I am interested in. How can I optimize settings to get the size range of interest?
You should look at the settings that bracket the DNA fragment size of interest. Take the treatment conditions for the larger fragment size and increase the treatment time until the desired fragment size is reached. Conversely, you can take treatment conditions for the smaller size fragment and decrease the treatment time.
Why does the size distribution of fragments seem to get wider with increasing size range?
The scale on an electropherogram generated by the Agilent Bioanalyzer DNA chip is logarithmic, hence the shape of the peak. A peak at 2000bp, with the same base width in seconds as a peak at 500 bp, will cover much wider bp range because of the logarithmic scale. Below is a picture of electropherogram of the 500bp DNA fragment and the same data shown using a linear scale.
I have an Illumina protocol for creating 400bp fragments that uses T6 round bottom glass tubes. What alternatives are recommended?
Covaris has developed DNA shearing protocols using a microTUBE-500 that generates an equivalent DNA fragment size distribution as the one generated in T6 tubes. These protocols are available for all Covaris focused-ultrasonicators. Click here and follow the link “DNA Shearing” for the latest information.
Aside from the phosphodiester bonds, are there any other bonds broken during processing of my samples with the Covaris AFA?
To date there has not been a study to show the effect of acoustic fragmentation on the bonds within the DNA molecule. Indirect evidence from billions of bases sequences so far indicate to a great extent that only phosphodiester bonds are broken during AFA fragmentation of DNA, generating a population of blunt and overhangs of varying lengths which are easily repaired during the initial steps of library preparation.
In what buffer type can I shear my DNA?
We recommend Tris-EDTA pH 8.0 buffer (TE) to give consistent results for a broad range of DNA fragment sizes.
Can I use the Covaris instrument to fragment RNA? If so, what conditions should I use?
Yes, it is possible to fragment RNA using Covaris AFA technology. To fragment mRNA or total RNA into 200nt fragments click here and follow the link for “RNA Fragmentation” to find instrument specific protocols.
Can I use the published protocols to shear PCR amplicons?
Yes, the PCR amplicons can be fragmented using standard protocols as long as the size difference between starting material and desired fragment size is tenfold. When the size difference is smaller the treatment time needs to be adjusted. We recommend running a time course experiment to determine the length of treatment required. As a rule of thumb, if the size of your starting material is within the size range of the generated DNA fragments, you need to extend the treatment time (e.g. if you are trying to get DNA fragments with a mean size of 200 bp the range of fragments that are generated is between 50 and 700 bp).
Does the DNA source have an effect on the shearing conditions and results?
The Covaris AFA random fragmentation is a DNA source independent process. The protocols on the Covaris web site were developed using human, lambda and E. coli genomic DNA. Shorter lengths of starting material may require a longer treatment time to reach the desired fragments sizes.
What is the minimum concentration of input DNA for the g-TUBE?
The g-TUBE has been tested with DNA input as low as 100 ng. For lower input, we recommend to reduce centrifugation speed if necessary.
Can I use a microcentrifuge tube to shear the DNA?
No, the sample vial is a critical component of the acoustic circuit of the Covaris AFA process; the sample vessel material, thickness, geometry and the solution in the vessel all play an important role in this circuit. Following the Covaris recommended protocols and using the appropriate Covaris sample vessels will provide the optimal results. This document illustrates the dramatic effect on thermal control of using a microcentrifuge tube instead of a microTUBE.
Can I use microTUBE or miniTUBE for DNA storage?
No, microTUBE or miniTUBE should not be used for storage, and samples should be transferred after processing.
I notice a fiber inside the microTUBE/milliTUBE. What is the purpose of this fiber, and should I be worried about contamination of my samples from this fiber during treatment?
The fiber inside the tubes serves a dual purpose. The first is providing nucleation sites for inertial cavitation. Cavitation is a process where a bubble in the liquid rapidly collapses creating a microjet that fragments the DNA. The second purpose is to allow for efficient mixing of the sample during processing. The acoustic fiber is thoroughly cleaned during the manufacturing process before being inserted inside the tubes and is free from organic contaminations.
Which centrifuges are compatible with the g-TUBE?
– Eppendorf MiniSpin plus
– Eppendorf Model 5415 R with temperature set at 30° C
I am not getting consistent results when shearing DNA. What might be the reason?
Inconsistent results might be caused by several different factors; however there are few important things to check first:
Sample volume: Each consumable has been optimized for a specific sample volume in order to allow optimal performance. For example, the presence of large headspace allows for the occasional formation of an air gap in the tubes, disrupting the consistent fragmentation of DNA to the desired size range. Click here and follow the link “DNA Shearing” to see information on instrument specific recommended sample vessels for different volumes.
Water level: The water level is critical for DNA shearing. Not only does the water allow for the acoustic energy to couple from the transducer into the tube, it is also important in keeping your sample at the appropriate temperature during processing and minimizing vibrations which could lead to glass tube breakage. Exact water levels to follow are published for each instrument and consumable, click here and follow the link “DNA Shearing” for the latest information.
Water bath temperature: Water temperature should be closely controlled and matched to the application. Warmer temperatures promote less forceful collapse of acoustic cavities within the sample fluid, causing a shift toward larger mean fragment size, therefore the water bath temperature during DNA shearing should be tightly controlled.
Degas level of water: For S, E and LE- series instruments, insufficient degas levels within the bath may result in less efficient acoustic coupling and thereby shift the mean fragment size. System degas pumps should be run in advance and during AFA treatment. Prior to running a process the water bath should be degassed for at least 30 min to one hour, until the “degas” indicator in the SonoLab software can be checked without error.
Water purity: Foreign materials such as algae and particulates may scatter the high frequency focused acoustic beam, resulting in a shift to larger mean fragment size. Bath water should be pure distilled or DI water, changed daily or cleansed by a Covaris Water Conditioning System.
I am not getting the expected DNA fragment sizes. How can I check whether the instrument is working properly?
To check whether the instrument is working properly we recommend using the Covaris DNA Shearing Verification Kit (PN 520120). The user manual is available here.
When analyzing DNA fragments on the Bioanalyzer I am noticing tailing or even a split peak. What is the probable cause?
Loading too much DNA on the chip can distort the peak causing split or tailing. The split peak or shoulder on the right hand side of the peak can also be caused by an occasional formation of an air gap in a microTUBE or milliTUBE. This will result in partitioning of the liquid within the tube and thus the sample will not receive uniform acoustic treatment. As a result larger DNA fragments are observed in the upper region of the electropherogram. To avoid the air gap formation the correct sample volume for the tube type must be used and care should be taken to avoid introduction of air during pipetting.
You can also use Covaris Centrifuge microTUBE Adaptors (PN: 520059 or 500406) to remove any air bubbles introduced during pipetting of the sample into the microTUBE. This reusable adapter fits most bench top microcentrifuges.
When analyzing DNA fragments on the Bioanalyzer I am noticing a small peak near the lower marker. What is the probable cause?
The extra peak can be caused by the presence of RNA in the DNA preparation. The RNA can be sheared similar to DNA into small fragments. However the single stranded RNA runs faster than DNA fragments of the same length. If you are performing PCR prior to shearing, the smaller peak might also indicate primer dimer formation, an artifact of the PCR reaction not generated by shearing.
I followed settings provided in the DNA Shearing Quick Guide for 300bp, but my distribution is centered on 320 bp on a High Sensitivity Bioanalyzer chip. Why is this?
Mean DNA fragment size distributions published in Covaris DNA Shearing Guide are based on electropherograms generated from the Agilent Bioanalyzer with DNA 12000 Kit (cat# 5067-1509), with the exception of the 320 µl microTUBE-500 protocol (High Sensitivity DNA Kit, cat# 5067-4626). DNA fragment representation will vary with analytical systems, please carry out a time course based on settings provided in this document to reach desired fragment size distribution. Please see this document for an illustration.
When I check my sheared samples on a gel, the smear profile looks different to the size range you indicate on your protocol. Why is that?
The smear profile on a gel is dependent on the concentration of the loaded DNA sample, as well as the staining technique that is used. Overloading the gel will give an impression of a wider distribution. Also, staining the gel after the electrophoresis will avoid the gradient formation of Ethidium Bromide.