brilliantdye

Welcome to the world of BrilliantDye®

Brand introduction

BrilliantDye: The premium quality drop-in alternative for BigDye®

Format

The BrilliantDye kits are delivered as a 2.5x concentrated ready-reaction premix, fully optimized for a highly flexible chemistry, designed for all kinds of sequencing applications, including de novo sequencing and resequencing. The kits generate data with uniform peak heights and optimized signal balance to produce long, high-quality reads.

Compatibility
The kits are compatible with ABI genetic analyzers of the 310, 3100, 3130, 3500, 3730 and SeqStudio series as well as the Promega Spectrum system. No need for changing protocols, workflows, settings and calibrations.

BrilliantDye Terminator cycle sequencing kit (two versions available)

The BrilliantDye Terminator v1.1

The BrilliantDye Terminator v1.1 Cycle sequencing kit is designed for specialty applications that require optimal basecalling adjacent to the primer.

The BrilliantDye Terminator v3.1

The BrilliantDye Terminator v3.1. Cycle sequencing kit is the method of choice for longest reads.

The dGTP BrilliantDye kit

For sequencing through G- and GT-rich templates, NimaGen offers the dGTP BrilliantDye kits, optimized for challenging sequence templates, by replacing the dITP with dGTP.

Products

BrilliantDye products

dGTP BrilliantDye™ Terminator (v3.1) Cycle Sequencing kit (1000 rxn)

dGTP BrilliantDye™ Terminator (v3.1) Cycle Sequencing kit (100 rxn)

BrilliantDye™ Terminator 5X Sequencing Buffer (1 mL)

BrilliantDye™ Terminator 5X Sequencing Buffer (28 mL)

BrilliantDye™ Terminator (v1.1) Cycle Sequencing kit (24 rxn)

BrilliantDye™ Terminator (v1.1) Cycle Sequencing kit (1000 rxn)

BrilliantDye™ Terminator (v1.1) Cycle Sequencing kit (5000 rxn)

BrilliantDye™ Terminator (v1.1) Cycle Sequencing kit (25000 rxn)

BrilliantDye™ Terminator (v1.1) Cycle Sequencing kit (100 rxn)

BrilliantDye™ Terminator (v3.1) Cycle Sequencing kit (24 rxn)

BrilliantDye™ Terminator (v3.1) Cycle Sequencing kit (100 rxn)

BrilliantDye™ Terminator (v3.1) Cycle Sequencing kit (1000 rxn)

BrilliantDye™ Terminator (v3.1) Cycle Sequencing kit (5000 rxn)

BrilliantDye™ Terminator (v3.1) Cycle Sequencing kit (25000 rxn)

dGTP BrilliantDye™ Terminator (v1.1) Cycle Sequencing kit (100 rxn)

dGTP BrilliantDye™ Terminator (v1.1) Cycle Sequencing kit (1000 rxn)

May 12, 2021May 12, 2021

ZYMO RESEARCH RELEASES OPEN-SOURCE BIOINFORMATICS PIPELIN

ZYMO RESEARCH RELEASES OPEN-SOURCE BIOINFORMATICS PIPELINE FOR SARS-COV-2 VARIANT DETECTION IN WASTEWATER

EARLY RELEASE OF THE VIRSIEVE™ BIOINFORMATICS PIPELINE WILL PROMOTE COLLABORATION IN ENVIRONMENTAL SARS-COV-2 RESEARCH

Zymo Research releases the VirSieve™ Open-Source Bioinformatics Pipeline source code to the environmental microbiology community to promote global collaboration and support for the detection of SARS-CoV-2 variants in wastewater.

Zymo Research has released the VirSieve™ Bioinformatics Pipeline source code to the environmental microbiology community to promote global collaboration and support for the detection of SARS-CoV-2 variants in wastewater. VirSieve™ is an automated computational pipeline that analyzes sequencing reads from the SARS-CoV-2 virus in wastewater samples to better understand viral variants present in monitored communities. By rating the confidence interval in observed viral mutations, the software has the potential to significantly increase the accuracy of analyzing any changes in the viral genetic material.

According to the Centers for Disease Control and Prevention, while the deployment of vaccines has had a tremendous positive impact on curbing the SARS-CoV-2 pandemic within the USA, new viral strains are still emerging. Some of these strains, e.g., B.1.1.7, are classified as “variants of concern” due to evidence of increased spread, pathogenicity, or vaccine evasion. Wastewater Next-Gen Sequencing is emerging as the most viable solution for near real-time community surveillance.

Viral sequencing from wastewater is difficult due to fragmentation and degradation of the viral RNA, often resulting in sequencing errors that ultimately manifest as false mutations. VirSieve™ can identify these false variants and mark them as being low or no confidence, allowing researchers to filter mutations with a higher degree of support. Zymo Research also provides DNA/RNA Shield™, a reagent that safeguards viral RNA from further degradation after inactivation of any virus for safe, ambient temperature collection and transportation of wastewater samples.

“Zymo Research has previously developed public software tools like the Zymo Research Transmit Program, an open-source client for submitting COVID-19 test results through the CalREDIE public health reporting system,” said Dr. Michael Weinstein, Director of Laboratory Information Systems, and Project Lead on the VirSieve™ Pipeline project at Zymo Research. “VirSieve™ is part of Zymo Research’s ongoing effort to provide maximum support to pandemic response efforts around the globe. We feel VirSieve™ has the potential to be used not only for tracking variant strains of the SARS-CoV-2 virus in wastewater, but other viruses as well, for future public health efforts.”

“Getting accurate variants from clinical samples or sewage is key to proper placement of a viral strain in the context of all other viral variants and informing pathogen tracking efforts,” said Dr. Christopher Mason, who is a paid speaker for Zymo Research, is the co-director of the WorldQuant Institute for Quantitative Prediction and a professor of physiology and biophysics at Weill Cornell Medicine.

For more information about the VirSieve™ Pipeline visit Zymo Research’s website or contact us via email at customerservice@biolinkk.com.

IRVINE, Calif., (May. 11, 2021)

For more information about Zymo Research’s COVID-19 products, check out the following links:

April 27, 2021April 28, 2021

Primary Antibodies

Primary antibodies directly bind to a particular antigen of interest. The variable region of the primary antibody recognizes an epitope on the antigen and allows for the detection, quantification, or purification of the antigen.

While many primary antibodies are not directly conjugated to a fluorochrome or enzyme and a secondary antibody antibody is required for detection, SouthernBiotech offers a wide range of conjugated primary antibodies allowing for the direct detection of the target antigen without the need for a secondary antibody.

April 22, 2021June 22, 2021

Secondary Antibodies

Explore by Target

Secondary Antibodies

Secondary antibodies bind to primary antibodies and are commonly used to detect and visualise a primary antibody, which is bound to its protein of interest, in applications like western blotting or immunocytochemistry. Multiple secondary antibodies can bind to the same primary antibody, increasing the sensitivity and amplifying the signal. Secondary antibodies are commonly conjugated with reporter molecules, either enzymes like HRP or fluorophores like APC, to enable the fluorescent, colorimetric, or chemiluminescent detection of primary antibodies.

Secondary antibodies are raised against the host species and isotype of the primary antibody. For example, if you used a polyclonal primary antibody raised in goat, you will require an Anti-Goat IgG (Heavy & Light chains) secondary antibody raised in a different species.

Explore our comprehensive portfolio of conjugated and unconjugated secondary antibodies for the fluorescent, colorimetric, and chemiluminescent detection of primary antibodies in a diverse range of applications, including: flow cytometry, immunohistochemistry, immunofluroescence, immunocytochemistry, and western blotting.

Anti-African G. Monkey

Anti-Bovine

Anti-Canine

Anti-Chicken

Anti-Chimpanzee

Anti-Cynomolgus

Anti-Human

Anti-Equine

Anti-Feline

Technical Information

Non-Fluorescent Conjugates

Enzymes, like Horseradish Peroxidase (HRP) or Alkaline Phosphatase (AP), can conjugate to an antibody via covalent linkages. Enzyme conjugated antibodies are used in conjunction with a substrate, either fluorogenic, colorimetric, or chemiluminescent, to create a detectable signal. The non-detectable, soluble substrate is converted by the enzyme into a detectable (and normally insoluble) form. Enzyme conjugated antibodies enable signal amplification; as the enzyme is able to convert many molecules of the substrate, the length of time the reaction is allowed to continue determines the strength of the signal.

Horseradish Peroxidase

Horseradish peroxidase (HRP) is an enzyme derived from the root of Armoracia rusticana (a.k.a. the horseradish plant) that is commonly used as a reporter protein. HRP conjugated secondary antibodies can either be used for colorimetric detection, where the HRP catalyses the conversion of a chromogenic substrate to a coloured precipitate, or chemiluminescent detection, where the HRP produces a signal by oxidising a chemiluminescent substrate to a form which emits light.

Multiple HRP molecules can bind to a single secondary antibody, as such, HRP conjugated secondary antibodies can be utilised for signal enhancement and enable detection of proteins expressed at low levels. HRP also has a high turnover rate which enables HRP conjugated secondary antibodies to generate a strong signal in a short amount of time (often within five minutes).

Alkaline Phosphatase

Alkaline phosphatase (AP) conjugated secondary antibodies are commonly used for ELISA and western blotting. They are also sometimes used for immunohistochemistry, however, their large size may limit penetration into tissues. It is worth noting, that whilst HRP generates a maximum signal quickly, often within five minutes, the signal from AP gradually increases and peaks at around an hour. AP generates a very stable signal which can last several days; this is particularly useful if multiple exposures are needed, especially over a matter of days.

Trying to decide between HRP and AP for your blot?

Use our table below to help with the decision.

AP	HRP
Sensitive	Sensitive
Long lasting signal (24-48 hours)	Quick max signal (~5 minutes)
Useful for multiple exposures	Inhibited by azides
Inhibited by phosphate buffers	Economical

Biotin

Biotin is a small, water-soluble B vitamin that can be bound by avidin, NeutrAvidin, and streptavidin with high affinity. Due to its small size, binding with biotin does not typically affect a protein’s biological activity. Multiple biotin molecules can conjugate to a single secondary antibody, as such, biotin conjugated secondary antibodies can be utilised for signal enhancement and enable detection of proteins expressed at low levels. Visualisation happens through biotin / streptavidin interaction whereby the streptavidin is bound to either HRP or a fluorescent probe. By utilising a biotin conjugated secondary antibody, the same secondary antibody can be used in multiple applications simply by switching the streptavidin used.

Fluorescent Conjugates

Fluorescent-dye conjugated secondary antibodies allow for a brighter signal and provide the ability to distinguish between multiple proteins in a single sample, as such, they are a valuable tool for identifying proteins in immunocytochemistry, western blotting, immunofluorescence, immunohistochemistry, and many more applications. Common fluorescent dyes include the Alexa Fluor range, AMCA, Cy3, FITC, PE, and TRITC.

Examples of fluorescent detection:

Figure 1: Anti-GFAP Antibody (green) strongly labels astroglial cells, shown by IF in a section of mouse hippocampus.

Figure 2: Anti-FOX3 Antibody (A85403) (red) strongly stains the nuclei of neurons in the cerebellar granule layer, shown by IF in a section of mouse cerebellum.

Antibody Classes, Subclasses and Fragments

An antibody is a “Y-shaped” glycoprotein that is capable of binding to specific antigens. Each antibody is composed of four polypeptide chains, two identical heavy chains and two identical light chains, which vary in sequence and length between species and between isotype classes. An antibody’s structure can be broken down into: two F(ab) regions, the top sections of the “Y” which contain the variable region which binds specifically to a particular epitope on the antigen; a hinge region; and an Fc region, the bottom of the “Y” which provides a binding site for endogenous Fc receptors (and secondary antibodies).

In mammals, antibodies are classified into five main classes or isotypes according to the heavy chain they contain. These are: IgA (alpha), IgD (delta), IgE (epsilon), IgG (gamma), and IgM (mu). Each class differs in the sequence of constant domains, the number of constant domains, the hinge structure, and the valency of the antibody. The light chains of an antibody are classified as either kappa or lambda based on their polypeptide sequence. Typically, the two light chains in an individual antibody are the same type.

Whole antibodies can be digested by papain or pepsin to form F(ab), F(ab’) and F(ab’)₂ fragment antibodies, which have no Fc portion or a significantly reduced Fc portion. The structures of F(ab), F(ab’) and F(ab’)₂ fragments differ: F(ab) fragments are single antigen-binding F(ab) portions, F(ab’) fragments are single F(ab) portions with the hinge region present, and F(ab’)₂ fragments are two F(ab) portions linked via the hinge region. F(ab), F(ab’) and F(ab’)₂ fragment secondary antibodies are used to prevent the non-specific binding between the Fc region of an antibody and the Fc receptor on a cell.

Antibody Classes and Subclasses

Immunoglobulin Classes	IgA	IgD	IgE	IgG	IgM
IgG Subclasses – Goat	IgG1	IgG2
IgG Subclasses – Human	IgG1	IgG2	IgG3	IgG4	IgA1	IgA2
IgG Subclasses – Mouse	IgG1	IgG2a	IgG2b	IgG3
IgG Subclasses – Rat	IgG1	IgG2a	IgG2b	IgG2c
Light Chains	Kappa	Lambda
Heavy Chains	Alpha	Delta	Epsilon	Gamma	Mu

April 2, 2021April 2, 2021

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April 2, 2021April 2, 2021

Total-Rna-Seq-Library-Prep

Any Organism. One rRNA Depletion Solution.

Zymo-Seq RiboFree Total RNA Library Kit

Compatible with
any Organism

Suitable for
FFPE RNA

Boost High-Value
NGS Reads

Zymo-Seq RiboFree Total RNA Library Kit

Comprehensive Gene Detection for Every Transcriptome & Beyond

The Zymo-Seq RiboFree Kit Detects an Exceptional Number of Genes. Total RNA extracted from various species were used as inputs for library preparation using a single standard RiboFree protocol. The libraries were sequenced paired-end at a 100-bp read length. STAR-aligned reads were quantified at the gene level using StringTie.

Comprehensive Gene Detection for Every Transcriptome & Beyond

>90% rRNA Depletion Across Organisms

The Zymo-Seq RiboFree Kit Produces Dense Coverage of Protein Coding Genes. Classification of the STAR-aligned reads was based on Ensembl annotations and RepeatMasker rRNA tracks from UCSC genome browser when applicable.

Unparalleled Multi-Species Compatibility

RiboFree Universal Depletion Provides Cross-Species Compatibility. The Zymo-Seq RiboFree Total RNA Library Kit is unmatched in cross-species compatibility thanks to an innovative probe-free depletion strategy. Leveraging the robust depletion kinetics, scientists are exploring diverse applications of RiboFree using samples from vertebrates, plants and mo

Suitable for Degraded Samples*

The Zymo-Seq RiboFree Kit Effectively Depletes rRNA from Formalin-Fixed, Paraffin-Embedded (FFPE) Human Tissue RNA. Total RNA (500 ng) from FFPE tissues was used as input per sample with only one modification to the standard protocol: column cleanup using RNA Clean & Concentrator was adopted after depletion. Median values (n=20) are shown in the figure.

The Fastest RNA-Seq Library Workflow

Sample to Sequencer in a Single Day

The Zymo-Seq RiboFree Kit has the Fastest Workflow from Total RNA to rRNA-depleted, NGS Libraries. The Zymo-Seq RiboFree Total RNA Library Kit offers a ≥ 30% reduction in workflow completion time compared to other available RNA library prep kits. Workflow time considers the hours required to complete both rRNA depletion and library preparation.

Featured Citations

1. Omelchenko, D.O., et al., Assembly and Analysis of the Complete Mitochondrial Genome of Capsella bursa-pastoris. Plants (Basel), 2020. 9(4).

2. Licastro, D., et al., Isolation and Full-Length Genome Characterization of SARS-CoV-2 from COVID-19 Cases in Northern Italy. J Virol, 2020. 94(11).

3. Shtratnikova, V.Y., et al., Genome-Wide Transcriptome Profiling Provides Insight on Cholesterol and Lithocholate Degradation Mechanisms in Nocardioides simplex VKM Ac-2033D. Genes (Basel), 2020. 11(10).

4. Brussel, K.V., et al., Identification of Novel Astroviruses in the Gastrointestinal Tract of Domestic Cats. Viruses, 2020. 12(11).

5. Phylogenetic Characterization of Crimean-Congo Hemorrhagic Fever Virus Detected in African Blue Ticks Feeding on Cattle in a Ugandan Abattoir. Microorganisms, 2021. 9(2).

6. The FUR-like regulators PerRA and PerRB control a complex signaling network required for mammalian host-adaptation and virulence of Leptospira interrogans. BioRxiv, 2020.

7. Niche partitioning facilitates coexistence of closely related gut bacteria. BioRxiv, 2021.

February 18, 2021February 18, 2021

What is a Lateral Flow Immunoassay?

What is a lateral flow immunoassay?

The following article describes what a lateral flow immunoassay is used for. In addition, the article explains the different component parts used during manufacturing in order to make them work.

Common names for a lateral flow immunoassay

Different industry sectors and different countries use varying terminology to describe a lateral flow immunoassay. Common names include:

˃ Lateral flow test (LFT)
˃ Lateral flow device (LFD)
˃ Lateral flow assay (LFA)
˃ Lateral flow immunoassay (LFIA)
˃ Lateral flow immunochromatographic assays
˃ Dipstick
˃ Express test
˃ Pen-side test
˃ Quick test
˃ Rapid test
˃ Test strip

What is a lateral flow immunoassay?

So, what is a lateral flow immunoassay? Basically, it is a simple to use diagnostic device used to confirm the presence or absence of a target analyte, such as pathogens or biomarkers in humans or animals, or contaminants in water supplies, foodstuffs, or animal feeds. The most commonly known type of lateral flow rapid test strip is the pregnancy test.

LFDs typically contain a control line to confirm the test is working properly, along with one or more target or test lines. They are designed to incorporate intuitive user protocols and require minimal training to operate. They can be qualitative and read visually, or provide data when combined with reader technology, such as AppDx^®.

Lateral flow tests are widely used in human health for point of care testing. They can be performed by a healthcare professional or by the patient, and in a range of settings including the laboratory, clinic or home. In the medical diagnostic industry, there are strict regulatory requirements which must be adhered to for all products developed and manufactured and Abingdon Health can guide you through this process.

Due to their versatile nature, lateral flow rapid tests are used across a number of other industry sectors including pharma, environmental testing, animal health, food and feed testing, and plant and crop health.

Types of lateral flow tests

Lateral flow assays can be developed to be used in a dipstick format or in a housed cassette. Both dipsticks and housed tests will work in a similar way, it is just dependent on the industry, sample matrix, and the market requirement, as to which format is suitable.

Sandwich assays – A positive test is represented by the presence of a coloured line at the test line position.

Competitive assays – A positive test is represented by the absence of a coloured line at the test line position.

Sample matrice

The target analyte and the market requirements will determine the type of sample that will be used in the assay.

Some samples require running buffer to aid sample delivery e.g. animal feed. Other samples such as blood, serum, urine, or saliva may be able to be placed directly onto a test, while there are occasions where a dilution buffer is required.

For example, the Abingdon Health product Seralite^® – FLC, a rapid test for the quantitative measurement of kappa and lambda immunoglobulin free light chains (FLCs), uses serum and requires a buffer for sample dilution. FLCs are used as an aid in the diagnosis and monitoring of a type of blood cancer, Multiple Myeloma.

A rapid plant disease tests, however, require the mixing of plant material samples, such as leaves and wood, with a running buffer in order to detect commercially damaging plant disease.

LFDs are versatile enough to be developed to detect target analytes in sample matrices including:

Milk	Whole blood	Serum
Saliva	Urine	Tissue samples
Food	Drink	Animal feed
Plant material	Water	Fuel

Label types

Typically, lateral flow assays utilise conjugated gold, carbon, or coloured latex nanoparticles within the conjugate pad. Other labels include magnetic beads or coloured polystyrene beads.

Regardless of the label types, they all perform the same function to create a three-way bond with antibodies and targets in order to make visible the control and test lines.

Labels will be chosen during lateral flow assay development depending on several factors such as the target, sample matrix and antibody. The optimisation of the assay will ensure the label interacts correctly with the antibody and antigen to ensure efficiency and accuracy of results.

Multiplexed lateral flow assays

Both sandwich and competitive assays can be developed to include one or more test lines. For example, our nucleic acid lateral flow immunoassay, PCRD, is a multiplexed sandwich assay containing two test lines and one control line.

In addition, by using complementary reader technology, sandwich and competitive multiplexed assays can produce quantitative results.

A multiplexed assay may be used for:

˃ Detecting multiple targets in a single test rather than using many individual tests. In situations where only a small sample volume is available a multiplex assay allows you to maximise its use;
˃ To assist diagnosis where the presence of a number of markers together is required;
˃ Confirming the presence of multiple contaminants during high volume food and feed testing;
˃ Offering cost-saving benefits to end-users in a laboratory or in-the-field by testing for different targets simultaneously; and
˃ Remote or agricultural areas where resources are limited and where multiplexed testing will save time.

Quantitative rapid lateral flow devices

Using names such as rapid test or quick test can lead to myths about lateral flow devices that they are limited in their capability. However, lateral flow devices are compact, easy-to-use, and offer considerable flexibility.

Early versions of LFDs were predominantly qualitative assays. However, improvements in reagents, component materials, and reader technologies along with manufacturing processes mean quantitative results are achievable.

In addition, the developments in reader technology and advancements in raw materials, such as labels, means a lateral flow rapid test can match the sensitivity of an ELISA assay.

How does a lateral flow test work?

LFDs use immunoassay technology using nitrocellulose membrane, coloured nanoparticles (or labels), and typically antibodies, to produce results.

When a sample is added, the sample will flow along the test device passing through the conjugate pad into the nitrocellulose membrane and then onto the absorbent pad.

The bullet points below demonstrate how a sandwich assay works. Alternatively, click the play button on this image to watch a video.

˃ The sample pad acts as the first stage of the absorption process, and in some cases contains a filter, to ensure the accurate and controlled flow of the sample.
˃ The conjugate pad, which stores the conjugated labels and antibodies, will receive the sample. If the target is present, the immobilised conjugated antibodies and labels will bind to the target and continue to migrate along the test.
˃ As the sample moves along the device the binding reagents situated on the nitrocellulose membrane will bind to the target at the test line. A coloured line will form and the density of the line will vary depending on the quantity of the target present. Some targets may require quantification to determine target concentration. This is where a rapid test can be combined with a reader to provide quantitative results.
˃ The sample will pass through the nitrocellulose membrane into the absorbent pad. The absorbent pad will absorb the excess sample. The specification of the absorbent pad will have an impact on the volume of sample the test can incorporate.

February 17, 2021February 17, 2021

Intro Solisbiodyne

PCR, qPCR and RT-qPCR reagents

Solis BioDyne has been developing and producing life science reagents since 1995, having become one of the leading reagent suppliers in Europe today. High standards for production and service have made Solis BioDyne a trusted trademark worldwide. Our DNA polymerases, PCR Master Mixes, qPCR Mixes and reverse transcription reagents are used by a quickly growing number of customers across the globe, including top research institutes and biotech-companies. Solis BioDyne has partners in both private and state sectors, with cooperation projects ranging from OEM production to scientific research.

Product Categories

Endpoint PCR

qPCR and RT-qPCR

cDNA Synthesis

Additional Enzymes and Reagents

February 17, 2021February 17, 2021

Bisulfite Conversion for Illumina Methylation Arrays

Troubleshooting & Best Practice Guidelines

Methylation arrays are common platforms for analyzing 5-methylcytosine. The Infinium™ MethylationEPIC BeadChip and Infinium™ HumanMethylation450 BeadChip (commonly referred to as the EPIC and 450K arrays respectively) as well as the recently launched Infinium™ Mouse Methylation BeadChip from Illumina^® all utilize Zymo Research’s bisulfite conversion technologies to distinguish 5mC from unmodified cytosines. In addition to the test probe sets, the arrays include bisulfite conversion quality control probes. In some instances, the analysis software will flag samples for low bisulfite conversion efficiency. Possible causes for these warnings are low bisulfite conversion efficiency, low DNA input/quality, and chip failure.

Validated Protocols

The only approved and validated bisulfite conversion kit for the Infinium™ Methylation arrays is the EZ DNA Methylation kit (catalog numbers: D5001, D5002, D5004). It is critical to follow the Illumina^® recommended incubation protocol (16 cycles of 95°C for 30 seconds, 50°C for 60 minutes). Additional details can be found in the appendix of the respective bisulfite conversion protocols. While other bisulfite conversion kits will result in high-quality bisulfite converted DNA, only samples processed using the validated kits are supported by Illumina^®.

Along with using the validated bisulfite conversion kits, it is important to follow the rest of the BeadChip protocol as written, including the downstream software analysis. Illumina^® periodically reviews the performance of the analysis software and probe performance. Use the most up-to-date software and manifest files to ensure that downstream analysis is correct.

DNA Input & Quality

The amount and quality of genomic DNA used in these assays is critical to success. For best results, DNA should be as intact and high quality as possible. The arrays rely on a signal to noise ratio to determine the conversion efficiency. When low input or low quality DNA is used, the signal to noise ratio is much lower and can affect the reliability of the bisulfite conversion control signals.

The required minimum amount of DNA is 250 ng for the manual protocol and 1000 ng for the automated protocol. Genomic DNA should be quantified using a dsDNA specific method such as Picogreen^® or Qubit^®. NanoDrop^® or other spectrophotometric methods are not recommended for quantification due to issues with successfully distinguishing DNA from RNA. RNase treatment can also help ensure that DNA quantification is accurate.

When working with DNA isolated from FFPE samples or other degraded DNA samples, 500 ng DNA inputs or higher are highly recommended. Additionally, single-column bisulfite conversion (vs. the 96-well plate) is recommended for degraded samples due to the ability to use smaller elution volumes. Bisulfite-converted FFPE DNA should then be treated with the Illumina Infinium™ FFPE DNA Restoration Kit (as described in the manual) before processing the array. The entire sample should be used.

Low Bisulfite Conversion Efficiency

Bisulfite conversion can be impacted by a variety of factors. The quality of the CT Conversion Reagent is critical for successful conversion. It is recommended to prepare the reagent fresh before each conversion if possible. Otherwise, the prepared reagent should be stored according to the guidelines listed in the protocol. The conversion reagent should not be exposed to light or oxygen any more than necessary. If processing 96-well plates, a multichannel pipette should be used and the prepared conversion reagent should be added last to the plate.

Conversion should be performed in a thermal cycler with a heated lid. Samples and conversion reagent should be mixed thoroughly (no observed mixing lines) and fully spun down before placing tubes in the thermal cycler. If the tubes are not fully spun down or the lid is not heated properly, precipitation may form around the lid of the PCR tube. After the incubation, any precipitation that is observed should be avoided when transferring the sample as it could contain unconverted DNA.

The desulphonation incubation should be stopped at 15 minutes (20 minutes is the absolute maximum). Leaving the desulphonation buffer on the column longer than the recommended time can result in additional degradation to the sample.

A bisulfite conversion quality control check is highly recommended. Bisulfite conversion can be assessed using a variety of methods. Bisulfite converted DNA can be quantified by qPCR or using the RNA setting on NanoDrop^® (or similar spectrophotometric method). The expected DNA yield after bisulfite conversion is approximately 70-80%. This quantification step will help ensure that there is enough bisulfite converted DNA recovered for downstream processing. Bisulfite conversion efficiency can be assessed via colony Sanger Sequencing or via probe/TaqMan^® assay. In rare instances, sample purity may be responsible for low bisulfite conversion efficiency. In these cases, re-extraction or further cleanup is recommended.

Chip Failure

On occasion, chip failures may occur. If multiple samples on a single chip have low bisulfite conversion efficiency as determined by the BeadArray Controls Reporter, this might indicate a chip failure rather than a bisulfite conversion issue. In these instances, re-running leftover bisulfite-converted sample on a new chip can resolve the issue. To further troubleshoot, a post-bisulfite conversion/ pre-array QC is helpful to determine whether to rerun the samples.

Chip Failure

Methylation arrays such as the 450K and EPIC array are helpful tools for analyzing methylation changes in a variety of human and mouse samples. Whenever a sample is flagged for low bisulfite conversion, it is best to confirm that the following is true for your project:

●Validated and updated protocols were used
● DNA was as high-quality and intact as possible. Higher DNA input amounts were used for fragmented/low-quality samples
● Bisulfite conversion was performed according to best practices

Following these simple steps will help determine the reason for the bisulfite conversion probe failure.

Bisulfite conversion kit validated for Illumina^® Methylation Arrays
EZ DNA Methylation Kit (50 Rxns)	D5001
EZ DNA Methylation Kit (200 Rxns)	D5002
EZ-96 DNA Methylation Kit (Deep-Well)	D5004

DNA Methylation Analysis
Bisulfite Methods
Bisulfite conversion	EZ DNA Methylation Kits
NGS platforms	Pico Methyl-Seq Library Prep Kit Zymo-seq RRBS kit Zymo DNA methylation NGS service Targeted Bisulfite Service
PCR platforms	ZymoTaq DNA Polymerase ZymoTaq PreMix
Bisulfite Free Methods
Antibody Methods	5-mC DNA ELISA Kit Quest 5-hmC DNA ELISA Kit Methylated-DNA IP Kit
Enzymatic Methods	OneStep qMethyl Kits
Chromatin Analysis
ChIP	Zymo-Spin ChIP Kit ChIP DNA Clean & Concentrator dsDNA Shearase™ Plus ChIP-Seq service
Chromatin Structure	Zymo-seq ATAC

February 11, 2021

February 11, 2021February 11, 2021

Epigenetic clock, Zymo leading the way

With the increasing number of refugees seeking to settle in the European Union, and with potential benefits for refugees being significantly increased for minors, European authorities are calling for better methods of verifying an individual’s age when reliable records are unavailable. Zymo Research, through an exclusive license to the Horvath Epigenetic Clock, is leading the way to a fast, reliable, commercially available epigenetic age test.

Can epigenetics help verify the age claims of refugees?

When local authorities in Hildesheim, Germany, didn’t believe an asylum seeker who claimed to be under 18 years old — and thus eligible for privileged treatment — police turned to a blood-based age test sold by a California company.

In a paper published online in May, a team led by forensic-medicine specialist Stefanie Ritz-Timme of the University of Dusseldorf in Germany said that these tests were not ready for use in sensitive forensic evaluations.

But now, in the charged political atmosphere that has accompanied the arrival of millions of refugees to Europe, forensic scientists across the continent are joining forces to improve epigenetic-clock-based tests — with a focus on whether they might be used to help determine the age of refugees whose claims to be under 18 are disputed. They hope that, with time, such tests could replace existing methods, which assess the maturity of bones or teeth to determine an individual’s age but are imprecise and can be controversial.

Read Full Nature Article

Photo Credit: Nemanja Pancic/SIPA/Shutterstock

September 11, 2019