Disclosed herein is a novel method for multisite saturation mutagenesis. Provided in the instant disclosure is a means of generating genetic diversity at each desired codon position within the coding sequence of a gene using a multi-oligonucleotide primer pool and Gibson assembly approach where a mutant DNA library can be cloned into a target vector.

Cancer immunology provides promising new avenues for cancer treatment but validation of potential neoantigens to target is costly and expensive. Analysis of MHC binding affinity, antigen processing, similarity to known antigens, predicted expression levels (as mRNA or proteins), self-similarity, and mutant allele frequency, provides screening method to identify and prioritize candidate neoantigens using sequencing data. Methods of the invention thereby save time and money by identifying the priority candidate neoantigens for further experimental validation.

This invention provides sets and libraries of short hairpin ribonucleic acid (shRNA) molecules comprising a double-stranded region of random sequence containing random mismatches, methods of generating same, sets and libraries of expression vectors for same, methods of generating same, and methods for identifying an RNA therapeutic or RNA molecule that has an ability to affect a biological parameter, for identifying a drug target for a disease or disorder of interest, and for identifying a variant of an RNA molecule that has an altered ability to affect a biological parameter of interest.

A method of diagnosing severe sepsis prior to definitive clinical diagnosis. A pattern of gene expression that correlates strongly with a future diagnosis of severe sepsis and organ failure was identified in patients who had their blood drawn at first clinical presentation. The methods comprise identifying a pattern of two or more polynucleotides, whereby the altered expression of these polynucleotides correlates with prospective and actual sepsis. Also methods of identifying agents for treating sepsis based on the characteristics of this gene expression pattern are provided.

Aspects of the invention relate to methods, compositions for synthesizing oligonucleotides having a predefined sequence.

Presented herein are transposase enzymes and reaction conditions for improved fragmentation and tagging of nucleic acid samples, in particular altered transposases and reaction conditions which exhibit improved insertion sequence bias, as well as methods and kits using the same.

A multi-position double-tag connector set for detecting gene mutation and preparation method therefor and application thereof, the multi-position double-tag connector set comprising a double-tag connector A, a double-tag connector B and a double-tag connector C. The double-tag connector A, the double-tag connector B and the double-tag connector C are obtained respectively by means of synthesizing a connector primer P5 with a connector primer P7-A, a connector primer P7-B and a connector primer P7-C of 5 ends which are all modified with biotin. Using the multi-position double-tag connector set, the mutation rate of 110.sup.5 genes may be accurately detected and the sensitivity of gene mutation detection may be effectively improved. A plurality of mutation sites of a plurality of genes may be detected by one-time sequencing in combination with throughput of high-throughput sequencing.

Provided are compositions, methods and systems for determining the sequence of a template nucleic acid using a polymerase-based, sequencing-by-binding procedure. An examination step involves monitoring the interaction between a polymerase and template nucleic acid in the presence of one or more nucleotides. Identity of the next correct nucleotide in the sequence is determined without incorporation of any nucleotide into the structure of the primer by formation of a phosphodiester bond. An optional incorporation step can be used after the examination step to extend the primer by one or more nucleotides, thereby incrementing the template nucleotides that can be examined in a subsequent examination step. The sequencing-by-binding procedure does not require the use of labeled nucleotides or polymerases, but optionally can employ these reagents.

This application provides a bead with a covalently attached chemical compound and a covalently attached DNA barcode and methods for using such beads. The bead has many substantially identical copies of the chemical compound and many substantially identical copies of the DNA barcode. The compound consists of one or more chemical monomers, where the DNA barcode takes the form of barcode modules, where each module corresponds to and allows identification of a corresponding chemical monomer. The nucleic acid barcode can have a concatenated structure or an orthogonal structure. Provided are method for sequencing the bead-bound nucleic acid barcode, for cleaving the compound from the bead, and for assessing biological activity of the released compound.

Provided are methods for characterizing nucleotide accessibility and nucleic acid structure at single nucleotide resolution, using a combination of low-yield bisulfite conversion and next-generation sequencing (NGS). Cytosine (C) nucleotides that are in base-paired states or bound to proteins, etc. are less accessible to chemical reactions and thus exhibit lower bisulfite conversion yields. Analysis of NGS results of a low-yield bisulfite conversion product can thus inform nucleotide accessibility and nucleic acid structure. Compared to other methods for chemical probing of nucleic acid structure, the present methods provide higher information throughput, because each NGS read simultaneously reports on the base pair status of multiple nucleotides.