Abstract: Disclosed are methods of targeted sequencing by using gRNA-endonuclease complexes and methods of designing pools of gRNAs. The disclosure also provides sequencing adapters that that comprise a double-stranded nucleic acid having a single-stranded overhang with degenerate overhanging bases. In a first aspect, methods for Dephosphorylate targeted sequencing of double-stranded nucleic acids comprises cleaving dephosphorylated double-stranded nucleic acids with a plurality of endonuclease-guide ribonucleic acid (gRNA) complexes to generate double-stranded nucleic acid fragments having phosphorylated 5? end overhangs at targeted sites.

Abstract: The present disclosure is concerned with compositions, methods, and kits for preparing a sequencing library. In one embodiment, methods include producing a library of target nucleic acids having the same adapter at each end and then switching the identity of one adapter to result in target nucleic acids flanked by distinct adapters.

Abstract: Provided herein are methods for preparing sequencing libraries for determining the methylation status of nucleic acids from a plurality of single cells. The present methods combine split-and-pool combinatorial indexing and bisulfite treatment techniques to characterize the methylation profiles of large numbers of single cells quickly, accurately and inexpensively.