Xactly DNA Overhang profiling

Exact ends of fragmented DNA molecules tell a story

XACTLY provides comprehensive information about the native-state of fragmentary DNA termini including the type (5´, 3´ , or blunt), length, and sequence of all single-stranded overhangs. The generation of blunt ends or overhanging termini can result from physical, chemical, or enzymatic cleavage, physiologic processes or pathological mechanisms. The native termini of DNA molecules reveal information about where they have been and what they have interacted with.

We initially developed XACTLY to interrogate cell-free DNA degradation. While our internal cfDNA research is ongoing, we’ve also found many other exciting applications for XACTLY including: identifying restriction enzyme cut sites, nuclease digestion preferences, and better understanding DNA degradation over time.

 

 

Xactly overview

XACTLY works by ligating a pool of end-informative NGS adapters to substrate DNA without the conventional end-polishing step. By incorporating different Unique End Identifiers (UEI) for adapters of each length and type, XACTLY encodes the exact type, length and overhang sequence, if present, for every DNA molecule in a massively parallelized reaction. The result is a standard dsDNA sequencing library that adds novel information about the fragment termini.


Xactly in Practice

In this illustrated example we elucidate the cut site left by an uncharacterized restriction enzyme using XACTLY. HMW DNA is first digested with the restriction enzyme and then used as input into XACTLY. After the XACTLY protocol the library is sequenced and the data is analyzed with respect to their overhangs. The results show a high prevalence of reads contain 5´ 4 bp overhangs with a predominant base composition of roughly equal proportion of Guanine and Cytosine at the 5´ 4 bp overhang. Further analysis of the overhang motifs revel the 5´ 4 bp cut site to be GGCC.

 

 

OUr Vision for XACLTY

cfDNA fragment termini contain information about the molecular history and origin of the molecule. This wealth of information is currently overlooked or lost due to the limitations of existing library preparation technologies. XACTLY fills this precise gap and helps elucidate preceding events that lead to cfDNA formation. These mechanisms may differ in healthy and diseased conditions: for example, differential nucleosome positioning, or differences in nuclease type and function. These events can result in unique DNA fragmentation patterns and generate a cfDNA pool with mixture of discrete and complex overhang profiles specific to a biological state. Following capture of this information with XACTLY, sophisticated machine learning algorithms are used to identify termini biomarkers that distinguish diseased from healthy states.

 

 

Want More Xactly?

While XACTLY is not currently publicly available, we are always looking for research collaborations and partnerships. Please contact us if XACTLY sounds right for your research question! And check back for updates on our ongoing research with XACTLY. In the meantime check out our current literature.

 
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Check out our XACTLY White Paper