Maximize Yeast Transformation Efficiency



Introducing foreign DNA to yeast cells is essential for two-hybrid system screening and many other genetic manipulation techniques. The transformation efficiency of yeast cells is typically magnitudes lower than that of Escherichia coli due to the added challenges associated with permeating the fungal cell wall. However, implementing the following tips can help maximize the transformation efficiency of yeast species such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, and Candida albicans.

  1. Cell Growth State: Use cells in the mid-log phase to produce the most transformants. Early or late log-phase cells yield comparatively fewer transformants.
  1. Cell Density: The optimal cell density for transformation is between 5 x 106 and 2 x 107 cells/ml (0.8-1.0 OD600). Yeast cultures with cell densities at the high end of this range typically report the highest transformation efficiencies.
  1. Heat Shocking: Because of their hardy cell walls, yeast cells must be heat-shocked more intensively than E. coli. To ensure high transformation efficiencies, yeast should be heat-shocked for 45 minutes.
  1. Plating Media: Not all commercially available media are created equal. Our results show that Difco media are the most reliable for maximizing transformation efficiency.
  1. DNA Input: For circular DNA such as plasmids, the transformation efficiency stops increasing linearly for DNA inputs above 1 µg. For integrative transformation with linearized DNA, higher inputs of up to 5 µg of DNA are recommended. The DNA extraction method is crucial, as highly pure DNA should be used for transformation.

Transformation efficiency is also species-dependent, as some strains are more susceptible to transformation than others. The following table provides a range of transformation efficiencies to expect from four prominent yeast strains using traditional transformation protocols.


Transformation Efficiency (cfu/µg DNA)

S. cerevisiae

104 – 106

S. pombe

103 – 105

P. pastoris

103 – 106

C. albicans

102 – 104

It is important to note that the true transformation efficiency can vary significantly depending on the transformation method used, quality of the DNA, and other experimental parameters. For applications such as two-hybrid system screening or library screening that require high transformation efficiencies, it is imperative that researchers select a reliable transformation method such as the Frozen-EZ Yeast Transformation II Kit for optimal results. As an industry leader in yeast products, Zymo Research is dedicated to helping scientists streamline and elevate their transformations with innovative technologies.



RNA sequencing (RNA-Seq)


RNA sequencing (RNA-Seq) has revolutionized the study of gene expression and regulation, thus providing deep insights into the inner workings of living organisms. One common method for this technology, total RNA-Seq, profiles both coding and noncoding RNA with the depletion of the overly abundant ribosomal RNA (rRNA). With most existing rRNA depletion methods customized and established for human samples and the commonly studied mouse and rat model systems, the Zymo-Seq RiboFree® Total RNA Library Kit allows researchers to expand their horizons thanks to its novel rRNA depletion technology that is probe-free and species-independent. In this blog, you will find several recently published studies where researchers have leveraged the RiboFree® depletion strategy to explore the transcriptomes of organisms outside the common models.


As the scientific community transitions into a post-COVID era, there is an increased focus on epidemiological studies involving potential sources of zoonotic viruses. A recent study conducted by scientists in Uganda shed light on an insufficiently studied yet widespread tickborne pathogen called the Crimean-Congo hemorrhagic fever virus (CCHFV). Amidst their investigation, the researchers employed the Zymo-Seq RiboFree® Total RNA Library Kit to analyze a novel viral strain of the CCHFV.1

CCHFV is transmitted between livestock and humans through infected tick bites, causing severe outbreaks across Africa, Asia, the Middle East, and Eastern Europe.2,3 Despite being the most geographically widespread tick-borne viruses, strains of CCHFV remained poorly studied, especially true for the African strains, with most knowledge derived solely from severe human disease cases.

In this study, researchers took a novel approach by collecting RNA samples directly from the infected African blue ticks, Rhipicephalus (Boophilus) decoloratus. The aim was to expand the understanding of the natural variation of CCHFV strains from tick vectors and animal reservoirs directly. The Zymo-Seq RiboFree® Total RNA Library Kit enabled the authors to successfully generate total RNA libraries from tick RNA for sequencing. From there they were able to characterize the complete coding region of this novel CCHFV strain, contributing to the growing reservoir of data essential for the development of vaccines, diagnostic tools, and control strategies for combating viral infections.1


Coral reefs harbor 25% of the world’s biodiversity and unfortunately face significant challenges due to climate change-induced thermal stress.4, 5 This has led to a decline in global coral populations.5 While some reef species are highly sensitive to these thermal changes, others demonstrate remarkable resilience and even appear to thrive under these normally unfavorable conditions. One such resilient species is the mustard hill coral (Porites astreoides).6

To unravel the mechanisms behind the mustard hill coral’s resilience, researchers from the University of Rhode Island utilized the innovative Zymo-Seq RiboFree® technology to generate an ab initio reference transcriptome of P. astreoides, adding a useful resource for future studies.7

The researchers further leveraged the RNA-Seq data from the RiboFree® libraries to characterize the mapping potential of the draft reference genome they built from DNA sequencing. Remarkably, they achieved alignment rates of around or above 80% for unique sequences. This high alignment rate underscores the suitability of the new reference genome for future transcriptomic studies. As climate change continues to impact global biodiversity, understanding how resilient species like the mustard hill coral cope with these unprecedented challenges offers crucial insights into the future of our ecosystems.


Wine production is ironically very far from soothing; in fact, it’s quite a complex process. The quality and stability of the wine is dependent upon bacteria that are utilized for various biochemical reactions. One critical process in winemaking is malolactic fermentation (MLF)8, which plays a vital role in reducing wine acidity and enhancing microbiological stability9, both of which are highly desirable characteristics. Oenococcus oeni, a gram-positive heterofermentative lactic acid bacterium (LAB) species, is commonly used for controlled MLF in wine production due to its acid tolerance of high ethanol levels.10 However, its sensitivity to sulfur dioxide (SO2), an antiseptic compound commonly used in winemaking9, remains poorly understood.

To shed light on the transcriptional response of O. oeni during MLF under the stress of SO2 exposure, researchers from Australia conducted a comprehensive investigation.11 They utilized the Zymo-Seq RiboFree® Total RNA Library Kit to prepare total RNA-Seq libraries from O. oeni under various experimental conditions. Through sequencing and differential gene expression analysis, they unveiled key transcriptional changes induced by SO2 exposure, highlighting its potential as a target for the development of SO2-tolerant strains. These advancements are pivotal in enhancing wine production and expanding the knowledge within the winemaking community.


The significance of an RNA-Seq kit that is compatible across all species cannot be overstated. With the Zymo-Seq RiboFree® Total RNA Library Kit, scientists worldwide now have a powerful tool at their disposal to delve into the transcriptomics of less commonly studied organisms. As exemplified by the peer-reviewed research presented above, the possibilities for remarkable and unprecedented discoveries are unlimited. From unraveling the secrets of resilient coral species to untangling the intricate responses of bacteria in wine production, the Zymo-Seq RiboFree® Total RNA Library Kit has served as a key that opens the door to a wealth of knowledge and innovation.


  1. Wampande, E. M.; Waiswa, P.; Allen, D. J.; Hewson, R.; Frost, S. D. W.; Stubbs, S. C. B. Phylogenetic Characterization of Crimean-Congo Hemorrhagic Fever Virus Detected in African Blue Ticks Feeding on Cattle in a Ugandan Abattoir. Microorganisms 2021, 9 (2). DOI: 10.3390/microorganisms9020438.
  2. Bente, D. A.; Forrester, N. L.; Watts, D. M.; McAuley, A. J.; Whitehouse, C. A.; Bray, M. Crimean-Congo hemorrhagic fever: history, epidemiology, pathogenesis, clinical syndrome and genetic diversity. Antiviral Res 2013, 100 (1), 159-189. DOI: 10.1016/j.antiviral.2013.07.006.
  3. Hoogstraal, H. The epidemiology of tick-borne Crimean-Congo hemorrhagic fever in Asia, Europe, and Africa. J Med Ento mol 1979, 15 (4), 307-417. DOI: 10.1093/jmedent/15.4.307.
  4. Hughes, T. P.; Kerry, J. T.; Connolly, S. R.; Álvarez-Romero, J. G.; Eakin, C. M.; Heron, S. F.; Gonzalez, M. A.; Moneghetti, J. Emergent properties in the responses of tropical corals to recurrent climate extremes. Curr Biol 2021, 31 (23), 5393-5399. e5393. DOI: 10.1016/j.cub.2021.10.046.
  5. Hughes, T. P.; Anderson, K. D.; Connolly, S. R.; Heron, S. F.; Kerry, J. T.; Lough, J. M.; Baird, A. H.; Baum, J. K.; Berumen, M. L.; Bridge, T. C.; et al. Spatial and temporal patterns of mass bleaching of corals in the Anthropocene. Science 2018, 359 (6371), 80-83. DOI: 10.1126/science.aan8048.
  6. Loya, Y.; Sakai, K.; Yamazato, K.; Nakano, Y.; Sambali, H.; van Woesik, R. Coral bleaching: the winners and the losers. Ecology Letters 2001, 4 (2), 122-131.
  7. Wong, K. H.; Putnam, H. M. The genome of the mustard hill coral, Porites astreoides. GigaByte 2022, 2022, gigabyte65. DOI: 10.46471/gigabyte.65
  8. Ribereau-Gayon, P.; Dubourdieu, D.; Doneche, B.; Lonvaud, A. Handbook of Enology: The Microbiology of Wine and Vinifi cations; 2006.
  9. Lonvaud-Funel, A. Lactic acid bacteria in the quality improvement and depreciation of wine. Antonie Van Leeuwenhoek 1999, 76 (1-4), 317-331.
  10. Henick-Kling, T. Malolactic Fermentation. In Wine Microbiology and Biotechnology, Fleet, G. H. Ed.; 1993; pp 289-326.
  11. Onetto, C. A.; Costello, P. J.; Kolouchova, R.; Jordans, C.; McCarthy, J.; Schmidt, S. A. Analysis of Transcriptomic Response to SO2 by Oenococcus oeni Growing in Continuous Culture. Microbiol Spectr 2021, 9 (2), e0115421. DOI: 10.1128/ Spectrum.01154-21.




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


IRVINE, Calif., (May. 11, 2021) 

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


Any Organism. One rRNA Depletion Solution.

Zymo-Seq RiboFree Total RNA Library Kit

Compatible with
any Organism

Suitable for

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

Bisulfite Conversion for Illumina Methylation Arrays

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

Epigenetic clock, Zymo leading the way

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

Is an Epigenetic Clock the Key to Asylum?

Is an Epigenetic Clock the Key to Asylum?

A transcript of BBC World Service Radio’s Newsday interview with Keith Booher, PhD, of Zymo Research Corporation about their DNAge® test being used to determine the age of refugees seeking asylum in Europe as minors.

BBC: Could a finger prick test for blood make it easier for child refugees to have their age verified? That’s a question that California scientists have been thinking about. The field is known as Epigenetics and the test involves examining chemically modified DNA to create a sense of how old someone actually is. Keith Booher is a scientist in California and he has been part of the development of this device.

Booher: What we’re simply trying to do is to make an age determination or quantify aging. For example, in forensics cases when the provenance or age of a sample is unknown, we can make that assessment. There’s also some indications that it may be useful for biologically aging someone – so they may have been born 45 years ago, and are 45 years old but based on certain life decision they make – the diet they choose to eat, if they get enough exercise, do they live a stress-free lifestyle? Their biological age may be older or younger than their chronological age, just depending on the lifestyle they lead. And we should be able to quantify that as well.

BBC: So if I asked you now, how old you are, Keith, what would you tell me?

Booher: I’m 38 years old.

BBC: But that’s the chronological age, is that right?

Booher: Correct!


BBC: Which one is more important, biological or chronological?

Booher: You know, if you’re a young teenager trying to get your driver’s license, obviously your chronological age is very important. But if you are in advanced years of your life or you may have made some choices that are… poor when you were younger or over the course of your life, then biological age becomes much more critical.


BBC: How did you end up using your science with refugees? 

Booher: Our information was passed to law enforcement officials working in Germany. And they had a case where they were trying to accurately determine the age of a refugee asylum seeker and they determined that the methods available to them at that time were not precise enough to get a good age determination and they reached out to us and asked if we could help.

BBC: What have you found in the course of your work? How accurate is your science at the moment? 

Booher: Currently, epigenetic clocks are the most accurate, most precise way to age a sample. So our platform that we offer at Zymo Research has a median error of under two years – it’s about one and a half years. Other publications, people using different types of methods – you know, similar epigenetic aging, have shown median errors anywhere between two to four years. On aggregate, you have a precise age estimation method.

BBC: Has it helped clarify or clear doubt about age?

Booher: Yeah, I think so. In the case of the asylum seeker, I was told this person presented with no official documentation. The person claimed to be a minor, and therefore entitled to certain protections and assistance according to United Nations guidelines. So in this case, I think it definitely helped clear up any doubt regarding the age of the subject.

BBC: I can see it working with football and other sports as well. You know, where there have been all sorts of questions about ages.

Booher: Yeah, that’s correct! That’s a good point. So there’s certain rules about the age of participants engaged in these activities in different leagues.

BBC: Keith Booher from Zymo Research Corporation which is developing Epigenetics.


Alexandra, K. (Producer). (2018, September 24). Newsday [Radio Broadcast]. London, UK: BBC World Service Radio

A Piece of the Puzzle

A Piece of the Puzzle

Autism (autism spectrum disorder) is one of the most common lifelong neurodevelopmental disorders, affecting an estimated 1 in 59 children in the United States 1. Due to its prevalence, autism has become a heavily researched topic in the field of genomics, as medical professionals and advocacy organizations look for a deeper understanding of its wide spectrum of symptoms. Genome-wide studies have suggested the involvement of hundreds of genes and genetic pathways in the development of the disorder. An increased risk of autism development is also known to be influenced by environmental factors such as premature birth, oxygen deprivation, infection during pregnancy, and parental age at time of conception 3. Recent studies have shown that these factors influence what is known as our epigenetics.

But What is Epigenetics?

Epigenetic regulation – in the form of DNA methylation, histone modification, and chromatin remodelling – helps govern the proper expression of genes in the genome. One’s environment continuously shapes their epigenetics. Factors that influence epigenetics can include one’s diet, exercise, stress level, drug abuse, and exposure to toxic pollutants. Failure in epigenetic regulation can result in aberrant gene expression (mutations, deletions, copy number aneuploidies) that contributes to halted neural development 4. Thanks to progresses in Next-Gen Sequencing, scientists have been able to map over 600 human epigenes associated with neurodevelopmental disorders, including autism.

Ongoing Epigenetic Research 

Autism research is a continuously growing field, especially in the context of epigenetics. Currently, diagnosis and subsequent treatment is tough due to the degree of heterogeneity of traits those with autism can have. Fortunately, as sequencing technology becomes cheaper, scientists have more opportunities to analyze the epigenome with higher resolution. Without a high price point necessary for entry into autism research, smaller labs now have an opportunity to make significant discoveries.

There are many ongoing studies that aim to understand the epigenetic mechanisms behind the pathogenesis of autism spectrum disorder, as well as possible methods for treatment. A recent exciting study published in Nature Neuroscience demonstrated that mouse models of autism could be rescued via inhibition of histone deacetylase 8. These mice were deficient in the SHANK3 gene, another high-risk target linked to autism spectrum disorder. Mutations in the gene results in dysregulation of emotional and cognitive response in both humans and mice. The researchers found that low-dose treatment by anti-cancer drug romidepsin, a histone deacetylase inhibitor, fully restored gene function and completely reversed the social deficits of the mice after 3 days. This effect lasted 3 weeks in the mice, equivalent to the length of many years in humans.

Why Autism Research Is Important

Research that continues to find links between autism and epigenetics provides clues to the cause and mechanisms of the disorder, as well as invaluable biomarker discoveries. While it is currently thought that there is no single underlying cause of autism, epigenetic biomarkers can be used for risk assessment prior to diagnosis, diagnosis of the disorder itself, or even provide insight into its severity 9.

Studies show that early intervention leads to positive outcomes later in life for people with autism 1. By furthering research on such a prevalent disorder, we can better prepare families at an earlier timepoint. Considering autism’s broad range of characteristics, scientific findings into the disability may also provide insight into the inner workings of other related neurodevelopmental disorders.


2. Hans van Bokhover. “Genetic and epigentic networks in intellectual disabilities”. Annual Review of Genetics, 2011. 45: 81-104
4. Michelle Siu and Rosanna Weksberg. “Epigenetics of Autism Spectrum Disorder”. Neuroepigenomics in Aging and Disease, 2017. 978: 63-90
5. Luikenhuis, S., et al. “Expression of mecp2 in postmitotic neurones rescues rett syndrome in mice”. Proc Natl Acad Sci USA, 2004. 101: 6033-6038
6. Samaco, RC., et al. “Epigenetic overlap in autism-spectrum neurodevelopmental disorders: mecp2 deficiency cuases reduced expression of UBE3A and GABRB3”. Hum Mol Genet, 2005. 14: 483-492
7. Wong, C., et al. “Epigenome-wide DNA methylation analysis of monozygotic twins discordant for diurnal preference”. Twin Res Hum Genet, 2015. 18: 662-669
8. Qin, L., et al. “Social deficits in shank3-deficient mouse models of autism are rescued by histone deacetylase inhibition”. Nature Neuroscience, 2018. 21: 564-575
9. Loke, Yuk Jing, et al. “The role of epigenetic change in autism spectrum disorders”. Front Neurol, 2015. 6: 107

Where There’s a Whale There’s a Way

Where There’s a Whale There’s a Way

Their songs haunt the deepest depths of the ocean and their behaviors have intrigued scientists for generations. The humpback whale itself is a mystery in many ways, such as having no real visible age indicators after they are one year old.

Studying the age characteristics of sensitive animal populations can provide valuable ecological insights into questions of reproductive success, lifespan, survival trends, and more. In recent decades, marine biologists have used different methods to measure age in certain whale species. However, most have notable limitations.


For example, the age of deceased whales can be estimated using various invasive biopsy techniques, but doing so precludes the monitoring of living subjects over time. Alternatively, researchers attempted to measure the age of live whales using telomeric genetic markers, but those methods suffer from a lack of specificity and too much technical noise. By contrast, a number of recent research articles demonstrated that DNA methylation-based age estimators, commonly referred to as epigenetic clocks, are a highly accurate and technically robust means to measure aging in mammalian species as diverse as mice, canines, humans, and other primates1-3.

A group of researchers in Australia now show that DNA methylation analysis is an effective way to help predict the age of living whale populations, thus providing a new tool to study the demographics of these majestic ocean leviathans.

In their study, Polanowski et al.4 noted the development of DNA methylation-based age clocks and set out to develop a similar test for use in humpback whale (Megaptera novaeangliae) research. They focused on the evolutionarily conserved 5’ regulatory regions of genes whose changing DNA methylation patterns correlated with age. The authors first generated a calibration data set using a cohort of 45 humpback whales originating primarily from the Gulf of Maine and with known ages ranging from a few months up to 30 years. Importantly, the tissue sample source used in the analysis came from minimally invasive skin punch dart biopsies.

Humpback Epigenetic Age Assay (HEAA)

The scientists called their new experimental tool the Humpback Epigenetic Age Assay (HEAA). Application of their 3-gene test demonstrated a high positive correlation (R2 = 0.787) between DNA methylation change and age. Furthermore, a leave-one-out cross validation from the calibration cohort data set demonstrated precision and accuracy (Mean difference = 3.75 years; Standard deviation = 2.991 years) between the predicted and known ages of the samples.

Having established the HEAA, Polanowski et al. then set out to test their model on a humpback whale population of unknown age composition. Using skin biopsies once again, they measured the epigenetic ages of 63 humpback whales migrating north near Evans Head, Australia, and observed an average population age of 10 years, with a total age range from 0 to 52 years.

The authors further noted that the distribution of the measured ages resembled what would be expected for a sample population of that size and in that region after comparing their dataset to one obtained in the same area 50 years earlier, before the effects of whaling caused the fishery to collapse in the 1960s. One final test of the HEAA showed its ability to correctly pick the ordinal age-relationship between parent and progeny in both data simulations (<90% accuracy) as well as an empirical test of mother-calf pairs taken from the test and calibration data sets, respectively. The assay proved accurate in all 12 pairings tested.

With the HEAA, Polanowski et al. present an epigenetic clock similar to what has been shown to work so effectively in other mammalian species. Initial calibration and testing of the HEAA demonstrated its broad precision, accuracy, robust nature, and compatibility with a minimally invasive and readily obtainable sample source. Continued use of the HEAA, or similar DNA methylation-based age estimators, will help scientists track the overall health of whale populations well into the 21st century.


[1] Horvath S. DNA methylation age of human tissues and cell types. Genome Biology. 2013;14(10): R115. doi:10.1186/gb-2013-14-10-r115.
[2] Petkovich DA, Podolskiy DI, Lobanov AV, Lee SG, Miller RA, and Gladyshev VN. Using DNA methylation profiling to evaluate biological age and longevity interventions. Cell Metabolism. 2017;25(4);954-960.e6. doi:10.1016/j.cmet.2017.03.016.
[3] Thompson MJ, vonHoldt B, Horvath S, and Pellegrini M. An epigenetic aging clock for dogs and wolves. Aging. 2017;9(3):1055-1068. doi: 10.18632/aging.101211.
[4] Polanowski AM, Robbins J, Chandler D, and Jarman SN. Epigenetic estimation of age in humpback whales. Mol Ecol Resour. 2014;14(5):976-87. doi: 10.1111/1755-0998.12247.

Long-Read Epigenetics with Microbiome Standards

Long-Read Epigenetics with Microbiome Standards

Is m6A the new 5-mC?

Of the several chemical and structural modifications to DNA that are known to influence gene expression, 5-methylcytosine (5-mC), has garnered the most attention because of its role in transcriptional silencing and the readily available techniques to investigate it. For instance, bisulfite sequencing via short-read Next Generation Sequencing (NGS) has become the gold-standard for 5-mC detection due to its single-nucleotide resolution and the ever-decreasing cost of whole genome sequencing. The pairing of bisulfite to NGS has greatly expanded our understanding on the extent of cytosine methylation influence on gene expression.

But, the application of NGS technologies to analyze the genomes of complex microbial communities (e.g. microbiome and metagenomic samples) has caused other DNA modifications to gain more attention. For example, N6-methyladenine (m6A) plays important roles in bacterial survival and interactions with hosts 1. Like other epigenetic base modifications, m6A contributes to the regulation of gene expression as well other house-keeping functions in bacteria. Unfortunately, methylation detection by short-read bisulfite sequencing is based on cytosine to uracil conversion and cannot detect adenine modifications, such as m6A.

This is why many researchers are turning to long-read sequencing platforms (3rd generation sequencing technology), since they use differences in electronic signals as bases to pass through nanopores and detect other types of base modifications. Like any new technology, methylation detection with 3rd generation sequencing needed benchmarking with the use of well-defined standards and controls.

Not one, not two, but four sequencing platforms!

While the use of one sequencing platform is sufficient for most studies, some researchers incorporate two sequencing methods when verifying a new bioinformatics tool. However, to validate a new N6- methyladenine (m6A) detection tool named mCaller, McIntyre, et al. sequenced the ZymoBIOMICS Microbial Community Standard using PacBio, Oxford Nanopore, MeDIP-seq and whole-genome bisulfite sequencing 1. Single-molecule sequencing techniques, such as PacBio 2 and Oxford Nanopore Technologies (ONT) 3 have been used to detect m6A, but no method has had cross-validated results by using a well-characterized reference material on several sequencing platforms – until now.

Older m6A detection methods based on immunoprecipitation were limited in terms of nucleotide resolution, while previous single-molecule sequencing detection tools suffered from lower base modification calling (~70%) 3, 4. To improve accuracy, mCaller employs a neural network to learn and test different classifiers to detect m6A in Nanopore data generated from the E. coli MG1655 (a K-12 strain) genome.

To validate the tool, the bacterial genomes of the ZymoBIOMICS microbial community standard were sequenced via ONT, PacBio, and MeDIP-seq, and mCaller detection accuracy was compared across the different data sets. Remarkably, detection accuracy increased to 84.2% for high quality reads, and even 95.4% for single sites with at least 15x coverage, when compared to immunoprecipitation methods. Additionally, the methylome of the standard was verified with the use of the TruSeq DNA Methylation Kit.

Regulatory-Grade Genomes

As an additional control, the PacBio sequencing of the ZymoBIOMICS Microbial Community Standard was performed at two separate locations: the University of Florida and the Database for Reference Grade Microbial Sequences (FDA-ARGOS). The goal of FDA-ARGOS is to create a database of reference-grade microbial sequences available to the public. Because the sequencing of the ZymoBIOMICS Microbial Community Standards met the FDA-ARGOS quality requirements, they have been accepted as regulatory-grade genomes with the designations of FDARGOS_606 and FDAARGOS_612 under the BioProject number PRJNA477598.

“Overall, our results demonstrate a need for tool evaluation at a variety of sequence contexts, for which we propose the continued use of this well-validated microbial reference community.” – McIntyre, et al.

Team work makes the dream work

Although the ZymoBIOMICS Microbial Community Standard was developed as microbiome reference material, its well-defined and characterized composition has made it an excellent control for epigenetic sequencing. Such a cross-discipline use of reference materials and sequencing techniques demonstrates the power of inter-lab cooperation and the ability for different field leaders to push the capabilities of current technology.


[1] McIntyre ABR, Alexander N, Grigorev K, Bezden D, Sichtig H, Chiu CY, Mason CE. Single-molecule sequencing detection of N6-methyladenine in microbial reference materials. Nature Communications. 2019 10 (579).
[2] Fang G, Munera D, Friedman DI, Mandlik A, Chao MC, Banerjee O, Fengg Z, Losic B, Mahajan MC, Jabado OJ, Deikus G, Clark TA, Khai L, Murray IA, Davis BM, Keren-Pas A, Chess A, Roberts RJ, Korlach J, Turner SW, Kumar V, Waldor MK, Schadt EE. Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing. Nature Biotechnology 2012 30(12)
[3] Stoiber M, Quick J, Egan R, Lee JE, Celniker S, Neely RK, Loman N, Pennacchio LA, Brown J. De novo Identification of DNA Modifications by Genome-Guided Nanopore Signal Processing. BioRxiv 2017. doi:
[4] Rand AC, Jain M, Eizenga JM, Musselman-Brown A, Olsen HE, Akeson M, Paten B. Mapping DNA methylation with high-throughput nanopore sequencing. Nature Methods 2017 14(4)