Disclosed are an inline index based on Illumina sequencing, a DNA library labeled thereby and a method of constructing the same, where the method includes the steps of: breaking DNA sequence of a sample; repairing a flat end; ligating an inline index adapter to an end of the sequence; repairing a gap of the sequence and extending the sequence; and subjecting the sequence to PCR amplification to construct the DNA library. The inline index is a 6-bp DNA sequence free of ‘AAA’, ‘ACA’, ‘CCC’, ‘CAC’, ‘GGG’, ‘GTG’, ‘TTT’ and ‘TGT’. In addition, the inline index also has a minimum editing length equal to or less than 3, and includes bases of different laser colors, where the laser colors of adjacent bases are different.

The invention relates to a method for selecting a sequence set from a library of expressed nucleic acid sequences, wherein cells are provided, each cell comprises an expressed nucleic acid sequence expressed as a target protein. The cells are encapsulated by treating them with a cationic polysaccharide and subsequently treating them with an anionic polysaccharide, yielding encapsulated cells, perforating the membrane of the encapsulated cells, yielding solubilized compartments, contacting them with a ligand to said target protein, the ligand bearing a detectable label, and selecting a subset of solubilized compartments as a function of detectable label and isolating the expressed nucleic acid sequences from the selection as a selected sequence set.

The present invention concerns preparation of DNA molecules, such as a library, using a stem-loop oligonucleotide. In particular embodiments, the invention employs a single reaction mixture and conditions. In particular, at least part of the inverted palindrome is removed during the preparation of the molecules to facilitate amplification of the molecules. Thus, in specific embodiments, the DNA molecules are suitable for amplification and are not hindered by the presence of the palindrome.

Provided is an array including a solid support having a surface, the surface having a plurality of wells, the wells containing a gel material, the wells being separated from each other by interstitial regions on the surface, the interstitial regions segregating the gel material in each of the wells from the gel material in other wells of the plurality; and a library of target nucleic acids in the gel material, wherein the gel material in each of the wells comprises a single species of the target nucleic acids of the library. Methods for making and using the array are also provided.

Provided herein are methods of selective screening. In addition, various targeting proteins and sequences, as well as methods of their use, are also provided.

A method for identification of the most abundant oligonucleotide species in a library of oligonucleotides comprising more than 10.sup.6 oligonucleotide species, wherein the oligonucleotides of the library of oligonucleotides are not inherently capable of exponential amplification by PCR and include a coding sequence, which is characteristic of one oligonucleotide specie of the library of oligonucleotides and only one fixed sequence, which is present in a plurality of oligonucleotide species of the library of oligonucleotides, said fixed sequence being located on a 5′ side of the coding sequence, and wherein the method comprises specifically amplifying the sequence of a hybridised oligonucleotide species using the steps of: incubating the library of oligonucleotides under conditions of hybridization such as to allow complementary coding sequences to hybridize and form hybridized oligonucleotide species; extending a 3′ end of one or more of the hybridised oligonucleotide species, having only one fixed sequence, to provide extended sequences having an extended region, such that the extended region generates a second fixed sequence thereby forming extended sequences having two fixed sequences; amplifying extended sequences comprising two fixed sequences using PCR to provide amplified sequences, and optionally identifying an identity of the amplified sequences thereby revealing an identity of the hybridised oligonucleotide species in the step of incubating. The library of oligonucleotides is selected from the group of a library of encoded molecules, a library of aptamers, a library of reporter sequences derived from a library of encoded molecules, and a library of aptamers.

Non-naturally occurring polypeptides are disclosed that include (a) 3-3 secondary structure elements, wherein each secondary structure element is either an α-helix (H domain) of between 10-20 amino acid residues in length or a β-strand (E domain) of between 3-10 amino acid residues in length; and (b) 2-4 linkers of between 2 to 6 amino acid residues in length connecting adjacent secondary structure elements; wherein the polypeptide is between 25-50 amino acid residues in length; and wherein the polypeptide includes no cysteine residues.

The present disclosure provides ultrahigh-throughput single cell genomic sequencing methods, referred to herein as “SiC-seq”, which methods include encapsulating single cells in molten gel droplets to facilitate bulk cell lysis and purification of genomic DNA in microgels. Systems and devices for practicing the subject methods are also provided.

The disclosure provides methods and reagents for preparing DNA libraries from biological materials for targeted sequencing. The approach can enhance the efficiency and sensitivity of targeted sequencing applications, such as liquid biopsy analyses to assess genetically driven conditions. In an embodiment, the disclosed method comprises attaching the 5′ end of an oligonucleotide adapter to the 3′ end of double-stranded DNA fragment to produce an adapter/fragment chimeric molecule; producing at least one complementary strand of the adapter/fragment chimeric molecule by linear amplification; hybridizing the at least one complementary strand with an oligonucleotide probe, wherein the oligonucleotide probe comprises a hybridization domain with a sequence that hybridizes to a target sequence in the complement strand to produce a targeted complement strand/probe duplex; purifying the targeted complement strand/probe duplex; and extending the probe in the purified targeted complement strand/probe duplex and amplifying with PCR to produce a plurality of sequencing template molecules.

Disclosed herein is an efficient method of generating a library of variants of a sequence of interest, such as may be used in directed evolution, in one embodiment, the method includes an amplification reaction, e.g. error-prone PCR, to generate double-stranded DNA (dsDNA) variants of a sequence of interest, after which one strand of the dsDNA variants may be selectively degraded to produce single-stranded DNA (ssDNA) variants. The ssDNA variants may be hybridized to ssDNA intermediaries, e.g., uracilated circular ssDNA intermediaries, to form heteroduplex DNA, which may be transformed into cells, such as E. coli cells, yielding a library of variants. This method eliminates the inefficient sub-cloning steps and the need for costly primer sets required by many prior methods.