US Pat. No. 9,422,547

RECOMBINANT FUSION PROTEINS AND LIBRARIES FROM IMMUNE CELL REPERTOIRES

GigaGen, Inc., South San...

1. A method for preparing a recombinant immunoglobulin library, comprising:
providing primary immune cells from at least one mammalian donor;
isolating in a plurality of monodisperse droplets single immune cells from said primary immune cells;
generating a plurality of recombinant fusion polynucleotides each comprising a first polynucleotide encoding a heavy chain
variable domain and a second polynucleotide encoding a light chain variable domain, wherein said heavy chain variable domain
and light chain variable domain on each of said plurality of recombinant fusion polynucleotides are a cognate pair from one
of said isolated primary immune cells, wherein each of said plurality of recombinant fusion polynucleotides further comprise
a linker polynucleotide linking said first and second polynucleotides;

circularizing each of said plurality of linear recombinant fusion polynucleotides; and
inserting a third polynucleotide comprising a sequence encoding a promoter and a sequence encoding a constant region between
said first and second polynucleotide in each of said circularized recombinant fusion polynucleotides, thereby generating at
least 1,000 unique recombinant immunoglobulin expression constructs, thereby generating a recombinant immunoglobulin library.

US Pat. No. 9,695,474

SYSTEM AND METHODS FOR MASSIVELY PARALLEL ANALYSIS OF NUCLEIC ACIDS IN SINGLE CELLS

GigaGen, Inc., South San...

1. A method for analyzing at least two nucleic acid sequences in a single cell contained within a population of at least 10,000
cells, comprising:
isolating each of a plurality of single cells from the population of at least 10,000 cells in an emulsion microdroplet or
a reaction container;

introducing a unique barcode sequence affixed to a bead or a solid surface into the emulsion microdroplet or the reaction
container, wherein the unique barcode sequence comprises at least six nucleotides and wherein the unique barcode sequence
is selected from a pool of barcode sequences with greater than 1000-fold diversity in sequence;

for each of the plurality of single cells,
providing at least one set of nucleic acid probes, the set comprising (i) a first probe comprising a sequence that is complementary
to a nucleic acid sequence that is located at the 5? end of the unique barcode sequence, (ii) a second probe comprising a
sequence that is complementary to a nucleic acid sequence that is located at the 3? end of the barcode sequence and a second
region of sequence that is complementary to a non-human, exogenous sequence, (iii) a third probe comprising a sequence that
comprises the non-human, exogenous sequence and a sequence that is complementary to a first subsequence of a target nucleic
acid sequence, and (iv) a fourth probe comprising a sequence that is complementary to a second subsequence of the target nucleic
acid sequence;

amplifying the unique barcode sequence and the target nucleic acid sequence independently, wherein the unique barcode sequence
is amplified using the first probe and the second probe, and wherein the target nucleic acid sequence is amplified using the
third probe and the fourth probe;

hybridizing the non-human exogenous sequence to its complement;
amplifying the unique barcode sequence, the target nucleic acid sequence, and the non-human exogenous sequence using the first
probe and the fourth probe, thereby generating fused complexes;

performing bulk sequencing of the fused complexes; and
identifying a single cell for each of the fused complexes based on the unique barcode sequence.

US Pat. No. 9,738,699

RECOMBINANT FUSION PROTEINS AND LIBRARIES FROM IMMUNE CELL REPERTOIRES

GigaGen, Inc., South San...

1. A method for preparing a recombinant T cell receptor library, comprising:
providing primary immune cells from at least one mammalian donor;
isolating in a plurality of monodisperse droplets single immune cells from said primary immune cells;
generating a plurality of recombinant fusion polynucleotides each comprising a first polynucleotide encoding a beta chain
variable domain and a second polynucleotide encoding an alpha chain variable domain, wherein said beta chain variable domain
and alpha chain variable domain on each of said plurality of recombinant fusion polynucleotides are a cognate pair from one
of said isolated primary immune cells, wherein each of said plurality of recombinant fusion polynucleotides further comprise
a linker polynucleotide linking said first and second polynucleotides;

circularizing each of said plurality of linear recombinant fusion polynucleotides; and
inserting a third polynucleotide comprising a sequence encoding a promoter, a sequence encoding an internal ribosome entry
site, or a sequence encoding a constant region between said first and second polynucleotide in each of said circularized recombinant
fusion polynucleotides, thereby generating at least 1,000 unique recombinant T cell receptor expression constructs, thereby
generating said recombinant T cell receptor library.

US Pat. No. 10,106,789

SYSTEM AND METHODS FOR MASSIVELY PARALLEL ANALYSIS OF NUCLEIC ACIDS IN SINGLE CELLS

GigaGen, Inc., South San...

1. A method for creating a library of polynucleotides, comprising the steps of:introducing multiple sets of initial probes into a plurality of compartments under conditions selected such that more than a half of the plurality of compartments contain one or less than one set of the initial probes, wherein each set of the initial probes comprises (1) an initial forward probe, wherein the initial forward probe (i) is affixed to a bead or a solid surface, and (ii) comprises a sequence complementary to a first subsequence of a first target sequence and one of at least 1,000 unique barcode sequences, and (2) an initial reverse probe, wherein the initial reverse probe comprises (i) a sequence complementary to a second subsequence of the first target sequence and (ii) a sequence complementary to a non-human, exogenous sequence;
introducing multiple sets of second probes into the plurality of compartments, wherein each set of the second probes comprises (1) a second forward probe, wherein the second forward probe comprises (i) the non-human, exogenous sequence and (ii) a sequence that is complementary to a first subsequence of a second target sequence, and (2) a second reverse probe comprising a sequence that is complementary to a second subsequence to the second target sequence;
amplifying the first target sequence using the multiple sets of the initial probes;
amplifying the second target sequence using the multiple sets of the second probes;
hybridizing the non-human exogenous sequence to its complement; and
amplifying a fused sequence comprising one of the at least 1,000 barcode sequences, the first target sequence and the second target sequence, thereby generating a library of fused polynucleotides, wherein each of the fused polynucleotides comprises one of the at least 1,000 unique barcode sequences.

US Pat. No. 10,214,740

RECOMBINANT FUSION PROTEINS AND LIBRARIES FROM IMMUNE CELL REPERTOIRES

GigaGen, Inc., South San...

1. A method of generating a recombinant immunoglobulin library, comprising:mixing RNA transcripts from a single isolated cell with a first primer and a second primer, wherein each of the first primer and the second primer is attached to a bead;
isolating RNA transcripts bound to the first primer or the second primer;
generating from the isolated RNA transcripts, (i) a first polynucleotide comprising a region encoding a first variable domain from the single isolated cell, and (ii) a second polynucleotide comprising a region encoding a second variable domain from the single isolated cell;
generating a plurality of circularized polynucleotide constructs, each comprising the first polynucleotide, the second polynucleotide, and a linker polynucleotide linking the first polynucleotide and the second polynucleotide, wherein each of said plurality of circularized polynucleotide constructs comprises a cognate pair of linked first and second variable domains from said single isolated cell;
inserting a third polynucleotide comprising a sequence encoding a promoter and a sequence encoding a constant region between said first polynucleotide and said second polynucleotide in each of said plurality of circularized polynucleotide constructs, thereby generating a plurality of recombinant immunoglobulin expression constructs;
inserting said plurality of recombinant immunoglobulin expression constructs into a plurality of host cells; and
expressing said plurality of recombinant immunoglobulin expression constructs in said plurality of host cells, thereby generating the recombinant immunoglobulin library comprising a plurality of recombinant immunoglobulins, wherein each of said plurality of recombinant immunoglobulins comprises a linked heavy chain variable domain and a light chain variable domain which are a cognate pair from a single cell from a mammalian donor.