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Compositions and methods for negative selection of non-desired nucleic acid sequences

一种核酸序列、非期望的技术，应用在生物化学设备和方法、组合化学、重组DNA技术等方向，能够解决样品群体失真、不能用、丢失等问题

Active Publication Date: 2015-05-13
NUGEN TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First, it cannot be used in prokaryotes because prokaryotic mRNA does not contain a poly(A)-sequence at its 3' end
Second, even for eukaryotic RNA samples, many biologically important elements, such as regulatory transcripts, are not polyadenylated and thus lost from oligo-dT-primed libraries
Although NSR priming strategies may be effective when designed for specific organisms, NSR priming can introduce distortions in sample populations when used across a wider range of sample types with an underoptimized set of primers

Method used

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  • Compositions and methods for negative selection of non-desired nucleic acid sequences
  • Compositions and methods for negative selection of non-desired nucleic acid sequences
  • Compositions and methods for negative selection of non-desired nucleic acid sequences

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0219] Example 1 - Exclusion of bacterial ribosomal RNA fragments from directed (ie, strand-specific) whole-transcriptome libraries.

[0220] This example describes the exclusion of four directed cDNA libraries generated from total RNA from E. Bacterial rRNA fragments.

[0221] Probe Design and Synthesis

[0222]By comparing ribosomal operons from a phylogenetically distinct set of 40 bacterial strains and 10 archaeal strains using the ClustalW multiple sequence alignment program (European Bioinformatics Institute) InDA-C probe for prokaryotic rRNA transcripts. Candidate primer sequences were first selected from highly conserved sequences identified in the 16S rRNA (9 sites) and 23S rRNA (7 sites) subunits. These conserved regions were computationally fragmented and analyzed by Primer3 (Steve Rozen and Helen J. Skaletsky (2000), Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds.) Bioinformatics Methods and Protocols: Method...

Embodiment 2

[0228] Example 2 - Exclusion of Mitochondrial DNA Fragments from Genomic DNA Libraries.

[0229] This example describes the exclusion of mitochondrial DNA fragments from genomic DNA libraries using an Insert-Dependent Adapter Cleavage (InDA-C) probe targeting the mitochondrial genome.

[0230] Probe Design and Synthesis

[0231] InDA-C was selected for annealing to both strands of the hg19 version of the human mitochondrial genomic sequence within mitochondria-specific segments identified by the "Duke 20bp uniqueness" tracks provided by the UCSC Genome Browser probe. These sequences were then screened for the best predicted melting temperature and length. Oligonucleotides ranging in length from 20-25 nt were synthesized individually and combined in equimolar ratios. The resulting probe mix was diluted to 25 times the final concentration used in the InDA-C exclusion reaction (375 nM for each species, 15 nM final).

[0232] Genomic DNA library generation

[0233] DNA li...

Embodiment 3

[0236] Example 3 - Generation of Directed cDNA Libraries ( Figure 5 ).

[0237] This example describes the generation of a directional cDNA library using conventional blunt-end ligation starting with modified duplex adapters and 50 ng of poly(A)+ selected messenger RNA.

[0238] first strand synthesis

[0239] First-strand cDNA was generated using random hexamer priming. Use containing 10μM random hexamers, 3.0mM MgCl 2 The first-strand synthesis reaction was performed with Invitrogen SuperScript III Reverse Transcriptase Kit with 1.0 mM dNTP. cDNA synthesis reactions were performed in 10 μL volumes, incubated at 40°C for 60 min and cooled to 4°C.

[0240] Second strand synthesis utilizing dUTP incorporation

[0241] Second strand synthesis was performed using the New England Biolabs NEBNext Second Strand Synthesis Module in which the second strand synthesis (dNTP-free) reaction buffer was supplemented with a dNTP mix containing 0.2 mM dATP, dCTP and dGTP and 0.54 mM d...

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Abstract

The present invention provides methods, compositions and kits for the generation of next generation sequencing (NGS) libraries in which non-desired nucleic acid sequences have been depleted or substantially reduced. The methods, compositions and kits provided herein are useful, for example, for the production of libraries from total RNA with reduced ribosomal RNA and for the reduction of common mRNA species in expression profiling from mixed samples where the mRNAs of interest are present at low levels. The methods of the invention can be employed for the elimination of non-desired nucleic acid sequences in a sequence-specific manner, and consequently, for the enrichment of nucleic acid sequences of interest in a nucleic acid library.

Description

[0001] cross reference [0002] This application claims priority to US Provisional Patent Application Serial No. 61 / 661,293, filed June 18, 2012, which is hereby incorporated by reference in its entirety. Background technique [0003] A next-generation sequencing (NGS) library is a collection of DNA fragments whose nucleotide sequences are to be determined. The source of DNA for insertion into these libraries is typically either genomic DNA that has been fragmented to the desired length or a copy of the transcriptome from a given cell population. Transcriptome libraries are generated by making cDNA copies of the RNA population, generating the complements of each DNA strand to generate double-stranded DNA, which is then ligated to library-specific adapters. cDNA can be synthesized by priming a population of polyadenylated transcripts using random primers, sequence-specific primers, or primers containing an oligo-dT tail. Frequently, these fragment populations contain DNA tha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06
CPCC12Q1/6806C12N15/1093C12N15/1096C12Q1/6844C12Q2525/131C12Q2525/191C12Q2521/301C12Q2521/531C12Q2537/159
Inventor 克里斯多佛·阿穆尔道格·阿莫莱赛李斌努里斯·库恩
Owner NUGEN TECH
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