Methods are disclosed for adding adapters to fragmented nucleic acids for next generation sequencing, including providing numerical codes based on variable adapter molecular barcode lengths on both sides of the fragmented nucleic acids and identifying reads from the same fragment based on both barcodes. The methods and products allow for the amplification of the fragmented nucleic acids when there is a low yield of isolated fragmented nucleic acids and also for efficient and reliable detection of low-frequency mutations including in subpopulations of cells within a subject.

Methods are disclosed for adding adapters to fragmented nucleic acids for next generation sequencing, including providing numerical codes based on variable adapter molecular barcode lengths on both sides of the fragmented nucleic acids and identifying reads from the same fragment based on both barcodes. The methods and products allow for the amplification of the fragmented nucleic acids when there is a low yield of isolated fragmented nucleic acids and also for efficient and reliable detection of low-frequency mutations including in subpopulations of cells within a subject.

The present disclosure provides methods for determining oligonucleotide purity and/or characterizing small RNAs. The methods comprising ligating adapters comprising unique molecule identifiers (UMIs), amplifying ligation products to generate a library, and sequencing the library. The methods of the disclosure exhibit reduced or no bias in terms of discrepancies that can arise during the ligation and/or amplification steps of the methods.

The present invention provides methods and systems for encoding and decoding of synthesis steps and conditions of combinatorial synthesis of molecular library on carrier-beads. The encoding is performed at each step of synthesis by attachment of smaller fluorescently labelled beads (label-beads) to the surface of a carrier-bead (carrier-bead). The number of label-beads should be such that each is spatially resolvable on a surface of the carrier-bead. Alternatively label-beads are detachable, or the carrier-bead are dissolvable, so the label-beads could be dispersed over large enough distance to be resolved spatially. The fluorescent spectrum of each of the label-beads carries information of the synthesis step and synthesis, i.e., a spectral barcode or binary encoding system. During decoding of the spectrally identified label-beads, a fluorescent spectrum of each spatially resolvable label-bead is determined.

Provided herein are methods and composition for trackable genetic variant libraries. Further provided herein are methods and compositions for recursive engineering. Further provided herein are methods and compositions for multiplex engineering. Further provided herein are methods and compositions for enriching for editing and trackable engineered sequences and cells using nucleic acid-guided nucleases.

Aspects of the invention include methods for preparing sequencing libraries, performing sequencing procedures that can correct for process-related errors, and identifying rare variants that are or may be indicative of cancer.

Provided are methods and compositions for generating a deletion library, and methods and compositions for generating and identifying a defective interfering particle (DIP). Also provided are transposon cassettes. A subject method can include: inserting a transposon cassette comprising a target sequence for a sequence specific DNA endonuclease into a population of circular target DNAs to generate a population of transposon-inserted circular target DNAs; contacting the population of transposon-inserted circular target DNAs with the sequence specific DNA endonuclease to generate a population of cleaved linear target DNAs; contacting the population of cleaved linear target DNAs with one or more exonucleases to generate a population of deletion DNAs; and circularizing the deletion DNAs to generate a library of circularized deletion DNAs. The population of circular target DNAs can include viral genomic DNA. Also provided are human immunodeficiency virus (HIV) deletion mutants, e.g., interfering, conditionally replicating, HIV deletion mutants, and related constructs.

The technology relates in part to methods and compositions for analyzing nucleic acid. In some aspects, the technology relates to methods and compositions for preparing a nucleic acid library. In some aspects, the technology relates to methods and compositions for analyzing ends of nucleic acid fragments.

The present disclosure provides methods, systems, and kits for processing nucleic acid molecules. A method may comprise providing a template nucleic acid fragment (e.g., within a cell, cell bead, or cell nucleus) within a partition (e.g., a droplet or well) and subjecting the template nucleic acid fragment to one or more processes including a barcoding process and a single primer extension or amplification process. The processed template nucleic acid fragment may then be recovered from the partition and subjected to further amplification to provide material for subsequent sequencing analysis. The methods provided herein may permit simultaneous processing and analysis of both DNA and RNA molecules originating from the same cell, cell bead, or cell nucleus.

A method for identifying a sub-population within a mixed population of cells is disclosed. The method involves contacting the mixed population of cells with at least one unique binding agent, wherein the at least one unique binding agent is designed to bind to a target molecule present in the sub-population, and wherein the at least one unique binding agent is attached to an epitope specific barcode that represents the identity of the target molecule. The method further involves sequentially attaching two or more assayable polymer subunits to the epitope specific barcode to create unique cell origination barcodes that represent the identities of individual cells to which the at least one unique binding agent has bound; and decoding the epitope specific barcode and cell origination barcodes, thereby identifying the sub-population within the mixed population of cells.