القص الصوتي المركز: المسار الصوتي أهم من رقم القوة

A focused acoustic shearing protocol is a simple idea in principle.

Place sample in a tube. Select the method. Apply acoustic energy. Look at the distribution of fragments.

However, a lot has already taken place between the transducer and the end DNA or chromatin fragments. Acoustic energy has been transmitted through a coupling medium, into the sample vessel, interacted with the liquid and resulted in an endpoint of fragmentation.

All of the way is important.

Thus, laboratories testing the focused acoustic shearing should consider more than just a power setting. The workflow of Longlight describes controlled mechanical fragmentation as one of the essential steps of NGS sample preparation, in which nucleic-acid fragments are processed by focused ultrasonication to obtain more uniform nucleic-acid fragments for downstream workflow processes.

Shearing starts before the sound reaches the DNA:

There is no “power number” for the sample.

It is a physical system that receives acoustic energy after it has passed through the system.

With focused ultrasonication, the sample is contained within a closed vessel and the acoustic energy is directed through a controlled medium and focused towards the sample region. Longlight says that its BoFU systems are unlike the traditional bath and probe sonicators because the acoustic energy is directed at the sample container instead of the probe.

That takes the meaning of a protocol as a whole to a new level.

Transducer, acoustic medium, vessel geometry, sample position and liquid volume are not accessories that are independent around the experiment. These are combined together and make the acoustic path.

In this case, the sample tube is a component of the acoustic system

Sample tubes can be easily treated as generic consumables.

In the case of focused acoustic shearing, that assumption is one that is worth re-evaluating.

The geometry plays a role in the introduction of the acoustic energy into the vessel. Transmission and heat transfer is influenced by the material. The acoustic focal zone and the tube can also have an impact on the uniformity of sample exposure to the desired conditions.

Longlight's BoFU-UniTube is specifically developed for BoFU focused-ultrasonication systems and not as a generic assay tube. The acoustic coupling, controlled energy transmission and thermal management are highlighted in its product description as part of the tube design.

This results in an easy method development guideline:

When a fragmentation protocol has been validated, never take for granted that the acoustic protocol hasn't changed.

Cavitation should not be the operator's responsibility

Probe sonication allows for physical insertion of the sonicator into the sample.

There is another way of focused acoustic processing. The sample remains tightly closed during the ultrasound transmission in the acoustic medium.

That has a implications for two reasons.

First of all, it eliminates the probe depth and direct contact of the probe in the routine sample handling procedure. Secondly, it reduces the dependence of geometry of the delivery on the manual positioning of a probe from one run to another by an operator.

The non-contact processing technology in Longlight's BoFU-100 الموجات فوق الصوتية المركزة works by transmitting focused acoustic energy through an acoustic medium, but the biological sample is kept away from the probe.

This doesn't mean that protocol optimization goes away.

It signifies that the variables are shifted from the hand positioning towards any more well-defined acoustic system.

A Fragment Endpoint is preferable to an Energy Endpoint

A typical error in the method development is the following thought:

The same acoustic settings were used, hence the experiment was the same,” they say.

This may not be the most productive destination.

The focus of the researcher is usually on the outcome of the shearing, for example: fragment-size distribution, sample recovery, compatibility with library preparation, or chromatin suitability or another downstream criterion.

Therefore, protocol optimization should be done the other way around.

Decide on the desired end product of the biology or analysis first. Then determine the acoustic conditions that repeatedly produce it.

In the case of genomic DNA, it could be a fragment distribution appropriate for a particular NGS library preparation process. The acceptable endpoint for chromatin may be different as the downstream experiment may be asking a different biological question.

Both can be powered by the same instrument, for acoustic energy. That doesn't mean they are the same shearing method, however.

The acoustic experiment can be easily modified by changing the temperature, which can produce quiet changes.

Mechanical energy cannot be put into a sample without repercussions.

One of them is heat.

A protocol that rams up the temperature of the sample during the protocol cannot be run in exactly the same environment throughout the protocol. This is more important in the case of temperature-sensitive biological materials, such as nucleic acids, and chromatin.

ال BoFU-800 Focused Ultrasonicator according to Longlight is a non-contact, isothermal processing environment with temperature control that is designed to minimize the effects of heat generated during the ultrasonication process. The system can also accommodate 1–8 samples and have individually customized or batch conditions.

Thus, the temperature should be put in the method record along with the acoustic conditions and sample information.

It's not only a comfort functionality of the instrument.

Both DNA Shearing and Chromatin Shearing should not make assumptions!

There are several biological jobs for the "Acoustic shearing".

DNA fragmentation of genomic DNA can be optimized to achieve a desired distribution of nucleic-acid fragments to be sequenced. Biological material surrounding DNA as well as downstream chromatin workflow needs should also be taken into consideration when shearing chromatin.

Similarly, FFPE processing, tissue disruption and cell lysis create totally different sample structures.

This is one of the reasons why a broad acoustic platform can be beneficial, but it is also a reason why the laboratories must not work towards one universal sonication protocol.

Longlight's BoFU-1600 Focused Ultrasonicator includes a number of applications including genome fragmentation, genomics/proteomics preparation, cell and tissue disruption, and FFPE processing. It can also process samples using individual conditions or common batch parameters as it has a 16-position design.

Acoustic technology can be shared with various applications.

The acoustic settings should not be the same by default.

In a method transfer more than three numbers should be involved

In many cases, when a focused acoustic shearing method is transferred between laboratories, it is the obvious settings that are used.

That is just one portion of the protocol.

Sample type, amount or concentration of sample input, working volume, sample vessel, acoustic conditions, temperature conditions, processing mode and QC method are more useful transfer records.

This is particularly critical when operators or laboratories switch methods.

A method that is not able to be used without the instrument developers standing by the instrument is not a very portable method.

Shear check is performed at the end of the focused acoustic

Follow the complete energy pathway prior to calling an acoustic shearing protocol validated.

Are the proper containers being utilized? Are the positioning and coupling of the sample consistent? Are Input conditions specified? Is temperature controlled? Are reward/penalty criteria based on the fragment end point or just the instrument setting?

The most effective approach to focused acoustic shearing is to consider the acoustic energy as an element in a controlled sample-processing system.

It's not just a matter of how much energy the instrument produces.

It is the consistency with which the appropriate amount of that energy gets to the appropriate sample under the appropriate conditions.

الأسئلة المتداولة

Q1. What is the focused acoustic shearing?

Focused Acoustic Shearing is a method of mechanically shearing biological material, e.g., DNA, chromatin, using concentrated ultrasonic energy. Focussed systems can be used to deliver acoustic energy into a sealed sample vessel without the need for a probe to touch the sample.

Q2. Is there a need for focused acoustic shearing for NGS?

Yes. One of the applications listed by Longlight for its focused ultrasonication systems and its DNA/RNA Shearing workflow is controlled DNA fragmentation.

Q3. Is focused acoustic shearing the same as probe sonication?

There are two types of probe sonication: No. Probe Sonication: a sonication probe is placed inside the sample; Focused Acoustic Systems: they can sonicate a closed sample vessel through an acoustic medium.

Q4. Are there any effects of acoustic shearing on sample tubes?

It can. For a focused acoustic system, vessel geometry, acoustic coupling and thermal behaviour are part of the energy-transfer path and hence, purpose-designed acoustic tubes can be used.

Q5. Is it possible to use the same focused acoustic system for DNA and chromatin?

While a focused-ultrasonication platform can be used for both applications, it's important not to assume that the same protocols will work for both. Optimization of method should be based on the endpoint and sample structure.