COMPOSITIONS AND METHODS FOR HIGHLY SENSITIVE DETECTION OF TARGET SEQUENCES IN MULTIPLEX REACTIONS

    公开(公告)号:US20230374574A1

    公开(公告)日:2023-11-23

    申请号:US18340581

    申请日:2023-06-23

    CPC classification number: C12Q1/6858

    Abstract: Efficient methods for production of targeted libraries from complex samples is desirable for a variety of nucleic acid analyses. Provided are methods of selectively blocking abundant targets present in a sample for preparing libraries of target nucleic acid sequences, thereby allowing for rapid production of highly multiplexed targeted libraries and analysis of low frequency sequences, including sequencing applications. Methods optionally include use of unique tag sequences. Methods comprise contacting a nucleic acid sample with a plurality of target specific primers or adapters capable of amplification of one or more target nucleic acid sequences under conditions wherein the target nucleic acid(s) undergo a first amplification; digesting the resulting first amplification products; ligating the digested target amplicons or repairing the digested target amplicons; and amplifying the ligated or repaired products in a second amplification, thereby producing a library of target nucleic acid sequence. Each of the reactions further comprise target specific primers that are not capable of completely processing the workflow, resulting in non-useful amplicon production and thereby blocking selected target sequences, e.g., those present in high abundance in the sample. Provided methods may be carried out in a single, addition only workflow reaction, allowing for rapid production of highly multiplexed targeted libraries, optionally including unique tag sequences, which are optimized for detection of low frequency target sequences.

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