The invention relates to a sequencing method, able to detect chromatin changes, specifically heterocromatin. Alterations in heterochromatin are associated with developmental defects and cancer, while its proper conformation is a hallmark of healthy cells. As such, reliable methods to characterize heterochromatin and its alterations are essential for the biomedical scientific community.
Our technology can be used in the scientific research (SOM – serviceable obtainable market) because is a quick and economically-competitive protocol to analyze the chromatin accessibility. Our preliminary studies suggest also a potential market in the diagnostic field (SAM – serviceable addressable market). In fact our data support the possibility to use our technology to stratify oncological patients and we are working to find new DNA structural marker to predict the tumoral progression.
Chromatin within the cell nucleus has a complex structure which is fundamental for genome function (see figure below). The characteristic of the cells, their plasticity and the ability to respond to the environmental stimuli depend on the chromatin shape and remodeling. Electron microscopy imaging of eukaryotic nuclei shows chromatin compartments with different levels of compaction, known as euchromatin and heterochromatin. The more accessible and less condensed euchromatin is generally enriched in expressed genes. Instead, heterochromatin contains highly condensed DNA and is transcriptionally inactive. Alterations in heterochromatin are associated with developmental defects, genetic diseases and cancer, while its proper conformation is a hallmark of healthy cells. As such, reliable methods to characterize chromatin conformation and its alterations are essential for the biomedical scientific community.
Current Technology Limitations
In recent years, the widespread adoption of experimental techniques based on high-throughput sequencing (NGS) has been instrumental in advancing the knowledge of chromatin structure and function. Several of these techniques were originally designed to map active chromatin regions. On the other hand, a limited set of options is available for genome-wide mapping of heterochromatin and Lamin Associated Domains (LADs). The most commonly used ones include ChIP-seq or DamID-seq targeting nuclear lamin components or ChIP-seq for the heterochromatin associated histone marks (H3K9 methylation). Such techniques suffer major limitations as the exogenous expression of a transgene (DamID-seq) or crosslinking and antibodies (ChIP-seq). More recently proposed high-throughput sequencing methods for genome-wide mapping of LADs and heterochromatin, such as gradient-seq and protect-seq, also rely on crosslinking.
We are working on some new SAMMY-derived protocols:
- The entrapped RNA SAMMY-seq (eR-SAMMY-seq)
This protocol, already set up, allows the extraction of chromatin-associated RNA, with the advantage over the existing procols to further fractionate the chromatin (see figure below). Thus with this technology we can isolate eu- and heterochromatin-associated RNA. It is possible the production of a kit for the analysis of chromatin associated RNA, that may be used in scientific research field. Alternatively, a combo kit could allow the contemporary isolation of fraction-specific DNA and RNA.
The single cell SAMMY-seq (sc-SAMMY-seq)
The protocol was only designed. Actually, only the preexisting DamID-seq technology allows identification of Lamin Associated Domains at the single cell level, but it cannot be applied on primary cells or tissue. Thus the development of sc-SAMMY-seq can revolutionaze the sequencing field.
Our Technology and solutions
We present a high-throughput sequencing based method to map euchromatin and heterochromatin accessibility (see figure below). The method is based on the sequential extraction of distinct nuclear fractions containing: soluble proteins (S1 fraction); the surnatant obtained after DNase treatment (S2 fraction); DNase-resistant chromatin extracted with high salt buffer (S3 fraction); and the most condensed and insoluble portion of chromatin, extracted with urea buffer that solubilizes the remaining proteins and membranes (S4 fraction). We further adapted the method to leverage high-throughput DNA sequencing for genome-wide mapping of the distinct chromatin fractions. The insoluble fractions are reproducibly enriched in lamina-associated heterochromatic regions (LADs), while the more soluble one correlates with euchromatin (see figure below). Thus with a unique protocol we are able to describe the accessibility of euchromatin and heterochromatin.
Our protocol overcomes several major limitations of other methods for mapping lamina associated heterochromatic regions. First of all, the procedure can be applied on primary cells, as it doesn't require exogenous gene expression as in DamID-seq. Then, SAMMY-seq does not involve chemical modifications of chromatin, which might cause artifacts and biases in sequencing. Additionally, it does not rely on antibodies for enriching specific chromatin fractions, thus avoiding issues related to antibody specificity, production, lot-variability and cross reactivity. This is particularly important when studying epigenetic changes in cells where protein levels of chromatin associated factors could be altered, thus allowing more flexibility in terms of experimental design compared to antibody-based techniques. Finally, SAMMY-seq is robust, as it yields reproducible results even at lower sequencing depth or with a small number of starting cells (10K) and requires only about 3 hours of bench work, excluding DNA extraction and library preparation.
Some advantages of our technology are summarized in the table below.
Potential industrial product:
Kits for analysis of genomic solubility on a small number of cells, that may be used in biomedical field e.g. for diagnosis. The kit will contain: a high salt solution, reagents for isolating genetic material from cells, reagents for purifying genetic material, one or more restriction enzyme or a DNase, reagents for PCR amplifying target loci of interest, and reagents for sequencing the amplified target loci of interest using a DNA or RNA sequencing platforms.
Kit for single cell SAMMY-seq (sc-SAMMY-seq)
Set up of buffers and reagents for the kit assembly.
Set up of technology
Companies involved in genome sequencing. Companies involved in bioinformatic tools for genome functions analysis.
Companies involved in genome sequencing.