Enrichment and determination of nucleic acids targets

#Embodiments of the disclosure include at least the fields of molecular biology, cell biology, nucleic acid amplification, genomics, sequencing, and medical diagnostics.

#Genetic abnormalities are the root of many genetic diseases, such as cancers and hereditary diseases, for example. These abnormalities can be exploited for diagnosis to guide treatments and clinical and health managements. The present disclosure satisfies a long-felt need in the art by providing methods and compositions that allow for improved enrichment of certain sequences having a higher efficiency and reduced errors in the enrichment, compared to known techniques.

#The present disclosure allows for methods and compositions related to enrichment of particular target sequences within nucleic acids, such as from a sample including a biological sample. In specific embodiments, the initial step(s) of the methods employ linear amplification of target sequences that ensures greater fidelity of the amplified sequences compared to conventional methods.

#For a more complete understanding of the present disclosure, reference is now made to the following descriptions taken in conjunction with the accompanying drawing.

#FIG. 1 illustrates one example of a method of the disclosure in which (a) double stranded nucleic acids are dissociated to produce first and second strands in which the first strand comprises the target sequence (*); (b) primer extension initiates on the first strand or the second strand, independently; (c) extension occurs to the end of the fragment (at least in some embodiments); (d) the extended product dissociates from the first strand or the second strand, respectively; (d) the steps of (a) through (d) occur two or more times; and (f) particular adaptors are ligated to the linearly amplified extension products.

#FIG. 2A demonstrates that upon using 10 ng of Tur-Q7 reference sample (HorizonDx, 1.3% Tier MAF), with the library construction methods disclosed herein and a unique molecular identifier (UMI)-aware variant caller named Bin-based Genotyping (BinGo) for SNV/Indel/di-nucleotide (phase), all expected mutations (100% sensitivity) and no any other mutations (100% specificity) were detected. FIG. 2B shows a graphic presentation of FIG. 2A.

#As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more. In specific embodiments, aspects of the subject matter may “consist essentially of” or “consist of” one or more elements or steps of the subject matter, for example. Some embodiments of the subject matter may consist of or consist essentially of one or more elements, method steps, and/or methods of the subject matter. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

#The present disclosure provides methods for enrichment and determination of nucleic acids targets that are advantageous over other methods in the art.

#In general embodiments of the disclosure, certain nucleic acids comprising target sequence(s) of interest are enriched by increasing their number using primer extension methods in a linear amplification manner prior to affixing adaptors to the amplified products. The methods lack amplification by polymerase chain reaction (PCR) at least at the initial steps of the method. In specific cases, the adaptors are ligated to primer extension products only after multiple cycles of extension and dissociation have occurred, thereby producing linear amplification products. Such steps considerably increase the efficiency of target enrichment by the linear amplification in the present methods, because linear amplification enriches as many original template targets as possible with repeated attempts that maximizes the chance of all of the original targets being contacted and copied into second generation products, and lacks the tendency for copying already copied, i.e. second, third and so forth generations of products that are much more abundant than the original templates associated with non-linear, exponential amplification methods such as PCR. In specific embodiments a target sequence is copied from an original template, whereas other methods enrich targets based on copying of a copied version of the template. The linear amplification methods of the present disclosure keeps to a minimum the production of products with mistakes prior to tagging of the amplified products with adaptors. Such exceptional high efficiency and accuracy is particularly useful for applications that utilize samples in low abundance, although the method may be utilized for samples having abundant nucleic acids as well.