Methods and kits for reducing adapter-dimer formation

Abstract

The current teachings relate to methods for reducing adapter-dimer formation, particularly when preparing nucleic acids of interest for subsequent amplification and / or sequencing. Also described are kits for use in performing certain disclosed methods.

Description

REFERENCE TO RELATED APPLICATIONS[0001]This application is a divisional of U.S. patent application Ser. No. 15 / 354,491, filed Nov. 17, 2016, which claims priority from U.S. Provisional Application Ser. No. 62 / 256,662, filed Nov. 17, 2015, the entire content of both of which is incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH[0002]This work was performed in part with government support under NSF Phase II Grant No. 1431020. The Government may have certain rights in the claimed inventions.FIELD OF THE INVENTION[0003]The current teachings relate generally to the field of nucleic acid sequencing, particularly to reducing adapter dimer formation. More particularly, the current teachings are directed to improving the creation of sequencing libraries comprising small RNA molecules.BACKGROUND OF THE INVENTION[0004]Small RNA sequencing using next generation sequencing technologies (sRNA-seq) is invaluable for small RNA profiling and discovery in fields such as...

AI Technical Summary

Benefits of technology

The patent provides a method for reducing the formation of adapter-dimers, which can be a problem in small RNA library preparation. The method involves a two-step process. First, excess unligated 3′ adapter is depleted using a magnetic-bead based method. Then, any remaining 3′ unligated adapter is inactivated using an enzymatic method. This combination of depletion and inactivation results in a significant reduction of adapter-dimer formation, allowing for the preparation of gel-free or low input libraries. Overall, this method improves the quality and efficiency of small RNA library preparation.

Problems solved by technology

Small RNA sequencing using next generation sequencing technologies (sRNA-seq) is invaluable for small RNA profiling and discovery in fields such as cancer, stem cell biology, and epigenetic gene regulation. sRNA-seq library preparation has historically suffered from three major drawbacks; severe bias, the need for gel-based purification, and the lack of low-input protocols.

However, since insert-containing molecules are only ˜20 bp larger than adapter-dimer molecules in small RNA libraries, SPRI size selection is not feasible, and gel-based selection must be performed.

The need for gel-based size selection greatly limits both throughput and automation potential of small RNA library preparation, as only a limited number of libraries can be run on a single gel and it is a labor-intensive process that is not amenable to automation.

However, adapter-dimer present in the libraries will also be greatly amplified, which eventually leads to a library where adapter-dimer products are extremely abundant, making it difficult to isolate insert-containing products and leading to sequencing data where very few of the reads are useful.

A number of methods have been developed to reduce adapter-dimer formation in small RNA library preparation, but unfortunately none are effective at reducing adapter-dimer formation to such an extent that gel-free or low-input small RNA library preparation is possible

The double-stranded DNA is a poor substrate for the T4 RNA ligase 1 enzyme used in the subsequent reaction, resulting in reduced formation of adapter-dimer products.

Traditional methods of construction of sRNA libraries have been shown to suffer from severe bias, resulting in final sequencing results that do not accurately represent relative abundances of small RNAs in the starting material.

However, the strategy of hybridization of a complementary oligonucleotide does not work well to reduce adapter-dimer formation when using adapters with randomized ends.

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