Methods and kits for nucleic acid sample preparation for sequencing

Abstract

The present disclosure relates to methods and kits for DNA library construction, particularly for consistent and reproducible DNA sequencing.

Description

PRIORITY[0001]This Application claims priority to U.S. Provisional Application No. 61 / 721,843, filed Nov. 2, 2012, which is heerebv incorporated by reference in its entiretyFIELD OF THE INVENTION[0002]The present disclosure relates to methods and kits for nucleic acid sample preparation, particularly for consistent and reproducible nucleic acid sequencing.BACKGROUND[0003]For nucleic acid sequencing, high throughput sample preparation products are generally shipped with significant quantities of dry ice (up to 50 lbs) to ensure that the reagents remain active upon receipt at the sequencing facility. Most of the enzymes required for sample preparation for next generation sequencing platforms are derived from mesophilic sources which are more sensitive to broad temperature fluctuations that may be observed during shipping. Even thermophilic sources, although they are more stable to temperature ranges, exhibit residual activities that may cause side reactions during shipping that negati...

AI Technical Summary

Benefits of technology

[0005]An object of this disclosure is to develop improved reagents for next generation sequencing library construction, In the various aspects, the invention provides methods and kits for library construction, which employ at least one lyophilized reaction mixture. The process of lyophilization yields a product that is inactive at room temperature until rehydrated with water. As a result of lyophilization the enzymes become essentially immobilized and thus are stable to conditions that would previously have caused significant decrease in enzyme activity. Such a formulation, upon lyophilization, yields a sequencing sample preparation kit that can be shipped under ambient conditions, negating the need for dry or wet ice-containing shipping.

[0006]In various embodiments, the invention provides for several advantages, including others that will be apparent from the following detailed disclosure. First, the invention enables simplified shipping that doesn't require coolants. Thus the reaction components and kits described herein can be shipped worldwide at room temperature with the simplest of infrastructure. Storage of the products are simplified and the cost and complexity of freezer validation for clinic applications is eliminated. Second, the invention provides enzyme products that are easier to use because the number of pipetting steps required for sample preparation is decreased by up to 30% compared to existing liquid based methods. Decreasing the number of pipetting steps also reduces the possibility of cross contamination. Third, the invention provides improved consistency across a microtiter plate or a broad number of samples due to consistent reagent dosing. Fourth, the invention provides consistent performance because each reaction can be single use in the lyophilized format. This prevents multiple rounds of freeze thawing of stock liquid solutions (typically stored at −20° C.) Which if done incorrectly can yield non-homogenous thawing of the product and thus variation in efficiencies of each step in the sample preparation workflow. Stock liquid solutions also increase the probability of cross contamination. The product according to the embodiments of the invention is room temperature stable and thus doesn't have to be thawed prior to use. Overcoming the need to thaw the solutions shortens sample prep time and will yield more consistent results.

Problems solved by technology

Which if done incorrectly can yield non-homogenous thawing of the product and thus variation in efficiencies of each step in the sample preparation workflow.

Stock liquid solutions also increase the probability of cross contamination.

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