dsDNA Quantification for NGS High quality Management

insights from {industry}Andrew Jones
Market Improvement SupervisorDeNovix Inc.

On this interview, Andrew Jones discusses the significance of high quality management in next-generation sequencing and the impact {that a} poorly adopted protocol can have on experimental outcomes.

How vital is high quality management of dsDNA samples for subsequent era sequencing? How can artefacts have an effect on the standard of subsequent knowledge analyses?

Amount and high quality of enter samples are two key concerns in profitable library preparation for subsequent era sequencing. The final word success or failure of a library preparation is usually strongly linked to utilizing an precisely quantified quantity of enter DNA.

NGS is integral to research today.

Picture credit score: Zita | Shutterstock

Top quality libraries present minimal bias and dependable sequence protection. A sturdy quantification and high quality management process for pattern enter is vital to make sure constant outcomes via the workflow and maximise sequencer throughput.

Most next-gen sequencer suppliers suggest the quantification of enter pattern by analyte-specific fluorometric quantification, for instance the DeNovix dsDNA fluorescence quantification assays in addition to PicoGreen and Qubit assays. These strategies make the most of intercalating dyes particular for dsDNA and don’t bind to different nucleic acid species that could be current.

Along with quantification, additionally it is vital that samples are free from potential enzymatic inhibitors. Contaminants similar to proteins, EDTA, phenol, salts and polysaccharides can all have a unfavorable influence on subsequent enzymatic reactions and result in decreased yield, uneven or poor protection and even failure of the library preparation. To evaluate the purity of a pattern, microvolume UV-Vis spectrophotometry is beneficial.

microvolume UV-Vis spectrophotometry

Picture credit score: DeNovix

What are some great benefits of having the ability to conduct absorbance and fluorescence measurements with one system?

The flexibility to mix each absorbance and fluorescence quantification strategies supplies a lot of vital benefits for researchers. For NGS labs, it allows researchers to acquire quantitative and qualitative data on a single platform for each pre- and post-PCR pattern QC whereas utilizing minimal quantities of pattern.

Combining outcomes from fluorescence quantification strategies with purity ratio and focus measurements gained via UV-Vis evaluation allows better-informed selections on whether or not your pattern is more likely to carry out nicely in sequencing.

For instance, a fluorometric quantification will affirm if there’s a enough dsDNA template for a library preparation and have in mind whether or not the 260/230nm and 260/280nm absorbance ratios will enable evaluation of any doubtlessly inhibitive contaminants.

The distinction between focus measurements utilizing absorbance and fluorescence may additionally give a sign of the effectivity of your extraction in isolating solely the nucleic acid of curiosity because the absorbance worth may even embrace absorbance of all nucleic acids (single nucleotides, oligos, RNA, dsDNA or ssDNA).

An instrument able to performing absorbance and fluorescence additionally extends the utility of the gadget for all times science labs with out the necessity for a number of purchases.

The DS-11 FX+ for instance, has a four-LED fluorescence optical core that, along with all frequent nucleic acid and protein quantification assays, may even measure a variety of different fluorophores. The DS-11 FX+ additionally has the choice to make use of conventional cuvettes for UV-Vis, preferrred for kinetic assays.

DeNovix DS-11 | Spectrophotometer | UV-Vis Spectrophotometry

Combining the 2 measurement modes additionally extends the dynamic vary over which samples will be measured on a single instrument. The broad dynamic vary of Microvolume absorbance (0.75ng/µL to 37,500 ng/µL dsDNA on the DS-11 Collection) allows nearly any pattern to be instantly quantified utilizing simply 1µL with out dilution.

With the addition of fluorescence, the decrease detection restrict is as little as 0.5 pg/µL (utilizing the DeNovix dsDNA Extremely Excessive Sensitivity Assay). One instrument can due to this fact measure throughout all focus ranges more likely to be encountered in a genomics or proteomics lab.

How have lowered detection limits been achieved? How has this affected the sensitivity and specificity of samples that may be examined?

The DeNovix crew has a depth of experience together with life scientists, engineers, software program builders and manufacturing. All are primarily based in our headquarters in Wilmington, Delaware, USA.

To set industry-leading detection limits in microvolume UV-Vis and fluorescence on one instrument, it was crucial to mix the event of novel measurement algorithms, distinctive {hardware} and fluorescence chemistry.

Over the past 12 months, DeNovix has launched new assays which have elevated the fluorescence sensitivity of dsDNA twenty fold over the earlier {industry} commonplace and improved the decrease detection restrict of microvolume UV-Vis by 60%.

Necessary to the DeNovix method is that the crew that developed these merchandise can also be the crew that assist them.

What functions are there for dsDNA samples beneath 5pg/ul?

Genomics methods are quickly transferring in the direction of lowering the complexity of samples and uncovering the roles of uncommon variants in bigger populations. Inspecting the genome or transcriptome of single cells or small populations reveals data on mobile variations which will stay obscured by bigger cell inhabitants research.

Examples embrace tumour heterogeneity research, isolation of circulating tumour cells, ChIP-Seq, DNA from formalin-fixed paraffin-embedded (FFPE) tissues, laser-capture microdissected (LCM) samples, stem cells, and examination of unculturable microorganisms.

Please describe the optical core within the fluorescence part. What wavelength ranges will be detected and the way is that this affected by the ultrasensitive photodiodes?

The optical core of the DS-11 FX is a singular, patent-pending innovation from DeNovix. Utilizing cutting-edge design and manufacturing methods, DeNovix fluorometers include 4 LED-light sources every with an impartial filter channel and photodiode detector.

This replaces single or twin channel techniques that had been pre-existing available in the market. The tempo of improvement of recent assays and fluorophores in life science is speedy. Offering excitation in UV, Blue, Inexperienced and Purple wavelengths (see desk for particulars) maximises flexibility of the instrument and supplies a platform able to assembly present and future calls for.

Fluorometer Mode (DS-11 FX, DS-11 FX+ and FX Module)
Gentle Sources

UV LED (~375 nm), Blue LED (~470 nm),

Inexperienced LED (~525 nm), Purple LED (~635 nm)

Excitation Filters

UV: 361-389 nm, Blue: 442-497 nm,

Inexperienced: 490-558 nm, Purple: 613-662 nm

Emission Filters 435-485 nm , 514-567 nm, 565-650 nm, 665-740 nm

What are the benefits and downsides of utilizing fluorescence vs. absorbance?

Each fluorescence and absorbance have their benefits and their drawbacks when quantifying samples.

Absorbance quantification of nucleic acids is carried out by analysing the quantity of sunshine throughout a UV spectrum transmitted via a pattern. The absorbance peak for nucleic acids is 260nm and the quantity of sunshine absorbed at this wavelength can be utilized to instantly calculate the focus of the pattern utilizing the Beer-Lambert equation. The primary benefits of utilizing absorbance are:

  • It’s fast and direct. The pattern is measured instantly without having to arrange assays or measure commonplace curves.
  • Vast dynamic vary. By shortening the pathlength, more and more concentrated samples will be measured. At the moment, the higher detection restrict for microvolume absorbance is 37,500ng/µL dsDNA.
  • By assessing the ratios of 260/280nm and 260/230nm, data on the extent and the character of impurities within the pattern will be inferred.
  • Low price. No assay prices, simply pipette a pattern onto the measurement floor and measure.

A downside of utilizing absorbance strategies for quantification of nucleic acids is that the height absorbance at 260nm is frequent for all nucleic acids. As such, any contaminating ssDNA, RNA, free nucleotides or oligo in a pattern will enhance the reported focus.

Fluorescence strategies then again, usually use a dye selective for the biomolecule of curiosity. The dye fluoresces when sure to the goal molecule and won’t detect different molecules within the pattern.

The quantity of fluorescence emitted is proportional to the focus of the pattern. The selectivity of fluorescence assays is a key benefit of fluorescence enabling the researcher to make sure quantitation knowledge is restricted for his or her analyte.

Fluorescence assays are additionally usually extra delicate than absorbance. Utilizing the DeNovix DS-11 FX and dsDNA quantification assays, concentrations as little as 0.5pg/µL of dsDNA in your unique pattern will be detected. This compares to 0.75 ng/µL utilizing microvolume UV-Vis.

Limitations of fluorometric strategies embrace:

  • There’s an elevated time and value because the assay requires establishing, and pipetting errors should be prevented to make sure accuracy.
  • It’s an oblique technique and so requires measurement towards a typical curve. Requirements should be correct, handled nicely, and be acceptable to your pattern kind and focus.
  • The dynamic vary of assays is extra restricted and a few a priori data of anticipated focus is required to keep away from dilution.

Please are you able to describe how ‘pathlength’ impacts spectroscopic measurements of microvolume samples? What issues can come up with out exact calibration and pathlength management? How is that this drawback solved in DeNovix techniques?

Small quantity spectrophotometers, such because the DS-11 Collection, allow the evaluation of microliter volumes of pattern by making a liquid bridge of the pattern between an higher and decrease measurement floor.

To be able to acquire quantitative measurements, the Beer-Lambert equation is used to correlate the absorbance with the focus and the pathlength via which the sunshine travels via the pattern.

A=ε*b*c

A: absorbance worth

ε: absorptivity coefficient with items of L/mol*cm

b: pathlength of the pattern expressed by way of cm.

c: focus of the pattern in answer, expressed in mol/L.

The accuracy of outcomes is due to this fact depending on the accuracy of a calibrated distance between the 2 measurement surfaces. The DS-11 makes use of a patented opto-mechanical system with proprietary algorithms that collectively management the pathlength to inside 1 micron.

The DS-11 makes use of real-time absorbance knowledge to find out the optimum pathlength for every pattern, mechanically adjusting the space between the measurement surfaces to ship optimum outcomes.

Increased focus samples require more and more smaller pathlengths and the high-quality management that DeNovix has over the optical path allows us to precisely set extraordinarily small pathlengths reliably and reproducibly with out requiring calibration.

Different microvolume techniques contain calibration of the pathlength via periodic verification and recalibration by a service engineer. The potential for drift away from a exact pathlength might trigger a scientific error over time in all samples measured. Throughout use, the DS-11 routinely screens pathlength accuracy and mechanically performs any crucial changes.

What issues are there with samples not bridging appropriately? How does this have an effect on the outcomes? How does the DeNovix Bridge Testing ® take away the potential for column breakage?

Microvolume spectrophotometers rely to a larger or lesser extent on the floor pressure of a pattern to bridge the air hole between two measurement surfaces.

Inaccurate outcomes will likely be obtained if the pattern fails to bridge the hole as the sunshine fails to journey uniformly via the pattern. Widespread causes of unbridged measurements are failing to pipette sufficient pattern, floor unconditioning or low floor pressure buffers.

The DS-11 makes use of a pathlength of 0.5 mm and compresses a pattern throughout the measurement cycle. That is an inherent benefit over techniques utilizing longer, 1.0mm pathlengths that must stretch the pattern and threat column breakage.

The DS-11 software program contains an algorithm to check for an unbridged situation and mechanically compensates to shorten the pathlength to attach with the pattern, with no person enter required.

DS 11 Collection | Spectrophotometer | Fluorometer

What does the longer term maintain for DeNovix? Will future fashions be capable to detect decrease pattern concentrations?

DeNovix has all the time labored alongside the first analysis neighborhood to know key workflow necessities and the constraints of present strategies. We’re regularly creating the performance and efficiency of present devices to set new requirements in detection and allow new functions.

We even have a improvement pipeline that makes use of our experience in engineering, optics and life science to resolve present issues in key laboratory workflows.

The place can readers discover extra data?

About Andrew Jones

Andrew Jones - headshotAndrew is at the moment the Market Improvement Supervisor with DeNovix Inc. having initially joined the Technical Advisory board in 2012.

DeNovix Inc. is a personal firm primarily based in Wilmington, Delaware that develops, manufactures and sells revolutionary merchandise for the life science neighborhood primarily based on excessive expertise optics and detection techniques.

Previous to DeNovix Inc., Andrew was European Medical and Translation Medication Supervisor with Affymetrix and beforehand held gross sales and administration roles at Roche Diagnostics within the microarray and subsequent era sequencing markets.

A graduate of the College of Wales, Andrew is a Grasp of Analysis in Life Science and relies within the South of England.

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