U.S. patent application number 15/633871 was filed with the patent office on 2018-02-15 for high throughput sequencing of multiple transcripts.
The applicant listed for this patent is BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM. Invention is credited to Brandon DEKOSKY, Andy ELLINGTON, George GEORGIOU, Scott HUNICKE-SMITH.
Application Number | 20180044726 15/633871 |
Document ID | / |
Family ID | 48741552 |
Filed Date | 2018-02-15 |
United States Patent
Application |
20180044726 |
Kind Code |
A1 |
HUNICKE-SMITH; Scott ; et
al. |
February 15, 2018 |
HIGH THROUGHPUT SEQUENCING OF MULTIPLE TRANSCRIPTS
Abstract
The present disclosure generally relates to sequencing two or
more genes expressed in a single cell in a high-throughput manner.
More particularly, the present disclosure relates to a method for
high-throughput sequencing of pairs of transcripts co-expressed in
single cells (e.g., antibody VH and VL coding sequence) to
determine pairs of polypeptide chains that comprise immune
receptors.
Inventors: |
HUNICKE-SMITH; Scott;
(Austin, TX) ; DEKOSKY; Brandon; (Austin, TX)
; ELLINGTON; Andy; (Austin, TX) ; GEORGIOU;
George; (Austin, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM |
Austin |
TX |
US |
|
|
Family ID: |
48741552 |
Appl. No.: |
15/633871 |
Filed: |
June 27, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14407849 |
Dec 12, 2014 |
9708654 |
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PCT/US2013/046130 |
Jun 17, 2013 |
|
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15633871 |
|
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61660370 |
Jun 15, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6869 20130101;
C12N 15/1075 20130101; B01L 2200/0647 20130101; C12Q 1/6806
20130101; B01L 2200/0673 20130101; B01L 3/502784 20130101; C12Q
1/6806 20130101; C12Q 2535/122 20130101; C12Q 2563/149 20130101;
C12Q 2563/159 20130101; C12Q 1/6869 20130101; C12Q 2535/122
20130101; C12Q 2547/101 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; B01L 3/00 20060101 B01L003/00; C12N 15/10 20060101
C12N015/10 |
Claims
1. A method comprising: a) sequestering single cells and an mRNA
capture agent into individual compartments; b) lysing the cells and
collecting mRNA transcripts with the mRNA capture agent; c)
isolating the mRNA from the compartments using the mRNA capture
agent; d) performing reverse transcription followed by PCR
amplification on the captured mRNA; and e) sequencing at least two
distinct cDNA products amplified from a single cell.
2. The method of claim 1, further defined as a method for obtaining
a plurality of paired antibody VH and VL sequences wherein the
cells are B-cells.
3. The method of claim 1, wherein the mRNA capture agent is a
bead.
4. The method of claim 3, wherein the beads are magnetic.
5. The method of claim 3, wherein the bead comprises
oligonucleotides which hybridize mRNA.
6. The method of claim 5, wherein the oligonucleotides comprise at
least one of poly(T) and primers specific to a transcript of
interest.
7. The method of claim 2, further defined as a method for obtaining
paired antibody VH and VL sequences for an antibody that binds to
an antigen of interest.
8. The method of claim 3, wherein the beads are conjugated to the
antigen of interest and the oligonucleotides are only conjugated to
the beads in the presence of an antibody that binds to the antigen
of interest.
9-13. (canceled)
14. The method of claim 1, wherein steps (a) and (b) comprise
isolating single cells and an mRNA capture agent into individual
microvesicles in an emulsion and in the presence of a cell lysis
solution.
15-16. (canceled)
17. The method of claim 1, wherein step (e) comprises linking cDNA
by performing overlap extension reverse transcriptase polymerase
chain reaction to link at least 2 transcripts into a single DNA
molecule.
18. The method of claim 1, wherein step (e) does not comprise the
use of overlap extension reverse transcriptase polymerase chain
reaction.
19. The method of claim 2, wherein step (e) comprises linking VH
and VL cDNAs by performing overlap extension reverse transcriptase
polymerase chain reaction to link VH and VL cDNAs in single
molecules.
20. The method of claim 2, wherein step (e) does not comprise the
use of overlap extension reverse transcriptase polymerase chain
reaction and wherein the VH and VL cDNAs are separate
molecules.
21. The method of claim 2, wherein the VH and VL sequences are
obtained by sequencing of distinct molecules.
22. The method of claim 2, further comprising identifying the
paired antibody VH and VL sequences comprises performing a
probability analysis of the sequences.
23-27. (canceled)
28. The method of claim 1, wherein sequestering the single cells
comprises introducing the cells to a device comprising a plurality
of microwells so that the majority of cells are captured as single
cells.
29. The method of claim 1, wherein step (e) comprises sequencing of
two or more transcripts covalently linked to the same bead.
30. (canceled)
31. The method of claim 1, further comprising determining natively
paired transcripts using probability analysis.
32-33. (canceled)
34. A system comprising: a) an aqueous fluid phase exit disposed
within an annular flowing oil phase; and b) an aqueous fluid phase,
wherein the aqueous phase fluid comprises a suspension of cells and
is dispersed within the flowing oil phase, resulting in emulsified
droplets with low size dispersity comprising an aqueous suspension
of cells.
35-43. (canceled)
44. A composition comprising an emulsion having a plurality of
individual microvesicles, said microvesicles comprising a bead with
immobilized oligonucleotides for priming of reverse transcription
and individual B-cells, which have been disrupted to release mRNA
transcripts from individual B-cells.
45-64. (canceled)
Description
[0001] The present application is a continuation of U.S.
application Ser. No.14/407,849, filed Dec. 12, 2014, as a national
phase application under 35 U.S.C. .sctn. 371 of International
Application No. PCT/US2013/046130, filed Jun. 17, 2013, which
claims the priority benefit of U.S. provisional application No.
61/660,370, filed Jun. 15, 2012, the entire contents of each of
which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0002] The present invention relates generally to the field of
molecular biology and immunology. More particularly, it concerns
methods for high-throughput isolation cDNAs encoding immune cell
receptors and antibodies.
2. Description of Related Art
[0003] There is a need to identify the expression of two or more
transcripts from individual cells at high throughput. In
particular, for numerous biotechnology and medical applications it
is important to identify and sequence the gene pairs encoding the
two chains comprising adaptive immune receptors from individual
cells at a very high throughput in order to accurately determine
the complete repertoires of immune receptors expressed in patients
or in laboratory animals. Immune receptors expressed by B and T
lymphocytes are encoded respectively by the VH and VL antibody
genes and by TCR .alpha./.beta. or .gamma./.delta. chain genes.
Humans have many tens of thousands or millions of distinct B and T
lymphocytes classified into different subsets based on the
expression of surface markers (CD proteins) and transcription
factors (e.g., FoxP3 in the Treg T lymphocyte subset).
High-throughput DNA sequencing technologies have been used to
determine the repertoires of VH or VL chains or, alternatively, of
TCR .alpha. and .beta. in lymphocyte subsets of relevance to
particular disease states or, more generally, to study the function
of the adaptive immune system (Wu et al., 2011). Immunology
researchers have an especially great need for high throughput
analysis of multiple transcripts at once.
[0004] Currently available methods for immune repertoire sequencing
involve mRNA isolation from a cell population of interest, e.g.,
memory B-cells or plasma cells from bone marrow, followed by RT-PCR
in bulk to synthesize cDNA for high-throughput DNA sequencing
(Reddy et al., 2010; Krause et al., 2011). However, heavy and light
antibody chains (or .alpha. and .beta. T-cell receptors) are
encoded on separate mRNA strands and must be sequenced separately.
Thus, these available methods have potential to unveil the entire
heavy and light chain immune repertoires individually, but cannot
yet resolve heavy and light chain pairings at high throughput.
Without multiple-transcript analysis at the single-cell level to
collect heavy and light chain pairing data, the full adaptive
immune receptor, which includes both chains, cannot be sequenced or
reconstructed and expressed for further study.
SUMMARY OF THE INVENTION
[0005] In a first embodiment, the present invention provides a
method comprising (a) sequestering single cells and an mRNA capture
agent into individual compartments; (b) lysing the cells and
collecting mRNA transcripts with the mRNA capture agent; (c)
isolating the mRNA from the compartments using the mRNA capture
agent; (d) performing reverse transcription followed by PCR
amplification on the captured mRNA; and (e) sequencing at least two
distinct cDNA products amplified from a single cell. In certain
aspects, the cells may be B cells (e.g., plasma cells or memory B
cells), T cells, NKT cells, and cancer cells.
[0006] Thus, in a specific embodiment, the present invention
provides a method for obtaining a plurality of paired antigen
receptor sequences comprising: (a) isolating single mammalian cells
in individual compartments with immobilized oligonucleotides for
priming of reverse transcription; (b) lysing the cells and allowing
mRNA transcripts to associate with the immobilized
oligonucleotides; (c) performing reverse transcription followed by
PCR amplification to obtain cDNAs corresponding to the mRNA
transcripts from single cells; (d) sequencing the cDNAs; and (e)
identifying multiple mRNA transcripts (e.g., paired antigen
receptor sequences) for a plurality of single cells based on the
sequencing. For example, in some aspects, a method is provided for
obtaining a plurality of paired antibody VH and VL sequences
comprising (a) isolating single B-cells in individual compartments
with immobilized oligonucleotides for priming of reverse
transcription; (b) lysing the B-cells and allowing mRNA transcripts
to associate with the immobilized oligonucleotides; (c) performing
reverse transcription followed by PCR amplification to obtain cDNAs
corresponding to the mRNA transcripts from single B-cells; (d)
sequencing the cDNAs; and (e) identifying the paired antibody VH
and VL sequences for a plurality of single B-cells. In further
aspects, a method is provided for obtaining a plurality of paired
T-cell receptor sequences comprising (a) isolating single T-cells
in individual compartments with immobilized oligonucleotides for
priming of reverse transcription; (b) lysing the T-cells and
allowing mRNA transcripts to associate with the immobilized
oligonucleotides; (c) performing reverse transcription followed by
PCR amplification to obtain cDNAs corresponding to the mRNA
transcripts from single T-cells; (d) sequencing the cDNAs; and (e)
identifying the paired T-cell receptor sequences for a plurality of
single T-cells based on the sequencing.
[0007] In further aspects, the method comprises obtaining sequences
from at least 10,000, 100,000 or 1,000,000 individual cells (e.g.,
between about 100,000 and 10 million or 100 million individual
cells). Thus, in some aspects, a method comprises obtaining at
least 5,000, 10,000 or 100,000 individual paired antibody VH and VL
sequences (e.g., between about 10,000 and 100,000, 1 million or 10
million individual paired sequences). In certain aspect, obtaining
paired sequence, such as VH and VL sequences, may comprise linking
cDNAs (e.g., VH and VL cDNAs) by performing overlap extension
reverse transcriptase polymerase chain reaction to link cDNAs in
single molecules. In an alternative aspect, a method of the
embodiments does not comprise the use of overlap extension reverse
transcriptase polymerase chain reaction. For example, two (or more)
cDNA sequences can be obtained by sequencing of distinct molecules,
such as by sequencing distinct separate VH and VL cDNA
molecules.
[0008] In one aspect, the method may further comprise determining
natively paired transcripts using probability analysis. In this
aspect, identifying the paired transcripts may comprise comparing
raw sequencing read counts. For example, a probability analysis may
comprise performing the steps of FIG. 9. In a specific aspect, a
method may comprise identifying the paired antibody VH and VL
sequences by performing a probability analysis of the sequences. In
certain aspects, the probability analysis may be based on the
CDR-H3 and/or CDR-L3 sequences. In some cases, identifying the
paired antibody VH and VL sequences may comprise comparing raw
sequencing read counts. In a further aspect, the probability
analysis may comprise performing the steps of FIG. 9.
[0009] Certain aspects of the present embodiments concern mRNA
capture agents. For example, the mRNA capture agent can be a solid
support, such as a bead, comprising immobilized oligonucleotides or
polymer networks such as dextran and agarose. In one aspect, the
bead is a silica bead or a magnetic bead. The mRNA capture agent
may comprise oligonucleotides which hybridize mRNA. For example,
the oligonucleotides may comprise at least one poly(T) and/or
primers specific to a transcript of interest. In certain aspects, a
bead of the embodiments is smaller than the individual cells that
being isolated (e.g., B cells).
[0010] In some aspects, individual compartments of the embodiments
may be wells in a gel or microtiter plate. In one aspect, the
individual compartments may have a volume of less than 5 nL. In
some aspects, the wells may be sealed with a permeable membrane
prior to lysis of the cells or prior to performing RT-PCR. In yet a
further aspect, the individual compartments may be microvesicles in
an emulsion.
[0011] In further aspects aspect, sequestering single cells (and an
mRNA capture agent) and lysis of the cells (steps (a) and (b)) may
be performed concurrently. Thus, in some aspects, a method may
comprise isolating single cells and an mRNA capture agents into
individual microvesicles in an emulsion and in the presence of a
cell lysis solution.
[0012] In further aspects, a method of the embodiments may comprise
linking cDNA by performing overlap extension reverse transcriptase
polymerase chain reaction to link at least 2 transcripts into a
single DNA molecule (e.g., in step (e)). In alternative aspects,
step (e) may not comprise the use of overlap extension reverse
transcriptase polymerase chain reaction. In certain aspects, step
(e) may comprise linking cDNA by performing recombination.
[0013] In yet further aspects, sequestering the single cells may
comprise introducing cells to a device comprising a plurality of
microwells so that the majority of cells are captured as single
cells (along with an mRNA capture agent, such as a bead). In
further aspects, a method may comprise sequencing of two or more
transcripts covalently linked to the same bead.
[0014] Thus, in some embodiments, a method is provided for
obtaining a plurality of paired antibody VH and VL sequences
wherein the cells are B-cells. In one aspect, the method is a
method for obtaining paired antibody VH and VL sequences for an
antibody that binds to an antigen of interest. In certain aspects,
the beads may be conjugated to the antigen of interest and the
oligonucleotides only be conjugated to the beads in the presence of
an antibody that binds to the antigen of interest. For example,
beads may be coated with an antigen of interest and the mRNA
capture agent (e.g., oligo-T) may associate with the bead only in
the presence of an antibody that binds to the antigen (see e.g.,
FIG. 10). For instance, the mRNA capture agent may be associated
with protein-A or otherwise functionalized to bind to an antibody
if present.
[0015] Certain aspects of the embodiments may concern obtaining a
sample from a subject (e.g., a sample comprising cells for use in
the methods of the embodiments). Samples can be directly taken from
a subject or can be obtained from a third party. Samples include,
but are not limited to, serum, mucosa (e.g., saliva), lymph, urine,
stool, and solid tissue samples. Similarly, certain aspects of the
embodiments concern biological fluids and antibodies and/or nucleic
acids therefrom. For example, the biological fluid can be blood
(e.g., serum), cerebrospinal fluid, synovial fluid, maternal breast
milk, umbilical cord blood, peritoneal fluid, mucosal secretions,
tears, nasal, secretions, saliva, milk, or genitourinary
secretions. In certain aspects, cells for use according to the
embodiments are mammalian cells, such as mouse, rat or monkey
cells. In preferred aspects the cells are human cells.
[0016] In some aspects, cells for use in the embodiments B cells,
such as B cells from a selected organ, such as bone marrow. For
example, the B cells can be mature B cells, such as bone marrow
plasma cells, spleen plasma cells, or lymph node plasma cells, or
cells from peripheral blood or a lymphoid organ. In certain
aspects, B cells are selected or enriched based on differential
expression of cell surface markers (e.g., Blimp-1, CD138, CXCR4, or
CD45). In some cases, sequences of a selected class of antibodies
are obtained, such as IgE, IgM, IgG, or IgA sequences.
[0017] In further aspects, a method of the embodiments may comprise
immunizing the subject (e.g., prior to obtaining a cell sample).
The method may further comprise isolation of a lymphoid tissue. The
lymphoid tissue isolation may at least or about 1, 2, 3, 4, 5, 6,
6, 8, 9, 10 days or any intermediate ranges after immunization. The
method may further comprise obtaining a population of nucleic acids
of lymphoid tissue, preferably without separating B cells from the
lymphoid tissue. The lymphoid tissue may be a primary, secondary,
or tertiary lymphoid tissue, such as bone marrow, spleen, or lymph
nodes. The subject may be any animal, such as mammal, fish,
amphibian, or bird. The mammal may be human, mouse, primate,
rabbit, sheep, or pig.
[0018] For determining the nucleic acid sequences (e.g., in the B
cells or in lymphoid tissues), any nucleic acid sequencing methods
known in the art may be used, including high-throughput DNA
sequencing. Non-limiting examples of high-throughput sequencing
methods comprise sequencing-by-synthesis (e.g., 454 sequencing),
sequencing-by-ligation, sequencing-by-hybridization, single
molecule DNA sequencing, multiplex polony sequencing, nanopore
sequencing, or a combination thereof.
[0019] In a further embodiment, the present invention provides a
system comprising (a) an aqueous fluid phase exit disposed within
an annular flowing oil phase; and (b) an aqueous fluid phase,
wherein the aqueous phase fluid comprises a suspension of cells and
is dispersed within the flowing oil phase, resulting in emulsified
droplets with low size dispersity comprising an aqueous suspension
of cells. In one aspect, the aqueous fluid phase exit is a needle.
In a further aspect, the aqueous fluid phase exit is a glass tube.
In certain aspects, the oil phase flows through a glass tube or
polymeric tubing. In certain aspects, the aqueous phase flows
through polymeric tubing. In still a further aspect, the
concentration of cells, aqueous fluid phase flow rate, and oil
phase flow rate allow for the formation of droplets, wherein each
droplet contains a single cell. In some aspects, the cells are
selected from the group consisting of: B cells, T cells, NKT cells,
and cancer cells. In certain aspects, the aqueous fluid phase
comprises beads for nucleic acid capture reverse transcription
reagents, polymerase chain reaction reagents, and/or combinations
thereof.
[0020] In yet a further embodiment, the present invention provides
a composition comprising (a) a bead; (b) an oligonucleotide capable
of binding mRNA; and (c) two or more primers specific for a
transcript of interest.
[0021] In still a further embodiments embodiment, the present
invention provides a composition comprising an emulsion having a
plurality of individual microvesicles, said microvesicles
comprising a bead with immobilized oligonucleotides for priming of
reverse transcription and individual B-cells, which have been
disrupted to release mRNA transcripts.
[0022] In certain embodiments, the present invention provides a
method comprising (a) adding a common sequence to the 5' region of
two or more oligonucleotides that are specific to a set of gene
targets; (b) performing nucleic acid amplification of the set of
gene targets by priming the common sequence; and (c) including in
the nucleic acid amplification oligonucleotides comprising the
common sequence immobilized onto a surface such that immobilized
oligonucleotides prime nucleic acid amplification, and resulting in
surface capture of amplified sequences.
[0023] As used herein the specification, "a" or "an" may mean one
or more. As used herein in the claim(s), when used in conjunction
with the word "comprising", the words "a" or "an" may mean one or
more than one.
[0024] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or." As used herein "another" may mean at least a second or
more.
[0025] Throughout this application, the term "about" is used to
indicate that a value includes the inherent variation of error for
the device, the method being employed to determine the value, or
the variation that exists among the study subjects.
[0026] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0028] FIG. 1 shows cells isolated into individual sealed wells.
The small, spherical objects within the wells are beads. This image
is taken through the dialysis membrane. Well diameter is
approximately 56 .mu.m.
[0029] FIG. 2 shows Left: An isolated single cell immediately prior
to lysis; Center: The cell in the process of lysing; and Right: The
microwell immediately after lysis, using time-lapse microscopy.
Well diameter is approximately 56 .mu.m.
[0030] FIG. 3 shows linked OE RT-PCR product. Letters indicate
approximate locations of constant, variable, joining, and diversity
regions, while numbers indicate approximate locations of
complementarity-determining regions.
[0031] FIGS. 4A-4C show an overview of the linkage (overlap
extension) RT-PCR process. 4A) V-region primers with a 5'
complementary heavy/light overlap region anneal to first strand
cDNA. 4B) Second strand cDNA is formed by 5' to 3' extension; the
overlap region is incorporated into all cDNA. 4C) After
denaturation, heavy and light chains with first strand sense anneal
to generate a complete 850 bp product through 5' to 3' extension.
The CDR-H3 and CDR-L3 are located near the outside of the final
linked construct, which allows CDR3 analysis by 2.times.250
paired-end Illumina sequencing.
[0032] FIG. 5 shows MOPC-21 cells viably encapsulated in droplets
formed via flow focusing. The two input streams to the flow
focusing device were comprised of equal parts MOPC-21 cells in PBS
(100,000 cells/mL, cell stream) and 0.4% trypan blue in PBS (dye
stream), and the cell stream and dye streams mixed together
immediately prior to the point of emulsion droplet formation.
MOPC-21 cells were shown to exclude trypan blue, demonstrating
viable encapsulation of single cells within the emulsion
droplets.
[0033] FIG. 6 presents an overview of high-throughput sequencing
technology for multiple transcripts applied toward the sequencing
of native antibody VH and VL mRNAs from B-cell populations. i)
B-cell populations are sorted for desired phenotype (e.g., mBCs,
memory B cells, naive BCs, naive B cells). ii) Single cells are
isolated by random settling into a microwell array; poly(dT)
microbeads are also added to the wells. iii) Wells are sealed with
a dialysis membrane and equilibrated with lysis buffer to lyse
cells and anneal VH and VL mRNAs to poly(dT) beads (blob represents
a lysed cell, circles depict magnetic beads, black lines depict
mRNA strands). iv) Beads are recovered and emulsified for cDNA
synthesis and linkage PCR to generate an .about.850-base pair VH:VL
cDNA product. v) Next-generation sequencing is performed to
sequence the linked strands. vi) Bioinformatic processing is used
to analyze the paired VH:VL repertoire.
[0034] FIGS. 7A-7C shows amplification of heavy and light chain DNA
on oligoimmobilized magnetic beads for high-throughput sequencing.
7A) Beads display a mix of 3 immobilized oligonucleotides: poly(T)
for mRNA capture, AHX89 for heavy chain amplification, and BRHO6
for light chain amplification. 7B) Reverse transcription is
initiated from captured mRNA (represented by gray dashed lines)
that has annealed to immobilized poly(T) oligonucleotides.
Specially designed immunoglobulin constant region reverse
transcription primers have either AHX89 at the 5' end (for heavy
chain) or BRH06 (for light chain). Reverse transcription polymerase
chain reaction occurs inside emulsion droplets. 7C) V region
forward primers have either an <F3> sequence at the 5' end
(heavy chain) or <F5> sequence (light chain) which will be
used to initiate pyrosequencing. cDNA strands are displayed as
black lines.
[0035] FIG. 8 shows a diagram of the nozzle/carrier stream
apparatus. A glass capillary tube supplies an outer oil phase
carrier stream (arrows) that surrounds a needle exit. The needle
injects aqueous phase containing cells, and monodisperse droplets
are generated by shear forces from annular oil phase flow.
[0036] FIG. 9 shown a general decision tree algorithm for pairing
of VH and VL sequences.
[0037] FIGS. 10A-10C show an exemplary process of mRNA capture from
isolated single cells encoding high-affinity antibodies for a
particular antigen. (10A) Antibody-secreting B cells (top left) are
isolated into compartments containing beads with immobilized
antigen. Secreted antibody (gray) is captured by the beads if the B
cell encodes a high-affinity antibody for the antigen. (10B) Any
unbound cell-secreted antibodies are washed away and an anti-IgG
antibody (white) with linked poly(dT) ssDNA (black strands) is
added to the compartment. The anti-IgG:poly(dT) (or other mRNA
capture moiety) construct is immobilized on beads containing
captured antibody. poly(dT) ssDNA is co-localized only with cells
that secrete high-affinity antibody to the desired antigen. (10C)
The compartments are sealed and cells are lysed. mRNA strands
(small circles) released from cells which secreted high-affinity
antibody are captured via hybridization to the poly(dT) on
poly(dT):antibody:bead constructs. Next, beads can be recovered for
single-cell mRNA transcript analysis.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0038] The present disclosure generally relates to sequencing two
or more genes expressed in a single cell in a high-throughput
manner. More particularly, the present disclosure provides a method
for high-throughput sequencing of pairs of transcripts co-expressed
in single cells to determine pairs of polypeptide chains that
comprise immune receptors (e.g., antibody VH and VL sequences).
[0039] The methods of the present disclosure allow for the
repertoire of immune receptors and antibodies in an individual
organism or population of cells to be determined. Particularly, the
methods of the present disclosure may aid in determining pairs of
polypeptide chains that make up immune receptors. B cells and T
cells each express immune receptors; B cells express
immunoglobulins, and T cells express T cell receptors (TCRs). Both
types of immune receptors consist of two polypeptide chains.
Immunoglobulins consist of variable heavy (VH) and variable light
(VL) chains. TCRs are of two types: one consisting of an .alpha.
and a .beta. chain, and one consisting of a .gamma. and a .delta.
chain. Each of the polypeptides in an immune receptor has constant
region and a variable region. Variable regions result from
recombination and end joint rearrangement of gene fragments on the
chromosome of a B or T cell. In B cells additional diversification
of variable regions occurs by somatic hypermutation. Thus, the
immune system has a large repertoire of receptors, and any given
receptor pair expressed by a lymphocyte is encoded by a pair of
separate, unique transcripts. Only by knowing the sequence of both
transcripts in the pair can one study the receptor as a whole.
Knowing the sequences of pairs of immune receptor chains expressed
in a single cell is also essential to ascertaining the immune
repertoire of a given individual or population of cells.
[0040] Currently available methods to analyze multiple transcripts
in single cells, such as the two transcripts that comprise adaptive
immune receptors, are limited by low throughput and very high
instrumentation and reagent costs. No technology currently exists
for rapidly analyzing how many cells express a set of transcripts
of interest or, more specifically, for sequencing native lymphocyte
receptor chain pairs at very high throughput (greater than 10,000
cells per run). The present disclosure aims to correct these
deficiencies by providing a new technique for sequencing multiple
transcripts simultaneously at the single-cell level with a
throughput two to three orders of magnitude greater than the
current state of the art.
[0041] One advantage of the methods of the present disclosure is
that the methods result in a higher throughput several orders of
magnitude larger than the current state of the art. In addition,
the present disclosure allows for the ability to link two
transcripts for large cell populations in a high throughput manner,
faster and at a much lower cost than competing technologies.
[0042] In certain embodiments, the present disclosure provides
methods comprising separating single cells in a compartment with
beads conjugated to oligonucleotides; lysing the cells; allowing
mRNA transcripts released from the cells to hybridize with the
oligonucleotides; performing overlap extension reverse
transcriptase polymerase chain reaction to covalently link DNA from
at least two transcripts derived from a single cell; and sequencing
the linked DNA. In certain embodiments, the cells may be mammalian
cells. In certain embodiments, the cells may be B cells, T cells,
NKT cells, or cancer cells.
[0043] In other embodiments, the present disclosure provides
methods comprising separating single cells in a compartment with
beads conjugated to oligonucleotides; lysing the cell; allowing
mRNA transcripts released from the cells to hybridize with the
oligonucleotides conjugated to the beads; performing reverse
transcriptase polymerase chain reaction to form at least two cDNAs
from at least two transcripts derived from a single cell; and
sequencing the cDNA attached to the beads.
[0044] In another embodiment, the present disclosure provides a
method comprising mixing cells with beads having a diameter smaller
than the diameter of the cells, wherein the beads are conjugated to
oligonucleotides, sequestering the cells and beads within
compartments having a volume of less than 5 nL, lysing the cells
and allowing mRNA transcripts to associate with the beads,
isolating the beads and associated mRNA from the compartments,
performing reverse transcription followed by PCR amplification on
the bead-associated mRNA, and sequencing the DNA product from each
bead to identify cDNA associated with each bead.
[0045] In other embodiments, the present disclosure provides a
system comprising an aqueous fluid phase exit disposed within an
annular flowing oil phase, wherein the aqueous phase fluid
comprises a suspension of cells and is dispersed within the flowing
oil phase, resulting in emulsified droplets with low size
dispersity comprising an aqueous suspension of cells.
[0046] In other embodiments, the present disclosure provides a
composition comprising a bead, an oligonucleotide capable of
binding mRNA, and two or more primers specific for a transcript of
interest.
[0047] In certain embodiments, the present disclosure also provides
for a device comprising ordered arrays of microwells, each with
dimensions designed to accommodate a single lymphocyte cell. In one
embodiment, the microwells may be circular wells 56 .mu.m in
diameter and 50 .mu.m deep, for a total volume of 125 pL. Such
microwells would normally range in volume from 20-3,000 pL, though
a wide variety of well sizes, shapes and dimensions may be used for
single cell accommodation. In certain embodiments, the microwell
may be a nanowell. In certain embodiments, the device may be a
chip. The device of the present disclosure allows the direct
entrapment of tens of thousands of single cells, with each cell in
its own microwell, in a single chip. In certain embodiments, the
chip may be the size of a microscope slide. In one embodiment, a
microwell chip may be used to capture single cells in their own
individual microwells (FIG. 6). The microwell chip can be made from
polydimethylsiloxane (PDMS); however, other suitable materials
known in the art such as polyacrylimide, silicon and etched glass
may also be used to create the microwell chip.
[0048] Several beads or other particles conjugated with
oligonucleotides may also be captured in the microwells with the
single cells according to the methods of the present disclosure. In
certain embodiments, beads may comprise oligonucleotides
immobilized on the surface of the beads. In other embodiments, the
beads may be magnetic. In other embodiments, the beads may be
coated with one or more oligonucleotides. In certain embodiments,
the oligonucleotides may be a poly(T), a sequence specific for
heavy chain amplification, and/or a sequence specific for light
chain amplification. A dialysis membrane covers the microwells,
keeping the cells and beads in the microwells while lysis reagents
are dialyzed into the microwells. The lysis reagents cause the
release of the cells' mRNA transcripts into the microwell with the
beads. In embodiments where the oligonucleotide is poly(T), the
poly(A) mRNA tails are captured by the poly(T) oligonucleotides on
the beads. Thus, each bead is coated with mRNA molecules from a
single cell. The beads are then pooled, washed, and resuspended in
solution with reagents for overlap extension (OE) reverse
transcriptase polymerase chain reaction (RT-PCR). This reaction mix
includes primers designed to create a single PCR product comprising
cDNA of two transcripts of interest covalently linked together.
Before thermocycling, the reagent solution/bead suspension is
emulsified in oil phase to create droplets with no more than one
bead per droplet. The linked cDNA products of OE RT-PCR are
recovered and used as a template for nested PCR, which amplifies
the linked transcripts of interest. The purified products of nested
PCR are then sequenced and pairing information is analyzed (FIG.
6). In other embodiments, restriction and ligation may be used to
link cDNA of multiple transcripts of interest. In other
embodiments, recombination may be used to link cDNA of multiple
transcripts of interest.
[0049] The present disclosure also provides a method to trap mRNA
from single cells on beads, perform cDNA synthesis, link the
sequences of two or more desired cDNAs from single cells to create
a single molecule, and finally reveal the sequence of the linked
transcripts by High Throughput (Next-gen) sequencing. According to
the present disclosure, one way to increase throughput in
biological assays is to use an emulsion that generates a high
number of 3-dimensional parallelized microreactors. Emulsion
protocols in molecular biology often yield 109-1011 droplets per mL
(sub-pL volume). Emulsion-based methods for single-cell polymerase
chain reaction (PCR) have found a wide acceptance, and emulsion PCR
is a robust and reliable procedure found in many next-generating
sequencing protocols. However, very high throughput RT-PCR in
emulsion droplets has not yet been implemented because cell lysates
within the droplet inhibit the reverse transcriptase reaction. Cell
lysate inhibition of RT-PCR can be mitigated by dilution to a
suitable volume.
[0050] In another embodiment, cells are lysed in emulsion droplets
containing beads for nucleic acid capture. In certain embodiments,
the beads may be conjugated with oligonucleotide. In certain
embodiments, the oligonucleotide may be poly(T). In other
embodiments, the oligonucleotide may be a primer specific to a
transcript of interest. In certain embodiments, the bead may be
magnetic. An aqueous solution with a suspension of both cells and
beads is emulsified into oil phase by injecting an aqueous
cell/bead suspension into a fast-moving stream of oil phase. The
shear forces generated by the moving oil phase create droplets as
the aqueous suspension is injected into the stream, creating an
emulsion with a low dispersity of droplet sizes. Each cell is in
its own droplet along with several beads conjugated with
oligonucleotides. The uniformity of droplet size helps to ensure
that individual droplets do not contain more than one cell. Cells
are then thermally lysed, and the mixture is cooled to allow the
beads to capture mRNA. The emulsion is broken and the beads are
collected. The beads are resuspended in a solution for emulsion OE
RT-PCR to link the cDNAs of transcripts of interest together.
Nested PCR and sequencing of the linked transcripts is performed
according to the present disclosure. In certain embodiments, the
aqueous suspension of cells comprises reverse transcription
reagents. In certain other embodiments, the aqueous suspension of
cells comprises at least one of polymerase chain reaction and
reverse transcriptase polymerase chain reaction reagents. In other
embodiments, restriction and ligation may be used to link cDNA of
multiple transcripts of interest. In other embodiments,
recombination may be used to link cDNA of multiple transcripts of
interest.
[0051] In another embodiment, emulsion droplets which contain
individual cells and RT-PCR reagents are formed by injection into a
fast-moving oil phase. Thermal cycling is then performed on these
droplets directly. In certain embodiments, an overlap extension
reverse transcription polymerase chain reaction may be used to link
cDNA of multiple transcripts of interest.
[0052] In another embodiment, cDNAs of interest from a single cell
are attached via RT-PCR to beads as described below, and the
transcripts on the beads are sequenced directly using
high-throughput sequencing. An equal mixture of three species of
functionalized oligonucleotide primers may be conjugated to
functionalized beads. One of the oligonucleotides may be poly(T) to
capture the poly(A) tail of mRNAs. The other two oligonucleotides
may be specific primers for amplifying the transcripts of interest.
Beads prepared in this way are mixed with cells in an aqueous
solution, and the cell/bead suspension is emulsified so that each
cell is in its own droplet along with an excess of beads. In
certain embodiments an average of 55 beads may be contained in each
droplet. Cells are thermally lysed, and poly(T) oligonucleotides on
the beads bind mRNAs. The emulsion is broken, and beads are
collected, washed, and resuspended in a solution with reagents and
primers for RT-PCR that will result in amplification of the
transcripts of interest in such a way that the transcripts are
attached to the beads. The bead suspension is emulsified and RT-PCR
is performed. The beads are collected and submitted for
high-throughput sequencing, which directly sequences the two
transcripts attached to the beads by initiating multiple sequence
reads using at least two different primers, where each initiation
primer is specific to a transcript of interest. The two transcripts
are paired by bead location in the high-throughput sequencing grid,
revealing sequences that are expressed together from a single cell.
Sequencing can be performed, for example, on Applied Biosystem's
SOLiD platform, Life Technologies' Proton Torrent, or Illumina's
HiSeq sequencing platform.
[0053] Primer design for OE RT-PCR determines which transcripts of
interest expressed by a given cell are linked together. For
example, in certain embodiments, primers can be designed that cause
the respective cDNAs from the VH and VL chain transcripts to be
covalently linked together. Sequencing of the linked cDNAs reveals
the VH and VL sequence pairs expressed by single cells. In other
embodiments, primer sets can also be designed so that sequences of
TCR pairs expressed in individual cells can be ascertained or so
that it can be determined whether a population of cells
co-expresses any two genes of interest.
[0054] Bias can be a significant issue in PCR reactions that use
multiple amplification primers because small differences in primer
efficiency generate large product disparities due to the
exponential nature of PCR. One way to alleviate primer bias is by
amplifying multiple genes with the same primer, which is normally
not possible with a multiplex primer set. By including a common
amplification region to the 5' end of multiple unique primers of
interest, the common amplification region is thereby added to the
5' end of all PCR products during the first duplication event.
Following the initial duplication event, amplification is achieved
by priming only at the common region to reduce primer bias and
allow the final PCR product distribution to remain representative
of the original template distribution.
[0055] Such a common region can be exploited in various ways. One
clear application is to add the common amplification primer at
higher concentration and the unique primers (with 5' common region)
at a low concentration, such that the majority of nucleic acid
amplification occurs via the common sequence for reduced
amplification bias. Another application is the surface-based
capture of amplification products, for example to capture PCR
product onto a microbead during emulsion PCR. If the common
sequence oligonucleotides are immobilized onto a bead surface, the
PCR products of interest will become covalently linked to the bead
during amplification. In this way, a widely diverse set of
transcripts can be captured onto a surface using a single
immobilized oligonucleotide sequence.
[0056] For example, two different common regions may be immobilized
onto a bead surface at equal concentration (e.g., one common
sequence for heavy chain, and a different common sequence for light
chain). Following PCR amplification, the bead will be coated with
approximately 50% heavy chain amplification product, and 50% light
chain amplification product. This balance between heavy and light
chain representation on the bead surface helps ensure sufficient
signal from both heavy and light chains when the bead is submitted
to high throughput sequencing.
[0057] Accordingly, in certain embodiments, the present disclosure
provides methods comprising adding a common sequence to the 5'
region of two or more oligonucleotides that are specific to a set
of gene targets; and performing nucleic acid amplification of the
set of gene targets by priming the common sequence. In certain
embodiments, the common sequence n is immobilized onto a surface.
In other embodiments, the common sequence may be used to capture
amplification products.
[0058] The methods of the present disclosure allow for information
regarding multiple transcripts expressed from a single cell to be
obtained. In certain embodiments, probabilistic analyses may be
used to identify native pairs with read counts or frequencies above
non-native pair read counts or frequencies. The information may be
used, for example, in studying gene co-expression patterns in
different populations of cancer cells. In certain embodiments,
therapies may be tailored based on the expression information
obtained using the methods of the present disclosure. Other
embodiments may focus on discovery of new lymphocyte receptors.
EXAMPLES
[0059] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1--Construction of a High Density Microwell Plate
[0060] A grid of micropillars (56 .mu.m diameter, 50 .mu.m height)
are photolithographically patterned onto a silica wafer using SU-8
photoresist (Fisher Scientific) and the silica wafer is used as a
mold to print polydimethylsiloxane (PDMS) chips (Sylgard 184, Dow
Corning) with the dimensions of a standard microscope slide and
containing approximately 170,000 wells per chip. Dimensions of the
micropillar may range from about 5 .mu.m to about 300 .mu.m wide
and from about 5 .mu.m to about 300 .mu.m high. Molded PDMS chips
are silanized in an oxygen plasma chamber for 5 minutes to generate
a hydrophilic surface. The PDMS chips are then blocked in 1% bovine
serum albumin (BSA) for 30 minutes and washed in deionized water
and phosphate-buffered saline (PBS) to prepare for cell
seeding.
Example 2--Method for Linking Two Transcripts from a Single Cell in
a High Throughput Manner
[0061] The process for physically linking two or more transcripts
derived from a single cell in a high throughput manner uses the
sealed PDMS microwell device of Example 1 to trap single cells into
separate wells. Cell lysis also occurs, and poly(T) magnetic micron
size beads for mRNA capture are also introduced into the
microwells. Once cells and beads have been loaded, the device is
sealed with dialysis membrane and a lysis solution is introduced.
Subsequently, beads are recovered, resuspended in solution with
reagents, primers and polymerase enzyme for overlap extension (OE)
RT-PCR, and the solution is then emulsified so that each bead is
encapsulated within a single emulsion droplet. The emulsion is
subjected to thermal cycling to physically link the two transcripts
(e.g., immunoglobulin heavy and light chain cDNA), and the linked
products are recovered from the emulsion following cycling. A
nested PCR amplification is performed, and then the resulting DNA
is sequenced using Illumina or any other NextGen sequencing
technology that can yield reads of appropriate length to
unequivocally interpret the transcript pairing information (FIG.
6).
[0062] The method outlined above was employed to link the
immunoglobulin variable heavy (VH) and variable light (VL) chains
in mixtures comprising the mouse hybridoma cell lines MOPC-21 and
MOPC-315. The VH and VL sequences expressed by each of these cell
lines is known and hence these experiments served for method
validation. 5 mL each of MOPC-21 and MOPC-315 cells were separately
withdrawn from culture two days after passage (cells were grown in
Falcon vented T-25 culture flasks, 10 mL volume in RPMI-1640, 10%
FBS, 1% P/S) and placed in 15 mL tubes. Cell density of 150,000
viable cells/mL with >98% viability, as measured with a
hemocytometer and trypan blue exclusion, were determined. RNAse A
was added to each tube at a concentration of 30 .mu.g/mL and cells
were incubated at 37.degree. C. for 30 minutes. Then, cells were
washed three times with complete culture media and twice with PBS
(pH 7.4). Washes were accomplished by centrifugation at 250 g at
room temperature for 5 minutes followed by aspiration and
resuspension. Cell concentrations were counted again with a
hemocytometer, and MOPC-21 and MOPC-315 cells were mixed to form a
cell suspension with a total concentration of 35,000 cells/mL in
PBS, composed of 17,500 MOPC-21 cells/mL and 17,500 MOPC-315
cells/mL.
[0063] 500 .mu.L of the MOPC-21 and MOPC-315 cell mixture were
applied to a PDMS microwell device that had been incubated with BSA
to block non-specific adsorption. 17,500 total cells were added to
each chip. Four chips were used in parallel (70,000 total cells
distributed across four PDMS chips), and cells were allowed to
settle into wells by gravity over the course of 5 minutes with
gentle agitation. As each PDMS chip contains approximately 120,000
wells and cell loading efficiently is estimated at approximately
70%, approximately 1 in 10 wells contain isolated cells. The
incidence of two cells per well can be accurately estimated with
Poisson statistics, and under these conditions, >95% of wells
containing cells contained a single cell.
[0064] The surfaces of the microwell devices were then washed with
PBS to remove unadsorbed cells from the chip surfaces, and 25 .mu.L
of poly(T) magnetic beads (mRNA Direct Kit, 2.8 .mu.m diameter,
Invitrogen Corp.) was resuspended in 50 .mu.L PBS and applied to
each microwell device surface, for an average of 55 poly(T) beads
per well. After magnetic beads were allowed to settle into wells by
gravity, a BSA-blocked dialysis membrane (12,000-14,000 MWCO
regenerated cellulose, 25 mm flat width, Fisher Scientific) that
had been rinsed in PBS was laid over each chip surface. PBS was
removed from the chip and membrane surfaces using a 200 .mu.L
pipette. Then, the tapered end of a 1000 .mu.L pipette tip was cut
to form a flat cylinder that was dragged across the membranes,
pressing the membranes to the PDMS chips and eliminating excess PBS
from between the PDMS microwell devices and dialysis membrane,
which sealed the microwells and trapped cells and beads inside
(FIG. 1).
[0065] Cell lysis and mRNA binding to the poly(T) magnetic beads
trapped within microwells was accomplished by dialysis. 500 .mu.L
of cell lysis solution (500 mM LiCl in 100 mM tris buffer (pH 7.5)
with 0.1% sodium deoxycholate and 10 mM ribonucleoside vanadyl
complex) was applied to the dialysis membranes, and lysis occurred
at room temperature as reagents dialyzed into microwells. Cell
lysis was fully complete in <5 minutes as determined by
time-lapse microscopy (FIG. 2).
[0066] PDMS microwell chips were maintained for 20 minutes at room
temperature inside a Petri dish, then placed in a cold room at
4.degree. C. for 10 additional minutes. A Dynal MPC-S magnet was
placed underneath the PDMS microwell device to hold magnetic beads
inside microwells as the dialysis membrane was removed with forceps
and discarded. The magnet was then placed underneath another Petri
dish with 4 subdivisions, one of which contained 2 mL of cold mRNA
Direct Lysis/Binding Buffer (100 mM tris pH 7.5, 500 mM LiCl, 10 mM
EDTA, 1% LiDS, 5 mM DTT). The four PDMS microwell devices were
sequentially inverted and resuspended in the 2 mL of solution to
allow the magnet to draw beads out of microwells and into the mRNA
Direct Lysis/Binding buffer solution. Magnetic beads were then
resuspended in the 2 mL mRNA Direct Lysis/Binding Buffer and the
solution was divided into two Eppendforf tubes and placed on the
Dynal MPC-S magnetic rack. Beads were washed once without
resuspension using 1 mL per tube of Wash Buffer 1 (100 mM tris pH
7.5, 500 mM LiCl, 1 mM EDTA, 4.degree. C.). Beads were then
immediately washed again in Wash Buffer 1 with resuspension. Beads
were then immediately resuspended in Wash Buffer 2 (20 mM tris pH
7.5, 50 mM KCl, 3 mM MgCl) and replaced on the magnetic rack.
Finally, beads were suspended in 2.85 mL cold RT-PCR mixture
(Quanta OneStep Fast, VWR) containing 0.05 wt % BSA (Invitrogen
Ultrapure BSA, 50 mg/mL) and primer concentrations listed in Table
1. Amplification was accomplished with two common primers
(CHrev-AHX89 and CLrev-BRH06) at high concentration which anneal to
the reverse complement of the 5' end of CLrev and CHrev specific
primers. V-region primers also contain linker sequences at the 5'
end to effect VH-VL linkage. 25 .mu.L of cold the RT-PCR mixture
was previously reserved for cycling without beads or emulsification
as a non-template control. The cold RT-PCR mixture containing the
poly(T) magnetic beads was added dropwise to a stirring Ika
dispersing tube (DT-20, VWR) containing 9 mL chilled oil phase
(molecular biology grade mineral oil with 4.5% Span-80, 0.4% Tween
80, 0.05% Triton X-100, v/v % oil phase reagents from Sigma Aldrich
Corp.), and the mixture was agitated for 5 minutes at low speed.
The resulting emulsion was added to 96-well PCR plates, with 100
.mu.L emulsion per well, and placed in a thermocycler. The RT step
was performed under the following conditions: 30 minutes at
55.degree. C., followed by 2 min at 95.degree. C. PCR amplification
was then performed under the following conditions: three cycles of
94.degree. C. for 30 s denature, 57.degree. C. for 1 min anneal,
and 72.degree. C. for 3 min extend; then twenty-seven cycles of
94.degree. C. for 30 s denature, 59.degree. C. for 30 s anneal, and
72.degree. C. for 3 min extend; then a final extension step for 7
min at 72.degree. C. FIG. 3 shows a diagram of the final linked
products.
TABLE-US-00001 TABLE 1 Primers for MOPC-21/MOPC-315 emulsion
linkage RT-PCR. Conc. Primer ID 400 CLrev-BRH06 400 CHrev-AHX89 40
MOPC21-CHrev-AHX89 40 MOPC21-CLrev-BRH06 40 MOPC315-CLrev-BRH06 40
MOPC315-CHrev-AHX89 40 MOPC21-VH-OE2 40 MOPC21-VL-OE2 40
MOPC315-VH-OE 40 MOPC315-VL-OE
[0067] Following thermal cycling, the emulsion was collected and
divided into three Eppendorf tubes and centrifuged at room
temperature for 10 minutes at 16,000 g. The mineral oil upper phase
was discarded, and 1.5 mL diethyl ether was added to extract the
remaining oil phase and break the emulsion. The upper ether layer
was removed and two more ether extractions were performed. Then the
ether layer was discarded, and residual ether solvent was removed
in a SpeedVac for 25 minutes at room temperature. The remaining
aqueous phase was diluted 5:1 in DNA binding buffer, then split in
three parts and passed through three silica spin columns (DNA Clean
& Concentrator, Zymo Research Corp.) to capture the RT-PCR cDNA
product. After washing each column with 300 .mu.L wash buffer (Zymo
Research Corp), cDNA was eluted with 20 .mu.L in each column, and a
nested PCR reaction was performed (ThermoPol PCR buffer with Taq
Polymerase, New England Biosciences) in a total volume of 200 .mu.L
using 4 .mu.L eluted cDNA as template. After a 2 min denaturing
step at 94.degree. C., cycling was performed at 94.degree. C. for
30 s denature, 62.degree. C. for 30 s anneal, 72.degree. C. for 20
s extend, for 30 cycles. 400 nM of each nested primer (Table 2) was
used to amplify linked heavy and light chains, which generated an
approximately 800 bp linked product.
TABLE-US-00002 TABLE 2 Primers for MOPC-21/MOPC-315 nested PCR.
Conc. Primer ID 400 MOPC21-CHrev- seq 400 MOPC21-CLrev- seq 400
MOPC315- CHrev-seq 400 MOPC315- CLrev-seq
[0068] Nested PCR product was electrophoresed on a 1% agarose gel,
and the 800 bp band was excised and dissolved in agarose-dissolving
buffer for 10 minutes at 50.degree. C., then captured onto and
eluted from a silica spin column according to manufacturer
protocols (Zymo Research Corp.) to obtain purified nested PCR
product. Purified cDNA was submitted for base pair paired-end reads
with the Illumina HiSeq sequencing platform. Other NextGen
sequencing technology (e.g. Roched 454, Pacific Biosciences etc.)
capable of providing reads suitable for identifying the linked
transcript can also be used for this purpose. (FIG. 3). HiSeq data
output was mapped to known MOPC-21 and MOPC-315 sequences using the
SHort Read Mapping Package software (SHRiMP) and filtered for
high-quality reads with >90% identity to known transcript
sequences. In this manner, approximately 18,000 linked heavy and
light chain sequences were obtained (Table 3).
TABLE-US-00003 TABLE 3 Raw read counts for sequenced VH-VL pairs.
Light MOPC-21 MOPC-315 Heavy MOPC-21 9,689 426 MOPC-315 1,042
6,591
[0069] Correct transcript pairings were further determined from the
degree of pairing skewness of the raw DNA sequencing data. For any
given two transcripts, e.g., immunoglobulin heavy chain H.sub.i and
light chain L.sub.j, with overall heavy or light chain mapped
frequencies f.sub.Hi, and f.sub.Lj, a measure of pairing skewness,
s, is computed:
s = Observed reads Expected reads ( random pairing ) = [ ( # Hi ) ]
Lj pairs ) fHi .times. fLj .times. ( # total pairs )
##EQU00001##
[0070] The calculated value s compares VL frequency paired with a
particular VH to the VL frequency in the entire sequence set. A
value of s>1 indicates that a heavy-light pair is observed at a
frequency above that corresponding to random pairing. Natural
pairings are deduced from entries with a maximum value of s (Table
4). Pairing skewness, s, for sequenced heavy and light pairs,
calculated from approximately 18,000 sequenced VH-VL linked pairs
are is shown in Table 4. Native heavy-light pairings are predicted
by the maximal value of s for each heavy chain and are highlighted
in green. This table demonstrates the capacity of our method to
resolve native heavy and light chain pairings from a heterogeneous
mixture of cells.
TABLE-US-00004 TABLE 4 Calculated Pairing Skewness. Light MOPC-21
MOPC-315 Heavy MOPC-21 1.58 0.11 MOPC-315 0.23 2.18
Example 3--High-Throughput Transcripts Pairing Analysis Using
Defined Mixtures of 5 Cell Lines
[0071] Five immortalized B cell lines were mixed at different
ratios and used to examine pairing efficiency of the linked
products generated by OE-PCR. The five B cell lines used in this
experiment were: MOPC-21, MOPC-315, IM-9, ARH-77, and DB (see Table
5). DB expresses extremely low levels of VH and VL transcript and
was used as a negative control.
[0072] All cell lines were obtained from ATCC and cultured in
RPMI-1640 supplemented with 10% FBS and 1% penicillin/streptomycin
(see Example 2). Following a 30-minute RNAse treatment and
subsequent wash, cells were seeded into microwells at a density of
17,500 total cells per chip along with poly(T) magnetic beads
according to Example 2. Wells were sealed with a dialysis membrane,
cells were lysed, and mRNA was allowed to anneal to the beads
(Example 2). Beads were then recovered, resuspended in OE RT-PCR
mix, and placed in an emulsion (Example 2). OE RT-PCR primer
concentrations used are given in Table 6, and thermal cycling
conditions are presented in Table 7.
TABLE-US-00005 TABLE 5 An overview of the 5 cell lines. % in
Relative Ig Mix Cell Line ATCC ID Organism Ig Class Expression 65
IM-9 CCL-159 Homo sapiens IgG/IgK Low 35 MOPC-21 63035 Mus musculus
IgG/IgK Medium 6 ARH-77 CRL-1621 Homo sapiens IgG/IgK High 3
MOPC-315 TIB-23 Mus musculus IgA/IgL Medium 1 DB CRL-2289 Homo
sapiens IgG/IgL Very Low
TABLE-US-00006 TABLE 6 OE RT-PCR primers for the mix of cell lines.
Conc. Primer ID 400 CLrev-BRH06 400 CHrev-AHX89 40 MOPC21-CHrev-
AHX89 40 MOPC21-CLrev- BRH06 40 MOPC315-CLrev- BRH06 40
MOPC315-CHrev- AHX89 40 MOPC21-VH-OE2 40 MOPC21-VL-OE2 40
MOPC315-VH-OE 40 MOPC315-VL-OE 40 hIgG-rev-OE- AHX89 40
hIgKC-rev-OE- BRH06 40 hIgLC-rev-OE- BRH06 40 hVH1-fwd-OE 40
hVH157-fwd-OE 40 hVH2-fwd-OE 40 hVH3-fwd-OE 40 hVH4-fwd-OE 40
hVH4-DP63-fwd- OE 40 hVH6-fwd-OE 40 hVH3N-fwd-OE 40 hVK1-fwd-OE 40
hVK2-fwd-OE 40 hVK3-fwd-OE 40 hVK5-fwd-OE 40 hVL1-fwd-OE 40
hVL1459-fwd-OE 40 hVL15910-fwd-OE 40 hVL2-fwd-OE 40 hVL3-fwd-OE 40
hVL-DPL16-fwd- OE 40 hVL3-38-fwd-OE 40 hVL6-fwd-OE 40
hVL78-fwd-OE
TABLE-US-00007 TABLE 7 OE RT-PCR thermal cycling conditions. #
Cycles Temp (.degree. C.) Time (min) 1 55 30 94 2 4 94 0.5 50 0.5
72 3 4 94 0.5 55 0.5 72 3 22 94 0.5 60 0.5 72 3 1 72 7
[0073] Emulsion OE RT-PCR product was recovered by diethyl ether
extraction followed by capture on and elution from a silica spin
column (Example 2) for use as template in a nested PCR under the
following conditions: 94.degree. C. for 2 min initial denature,
94.degree. C. for 30 s denature, 62.degree. C. for 30 s anneal,
72.degree. C. for 20 s extend, 40 total cycles. Nested primer
sequences and concentrations are reported in Tables 2 and 8.
TABLE-US-00008 TABLE 8 Nested PCR primers to generate approximately
800 bp linked products. Conc. (nM) Primer ID 400 hIgG-all-rev-
OEnested 400 hIgKC-rev- OEnested 400 hIgLC-rev- OEnested
[0074] Nested PCR product was electrophoresed on a 1% agarose gel,
and a region from 650 to 1000 bp was excised and purified with a
silica spin column (Example 2). Recovered cDNA was submitted for
Illumina HiSeq 100 bp paired-end sequencing. HiSeq data was mapped
to a reference file containing heavy and light chain sequences for
all five clones, and data was filtered to obtain paired-end reads
with >90% match to reference sequences, as in Example 2. Natural
pairings were identified by interrogating skewness of pairing data.
For any given immunoglobulin heavy chain Hi and light chain Lj,
with overall heavy or light chain mapped frequencies fHi and fLj, a
measure of pairing skewness, s, was computed:
s = Observed reads Expected reads ( random pairing ) = ( # HiLj
pairs ) fHi .times. fLj .times. ( # total pairs ) ##EQU00002##
[0075] The calculated value s compares VL frequency paired with a
particular VH to the VL frequency in the entire sequence set. A
value of s>1 indicates that a heavy-light pair is observed at a
frequency above that corresponding to random pairing. Natural
pairings are deduced from entries with a maximum value of s for
each heavy chain. Table 9 shows the natural pairings identified and
pairing skewness, s, for sequenced heavy and light pairs,
calculated from approximately 66,000 sequenced VH-VL linked pairs.
Native heavy-light pairings were predicted by the maximal value of
s for each heavy chain and are highlighted in gray. Table 9
demonstrates the ability of our method to resolve native heavy and
light chain pairings from a heterogeneous mixture of cells with
high throughput.
Example 4--Method for Linking Two Transcripts from Single B Cells
Trapped within High Density Microwell Plates
[0076] A population of B cells is allowed to settle by gravity into
PDMS microwell plates, constructed as described in Example 1. In
this example, each PDMS slide contains 1.7.times.10.sup.5 wells so
that four slides processed concurrently accommodate 68,000
lymphocytes at a .gtoreq.1:10 cell/well occupancy, which gives at
least a 95% probability of there being only one cell per well based
on Poisson statistics. Poly(dT) magnetic beads with a diameter of
2.8 .mu.m are deposited into the microwells at an average of 55
beads/well and the slides are covered with a dialysis membrane.
Subsequently, the membrane-covered slides are incubated with an
optimized cell lysis solution containing 1% lithium dodecyl sulfate
that results in complete cell lysis within <1 min. mRNA anneals
to the poly(dT) magnetic beads which are collected, washed and
resuspended in solution with reagents, primers, reverse
transcriptase enzyme, and polymerase enzyme for overlap extension
(OE) RT-PCR. In this manner beads become isolated within the
droplets that comprise the water in oil emulsion. The emulsion is
subjected to thermal cycling to physically link the two transcripts
(e.g. immunoglobulin heavy and light chain cDNA), and the linked
products are recovered from the emulsion following cycling. A
nested PCR amplification is performed, and then the resulting DNA
is sequenced using Illumina or any other NextGen sequencing
technology that can yield reads of appropriate length to
unequivocally interpret the transcript pairing information. An
overview of the process is presented in FIG. 6.
[0077] The method outlined above was employed to link the
immunoglobulin variable heavy (VH) and variable light (VL) chains
in mixtures of human primary cells.
[0078] A healthy 30-year-old male was vaccinated with the 2010-2011
trivalent FluVirin influenza vaccine (Novartis) and blood was drawn
at day 14 after vaccination after informed consent had been
obtained. PBMCs were isolated and resuspended in DMSO/10% FCS for
cryopreservation. Frozen PBMCs were thawed and cell suspensions
were stained in PBS/0.2% BSA with anti-human CD19 (HIB19,
BioLegend, San Diego, Calif.), CD27 (O323, BioLegend), CD38 (HIT2,
BioLegend) and CD3 (7D6, Invitrogen, Grand Island, N.Y.).
CD19.sup.+CD3.sup.-CD27.sup.+CD38.sup.int memory B cells were
sorted using a FACSAria II sorter system (BD Biosciences, San
Diego, Calif.). Cells were either cryopreserved in DMSO/10% FCS for
subsequent high-throughput VH:VL pairing or single-cell sorted into
96-well plates containing RNAse Inhibitor Cocktail (Promega,
Madison, Wis.) and 10 mM Tris-HCl pH 8.0 for single-cell RT-PCR
analysis. cDNA was synthesized from single-sorted cells using the
Maxima First Strand cDNA Synthesis Kit (Fermentas, Waltham, Mass.)
followed by amplification of the immunoglobulin variable genes
using primer sets and PCR conditions previously described (Smith et
al., 2009). Variable genes were determined with in-house analysis
software using the IMGT search engine (Brochet et al., 2008).
[0079] Memory B cells frozen for high-throughput VH:VL pairing were
thawed and recovered by centrifugation at 250 g for 10 min. Cells
were resuspended in 200 .mu.l RPMI-1640 supplemented with
1.times.GlutaMAX, 1.times.non-essential amino acids, 1.times.sodium
pyruvate and 1.times.penicillin/streptomycin (Life Technologies)
and incubated at 37.degree. C. for 13 h in a 96-well plate.
Recovered cells were centrifuged again at 250 g for 10 min and
resuspended in 400 .mu.l PBS, and 6 .mu.l were withdrawn for cell
counting with a hemocytometer. Approximately 8,800 cells were
recovered from frozen stock. Memory B cells were then spiked with
.about.880 IM-9 cells (ATCC number CCL-159) as an internal control.
Cells were resuspended over two PDMS microwell slides (340,000
wells) and allowed to settle into wells by gravity over the course
of 5 min with gentle agitation. The cell seeding process has been
calculated to be 90% efficient by measuring cell concentration in
seeding buffers both pre- and post-cell seeding; thus 8,000 primary
cells were analyzed in this experiment. The fraction of cells
isolated in the single and multiple cell per well states was
calculated using Poisson statistics:
P ( k , .mu. ) = .mu. k e - .mu. k ! ##EQU00003##
[0080] where k equals the number of cells in a single microwell and
.mu. is the average number of cells per well, so that the 1:39
cell:well ratio used in this experiment corresponds to 98.7% of
cells deposited at an occupancy of one cell/well. 25 .mu.l of
poly(dT) magnetic beads (Invitrogen mRNA Direct Kit) were
resuspended in 50 .mu.l PBS and distributed over each PDMS slide
surface, (mean of 55 poly(dT) beads per well). Magnetic beads were
allowed to settle into wells by gravity for .about.5 min, then a
BSA-blocked dialysis membrane (12,000-14,000 MWCO regenerated
cellulose, 25-mm flat width, Fisher Scientific) that had been
rinsed in PBS was laid over each slide surface, sealing the
microwells and trapped cells and beads inside (FIG. 1). Excess PBS
was removed from the slide and membrane surfaces using a 200 .mu.L
pipette. 500 .mu.L of cell lysis solution (500 mM LiCl in 100 mM
TRIS buffer (pH 7.5) with 1% lithium dodecyl sulfate, 10 mM EDTA
and 5 mM DTT) was applied to the dialysis membranes for 20 min at
room temperature. Time-lapse microscopy revealed that all cells are
fully lysed within 1 min (FIG. 2). Subsequently, the slides were
incubated at 4.degree. C. for 10 min at which point a Dynal MPC-S
magnet was placed underneath the PDMS microwell device to hold
magnetic beads inside the microwells as the dialysis membrane was
removed with forceps and discarded. The PDMS slides were quickly
inverted in a Petri dish containing 2 mL of cold lysis solution and
the magnet was applied underneath the Petri dish to force the beads
out of the microwells. Subsequently, 1 ml aliquots of the lysis
solution containing resuspended beads were placed into Eppendorf
tubes and beads were pelleted on a Dynal MPC-S magnetic rack and
washed once without resuspension using 1 mL per tube of wash buffer
1 (100 mM Tris, pH 7.5, 500 mM LiCl, 1 mM EDTA, 4.degree. C.).
Beads were resuspended in wash buffer 1, pelleted and resuspended
in wash buffer 2 (20 mM Tris, pH 7.5, 50 mM KCl, 3 mM MgCl) and
pelleted again. Finally beads were suspended in 2.85 mL cold RT-PCR
mixture (Quanta OneStep Fast, VWR) containing 0.05 wt % BSA
(Invitrogen Ultrapure BSA, 50 mg/mL) and primer sets for VH and VL
linkage amplification (FIG. 4 and Tables 6 and 10). The suspension
containing the poly(dT) magnetic beads was added dropwise to a
stirring IKA dispersing tube (DT-20, VWR) containing 9 mL chilled
oil phase (molecular biology grade mineral oil with 4.5% Span-80,
0.4% Tween 80, 0.05% Triton X-100, v/v %, Sigma-Aldrich, St. Louis,
Mo.), and the mixture was agitated for 5 min at low speed. The
resulting emulsion was added to 96-well PCR plates with 100 .mu.L
emulsion per well and placed in a thermocycler. The RT step was
performed under the following conditions: 30 min at 55.degree. C.,
followed by 2 min at 94.degree. C. PCR amplification was performed
under the following conditions: four cycles of 94.degree. C. for 30
s denature, 50.degree. C. for 30 s anneal, 72.degree. C. for 2 min
extend; four cycles of 94.degree. C. for 30 s denature, 55.degree.
C. for 30 s anneal, 72.degree. C. for 2 min extend; 22 cycles of
94.degree. C. for 30 s denature, 60.degree. C. for 30 s anneal,
72.degree. C. for 2 min extend; then a final extension step for 7
min at 72.degree. C. After thermal cycling the emulsion was
visually inspected to ensure the absence of a bulk water phase,
which is a key indicator of emulsion stability. Following visual
verification, the emulsion was collected and centrifuged at room
temperature for 10 min at 16,000 g, the mineral oil upper phase was
discarded, and 1.5 mL diethyl ether was added to extract the
remaining oil phase and break the emulsion. The upper ether layer
was discarded, two more ether extractions were performed and
residual ether was removed in a SpeedVac for 25 min at room
temperature. The aqueous phase was diluted 5:1 in DNA binding
buffer and passed through a silica spin column (DNA Clean &
Concentrator, Zymo Research, Irvine, Calif.) to capture the cDNA
product. The column was washed twice with 300 .mu.L wash buffer
(Zymo Research Corp) and cDNA was eluted into 40 .mu.L
nuclease-free water. Finally, a nested PCR amplification was
performed (ThermoPol PCR buffer with Taq Polymerase, New England
Biosciences, Ipswich, Mass.) in a total volume of 200 .mu.L using 4
.mu.L of eluted cDNA as template with 400 nM primers (Tables 8 and
11) under the following conditions: 2 min initial denaturation at
94.degree. C., denaturation at 94.degree. C. for 30 s for 39
cycles, annealing at 62.degree. C. for 30 s and extension at
72.degree. C. for 20 s, final extension at 72.degree. C. for 7 min.
The approximately 850 bp linked product (FIG. 3) was extracted by
agarose gel electrophoresis and sequenced using the 2.times.250
paired end MiSeq NextGen platform (Illumina, San Diego,
Calif.).
TABLE-US-00009 TABLE 10 Primer sets for human VH and VL linkage
RT-PCR amplification. Conc. (nM) Primer ID 40 hIgA-rev-OE- AHX89 40
hIgM-rev-OE- AHX89
TABLE-US-00010 TABLE 11 Primer sets for human VH and VL nested PCR
amplification. Conc. (nM) Primer ID 400 hIgA-all-rev- OEnested 400
hIgM-rev- OEnested
[0081] For bioinformatic analysis, raw 2.times.250 MiSeq data were
filtered for minimum Phred quality score of 20 over 50% of
nucleotides to ensure high read quality in the CDR3-containing
region (approximately HC nt 65-115 or LC nt 55-100). Sequence data
were submitted to the International ImMunoGeneTics Information
System (IMGT) for mapping to germline V(D)J genes (Brochet et al.,
2008). Sequence data were filtered for in-frame V(D)J junctions,
and productive VH and V.kappa.,.lamda., sequences were paired by
Illumina read ID. CDR-H3 nucleotide sequences were extracted and
clustered to 96% nt identity with terminal gaps ignored, to
generate a list of unique CDR-H3s in the data set. 96% nt identity
cutoff was found to be the optimal cutoff to cluster sequencing
error in spiked control clones; the number of unique CDR-H3
sequences and hence the number of unique V genes reported refer to
the number of clusters recovered from the sample (Table 12). The
top read-count CDR-L3 for each CDR-H3 cluster was assigned as a
cognate pair and a list of recovered VH:VL pairs was generated. The
observed accuracy ratio of 942:1 demonstrated the preservation of
correct heavy and light chain pairings in the IM-9 spiked control
cell line (Table 12).
TABLE-US-00011 TABLE 12 Key experimental statistics for Example 4.
Immunization Influenza (2010-11 Fluvirin) Cell Type Day 14 memory B
cells Fresh Cells vs. Freeze/Thaw Freeze/Thaw Cell:Well Ratio 1:39
% cells as single cells 98.7% Unique CDR-H3 Recovered 240 Control
Cell Spike IM-9 Accuracy Ratio.sup.1 942:1 .sup.1For known spiked
cells, (reads correct VL):(reads top incorrect VL)
[0082] The VH:VL pairings identified using this high-throughput
approach to were compared those identified using the established
single-cell sorting method (Smith et al., 2009; Wrammert et al.,
2008); this analysis was conducted in a double-blinded manner.
Peripheral CD19.sup.+CD3.sup.-CD27.sup.+CD38.sup.int memory B cells
were isolated from a healthy volunteer 14 d after vaccination with
the 2010-2011 trivalent FluVirin influenza vaccine (Smith et al.,
2009). For the scRT-PCR analysis, 168 single B cells were sorted
into four 96-well plates, and 168 RT and 504 nested PCR reactions
were carried out individually to separately amplify the VH and VL
(.kappa. and .lamda.) genes. DNA products were resolved by gel
electrophoresis and sequenced to yield a total of 51 VH:VL pairs,
of which 50 were unique. A total of 240 unique CDR-H3:CDR-L3 pairs
were recovered. Four CDR-H3 sequences detected in the
high-throughput pairing set were also observed in the single-cell
RT-PCR analysis. A blinded analysis revealed that CDR-H3:CDR-L3
pairs isolated by the two approaches were in complete agreement
(DeKosky et al., 2013). The agreement between established
single-cell RT-PCR sequencing methods and the high-throughput
sequencing methods demonstrated high accuracy in VH:VL sequences
recovered according to the methods described in the present
disclosure.
Example 5--Isolation of High Affinity Antibodies Following
High-Throughput VH:VL Pairing
[0083] This example describes the isolation of high affinity
anti-tetanus antibodies from human peripheral B cells following
booster immunization. One female donor was booster immunized
against TT/diphtheria toxoid (TD, 20 I.E. TT and 2 I.E. diphtheria
toxoid, Sanofi Pasteur Merck Sharpe & Dohme GmbH, Leimen,
Germany) after informed consent by the Charite Universitatsmedizin
Berlin had been obtained (samples were anonymously coded and study
approved by the hospital's ethical approval board, number
EA1/178/11, and the University of Texas at Austin Institutional
Review Board, IRB# 2011-11-0095). At 7 d post TT immunization, EDTA
blood was withdrawn and PBMC isolated by density gradient
separation as described (Mei et al., 2009). PBMCs were stained in
PBS/BSA at 4.degree. C. for 15 min with anti-human CD3/CD14-PacB
(clones UCHT1 and M5E2, respectively, Becton Dickinson, BD),
CD19-PECy7 (clone SJ25C1, BD), CD27-Cy5 (clone 2E4, kind gift from
Rene van Lier, Academic Medical Centre, University of Amsterdam,
The Netherlands, labeled at the Deutsches Rheumaforschungszentrum
(DRFZ), Berlin), CD2O-Pac0 (clone HI47, Invitrogen), IgD-PerCpCy5.5
(clone L27, BD), CD38-PE (clone HIT2, BD) and TT-Digoxigenin
(labeled at the DRFZ) for 15 min at 4.degree. C. Cells were washed
and a second staining was performed with anti-Digoxigenin-FITC
(Roche, labeled at the DRFZ) and DAPI was added before sorting.
CD19.sup.+CD3.sup.-CD14.sup.-CD38.sup.++CD27.sup.++CD20.sup.-TT.sup.+
plasmablasts were sorted using a FACSAria II sorter system (BD
Biosciences). A portion of sorted cells were washed and
cryopreserved in DMSO/10%FCS for high-throughput VH:VL pairing.
[0084] One vial containing approximately 2,000 frozen TT.sup.+
plasmablasts was thawed and recovered by centrifugation at
250.times.g for 10 min; approximately 20-30% of the cells are
anticipated to be viable (Kyu et al., 2009). Cells were resuspended
in 300 .mu.L RPMI-1640 supplemented with 10% FBS, 1.times.GlutaMAX,
1.times.non-essential amino acids, 1.times.sodium pyruvate and
1.times.penicillin/streptomycin (all from Life Technologies) and
incubated at 37.degree. C. for 13 h in a 96-well plate. Recovered
cells were centrifuged again at 250.times.g for 10 min and
resuspended in 400 .mu.L PBS, and 6 .mu.L were withdrawn for cell
counting with a hemocytometer. Cells were spiked with approximately
30 ARH-77 cells as an internal control (ATCC number CRL-1621) and
VH:VL transcripts were linked as described in Example 4, omitting
IgM primers and using a 38-cycle nested PCR; the resulting product
was submitted for 2.times.250 MiSeq sequencing. VH and VL chains
were also amplified individually to obtain full VH and VL sequences
for antibody expression. Nested PCR product was diluted 1:9 and 0.5
.mu.L were used as template in a PCR reaction with the following
conditions: 400 nM primers (Tables 8, 11 and 13), 2 min initial
denaturation at 94.degree. C., denaturation at 94.degree. C. for 30
s for 12 cycles, annealing at 62.degree. C. for 30 s and extension
at 72.degree. C. for 15 s, final extension at 72.degree. C. for 7
min. The resulting .about.450 bp VH or .about.400 bp VL products
were purified by agarose gel electrophoresis and submitted for
2.times.250 MiSeq sequencing. Sequence data was processed as
described above; additionally ten VH and VL pairs were selected
from TT+ plasmablast pairings for antibody expression and testing.
For complete antibody sequencing of these ten genes, 2.times.250 bp
reads containing the 5' V gene FR1-CDR2 and 3' CDR2-FR4 were paired
by Illumina read ID and consensus sequences were constructed from
reads containing the exact CDR3 of interest. Antibody genes were
then cloned into the human IgG expression vectors pMAZ-VH and
pMAZ-VL, respectively (Mazor et al., 2007). 40 .mu.g each of
circularized ligation product were co-transfected into HEK293F
cells (Invitrogen, N.Y., USA). Medium was harvested 6 d after
transfection by centrifugation and IgG was purified by a protein-A
agarose (Pierce, Ill., USA) chromatography column.
TABLE-US-00012 TABLE 13 Linkers for VH and VL separate
amplification primers. Conc. (nM) Primer ID 400 Linker-VHfwd 400
Linker-VLfwd
[0085] Antigen affinities were determined by competitive ELISA
(Friguet et al., 1985) using different concentrations of IgG in a
serial dilution of antigen, ranging from 100 nM to 0.05 nM in the
presence of 1% milk in PBS. Plates were coated overnight at
4.degree. C. with 10 .mu.g/mL of TT in 50 mM carbonate buffer, pH
9.6, washed three times in PBST (PBS with 0.1% Tween 20) and
blocked with 2% milk in PBS for 2 h at room temperature.
Pre-equilibrated samples of IgG with TT antigen were added to the
blocked ELISA plate, incubated for 1 h at room temperature, and
plates were washed 3.times.with PBST and incubated with 50 pi of
anti-human kappa light chain-HRP secondary antibody (1:5,000, 2%
milk in PBS) for .about.2 min, 25.degree. C. Plates were washed
3.times.with PBST, then 50 .mu.l Ultra TMB substrate (Thermo
Scientific, Rockford, Ill.) was added to each well and incubated at
25.degree. C. for 5 min. Reactions were stopped using equal volume
of 1M H.sub.2SO.sub.4 and absorbance was read at 450 nm (BioTek,
Winooski, Vt.). Each competitive ELISA replicate was fit using a
four-parameter logistic (4PL) equation, with error represented as
the s.d. of 2-3 replicates for each IgG analyzed. All ten
antibodies showed specificity for TT and bound TT with high
affinity (0.1 nM.ltoreq.KD.ltoreq.18 nM; Table 14) (DeKosky et al.,
2013). The high affinity of anti-TT antibodies recovered
demonstrates the application of high-throughput VH:VL sequencing
methods in the present disclosure for antibody discovery from human
cell donors.
TABLE-US-00013 TABLE 14 Tetanus toxoid-binding affinities of IgG
isolated by high-throughput sequencing of VH:VL pairs. Affinities
were calculated from competitive ELISA dilution curves. Antibody ID
Gene Family Assignment.sup.1 Affinity (K.sub.D) TT1
HV3-HD1-HJ6:KV3-KJ5 1.6 .+-. 0.1 nM TT2 HV3-HD3-HJ4:LV3-LJ1 14 .+-.
3 nM TT3 HV1-HD2-HJ4:KV3-KJ5 3.6 .+-. 1.8 nM TT4
HV2-HD2-HJ4:KV1-KJ1 2.7 .+-. 0.3 nM TT5 HV4-HD2-HJ6:KV2-KJ3 18 .+-.
4 nM TT6 HV1-HD3-HJ4:KV1-KJ2 0.57 .+-. 0.03 nM TT7
HV4-HD3-HJ4:KV1-KJ2 0.46 .+-. 0.01 nM TT8 HV3-HD3-HJ4:LV8-LJ3 2.8
.+-. 0.3 nM TT9 HV4-HD2-HJ4:KV1-KJ1 0.10 .+-. 0.01 nM TT10
HV1-HD3-HJ5:KV3-KJ5 1.6 .+-. 0.1 nM .sup.1Each heavy and light
chain was distinct.
Example 6--Bioinformatic Identification of VH:VL Sequences via
Mutual Pairing Agreement
[0086] Examples 4 and 5 disclose the identification of correct
VH:VL sequence pairs from high throughput sequencing whereby the
highest read-count VL sequence for a given VH sequence revealed the
native cognate VH:VL pairs encoded by individual B cells.
Alternatively, this example describes a method to identify correct
VH:VL pairs in high-throughput VH:VL amplicon data via consensus
pairing of both VH and VL sequences.
[0087] Raw data pairings are collected and the highest frequency VL
for each VH sequence were tabulated into File 1. The top VH for
every VL were tabulated separately into
[0088] File 2. Many computational techniques can be used to
accomplish the tabulation step; for example "grep--m 1 CDR3
filename" in Bash/Linux shell can select the top-ranked cognate
pair for a CDR-H3 or CDR-L3 sequence (CDR3) from a file (filename)
containing raw pairing data that has been pre-sorted to contain
sequences ordered by descending read counts. Other solutions for
data tabulation include the use of a hash to collect sequences and
sequence read counts (e.g. Perl computing language), or the use of
a dictionary to collect sequences and read counts (e.g. Python) or
other data storage structures (e.g. associative memories or
associative arrays). File 1 and File 2 were compared and any VH:VL
pairs appearing in both files showed "consensus" in that the pair
described by the top-ranked VL for a given VH agreed with the
top-ranked VH for a given VL. Many computational techniques can be
applied to accomplish file comparisons; one solution for file
comparison uses the "join" command in Bash/Linux where lines
containing desired fields that match across documents are printed
to standard output. The algorithm described in the present example
was effective at both identifying correct VH:VL pairs and at
reducing minor sequence errors because VH:VL pairs containing
sequence errors are often filtered out by mutual agreement
criteria. A general decision tree of the algorithm used for pairing
is provided as FIG. 9.
Example 7--VH:VL Pairing of Expanded Memory B Cells
[0089] Memory B cells were isolated and expanded in vitro, and two
aliquots of the expanded cells were processed for high-throughput
pairing. In vitro clonal expansion results in multiple copies of
cells containing the same VH:VL pairs, thus increasing the
probability of sequencing the same VH:VL pair in separate aliquots
derived from the same B cell sample.
[0090] PBMC were isolated from donated human blood and stained with
CD20-FITC (clone 2H7, BD Biosciences, Franklin Lakes, N.J., USA),
CD3-PerCP (HIT3a, BioLegend, San Diego, Calif., USA), CD19-v450
(HIB19, BD), and CD27-APC (M-T271, BD). CD3.sup.-
CD19.sup.+CD20.sup.+CD27.sup.+ memory B cells were incubated four
days in the presence of RPMI-1640 supplemented with 10% FBS,
1.times.GlutaMAX, 1.times.non-essential amino acids, 1.times.sodium
pyruvate and 1.times.penicillin/streptomycin (all from Life
Technologies) along with 10 .mu.g/mL anti-CD40 antibody (5C3,
BioLegend), 1 .mu.g/mL cPg ODN 2006 (Invivogen, San Diego, Calif.,
USA), 100 units/mL IL-4, 100 units/mL IL-10, and 50 ng/mL IL-21
(PeproTech, Rocky Hill, N.J., USA). 91,000 expanded B cells were
seeded over 12 chips, and after a 90% estimated well seeding
efficiency ratio approximately 41,000 expanded B cells were
analyzed per group (1:25 cell:well ratio) according to the methods
described in Example 4. Bioinformatic analysis was performed as
described in Example 6. 1,033 CDR-H3 sequences with .gtoreq.1 read
were sequenced in both groups, and 972/1,033 displayed matching
CDR-L3 pairs to yield a 94.09% matching fraction. Pairing accuracy,
A.sub.P can be estimated from the CDR-L3 matching fraction,
f.sub.match, of the two independent groups:
f.sub.match=A.sub.P,Group1.times.A.sub.P,Group2=A.sub.P.sup.2
A.sub.P=f.sub.match.sup.1/2
[0091] which yielded an overall accuracy of 97.0%. The theoretical
limit of accuracy from the rate of single cells per well by Poisson
distribution (98% for the 1:25 cell:well ratio utilized in this
experiment) correlated very closely with experimentally determined
accuracy of VH:VL pairings.
Example 8--The Use of Leader Peptide Primers for VH:VL Pairing
[0092] In this example, primers which anneal to the leader peptide
region of antibody cDNAs (as opposed to primers specific for the
framework 1 of the VH and VL domains, disclosed in Example 4) were
used to sequence antibody VH:VL pairs. Memory B cells were isolated
from donated human PBMC, and cells were split in two groups: Group
1 consisted of 29,000 cells and was analyzed immediately (using a
total of 510,000 wells, 1:16 cell:well ratio), while Group 2 was
expanded as described in Example 7 and 28,000 cells were analyzed
after in vitro expansion (using a total of 680,000 wells, 1:24
cell:well ratio). Both experiments were conducted as described in
Example 7 using leader peptide overlap extension primers reported
in Table 15 and emulsion linkage RT-PCR cycling with the following
conditions: 30 min at 55.degree. C., followed by 2 min at
94.degree. C.; four cycles of 94.degree. C. for 30 s denature,
54.degree. C. for 30 s anneal, 72.degree. C. for 2 min extend; 29
cycles of 94.degree. C. for 30 s denature, 60.degree. C. for 30 s
anneal, 72.degree. C. for 2 min extend; then a final extension step
for 7 min at 72 .degree. C. An additional barcoded region was also
included in the VL linkage primers (16N region) which was used to
identify multiple sequence reads of individual linkage events
(Table 15). Nested PCR was performed as in Example 5, with 25 PCR
cycles for each group.
TABLE-US-00014 TABLE 15 Overlap extension RT-PCR primers targeting
the leader peptide region of antibody mRNA. Conc. (nM) Primer ID
400 CHrev-AHX89 400 CLrev-BRH06 40 hIgG-rev-OE- AHX89 40
hIgA-rev-OE- AHX89 40 hIgM-rev-OE- AHX89 40 hIgKC-rev-OE- BRH06 40
hIgLC-rev-OE- BRH06 40 VH1_L 40 VH3_L 40 VH4/6_L 40 VH5_L 40
hV.lamda.1for_L 40 hV.lamda.2for_L 40 hV.lamda.3for_L 40
hV.lamda.3for-2_L 40 hV.lamda.3for-3_L 40 hV.lamda.4/5for_L 40
hV.lamda.6for_L 40 hV.lamda.7for_L 40 hV.lamda.8for_L 40
hV.kappa.1/2for_L 40 hV.kappa.3for_L 40 hV.kappa.4for_L
[0093] After high-throughput Illumina 2.times.250 bp sequencing of
nested PCR products, 23/23 CDR-H3 observed with .gtoreq.2 reads in
both leader peptide groups displayed matching CDR-L3. This example
demonstrates that various primer sets can be used to sequence
multiple transcripts using the methods in the present
disclosure.
Example 9--Low Dispersity, Single Cell Water-in-Oil Droplet
Formation Using a Nozzle and Annular Carrier Stream
[0094] In this example, the immortalized B cell lines MOPC-21 were
viably encapsulated in emulsion droplets of controlled size
consisting of a mixture of cells in PBS and Trypan blue stain for
cell viability visualization. This example demonstrates the
isolation of single cells into emulsion droplets of controlled size
distribution, furthermore the droplets being comprised of two
different aqueous streams which mix immediately prior to droplet
formation (FIG. 7).
[0095] MOPC-21 cells were resuspended at a concentration of 500,000
cells/mL of PBS. A coaxial emulsification apparatus was constructed
by inserting a 26-gauge needle (Hamilton Company, Reno, Nev., USA)
within 19-gauge hypodermic tubing (Hamilton) and the needle was
adjusted so that the needle tip was flush with the end of the
hypodermic tubing. The concentric needles were placed inside 3/8
inch OD glass tubing (Wale Apparatus, Hellertown, Pa., USA) with a
140 .mu.m orifice such that the needle exit is approximately 2 mm
from the nozzle orifice. The aqueous PBS/cell solution was injected
through the needle at a rate of 500 .mu.L/min, while a PBS/0.4%
Trypan blue solution (Sigma-Aldrich, St. Louis, Mo., USA) was
injected through the 19 ga hypodermic tubing, and an oil phase
(molecular biology grade mineral oil with 4.5% Span-80, 0.4% Tween
80, 0.05% Triton X-100, v/v %, Sigma Aldrich Corp.) was passed
through the glass tubing at a rate of 3 mL/min. Droplets suspended
in oil phase were collected into a 2 mL Eppendorf tube. A syringe
pump (KD Scientific Legato 200, Holliston, Mass., USA) was used to
control aqueous flow rates and a gear pump (M-50, Valco
Instruments, Houston, Tex., USA) was used to control oil flow
rates, and the resulting emulsions were analyzed via light
microscopy. Droplets with a mean diameter of approximately 85 .mu.m
were generated and encapsulated single cells displayed high
viability as measured by exclusion of trypan blue (FIG. 5).
Example 10--Sequencing Multiple Transcripts in B Cells via
Encapsulation in Emulsion Droplets
[0096] In this example the cell lysis and mRNA annealing to poly(T)
beads was accomplished within an emulsion generated using the
method outlined in Example 9. A population of memory B cells was
isolated and the cells expanded as in Example 7. Memory B cells
were resuspended in PBS at a concentration of 100 k/mL and passed
through the innermost, 26-gauge needle of the emulsion generator
device of Example 8 at a rate of 500 .mu.L/min. 450 poly(dT)
magnetic beads (1.0 .mu.m diameter, New England Biosciences,
Ipswich, Mass., USA) were pelleted with a magnet and resuspended in
5 mL of cell lysis/binding buffer (100 mM tris pH 7.5, 500 mM LiCl,
10 mM EDTA, 0.5% lithium dodecyl sulfate, 5 mM DTT), and the
resulting mixture was passed through the 19-gauge hypodermic tubing
at a rate of 500 .mu.L/min, while oil phase (molecular biology
grade mineral oil with 4.5% Span-80, 0.4% Tween 80, 0.05% Triton
X-100, v/v %, Sigma Aldrich Corp.) was passed through the outermost
glass tubing at a rate of 3 mL/min to generate an emulsion
consisting of aqueous droplets of approximately 85 .mu.m diameter
containing single cells. The emulsion stream was collected into 2
mL Eppendorf tubes, and cells were lysed by detergent as droplets
were generated to allow for mRNA capture onto poly(dT) magnetic
beads encapsulated within the emulsion droplets.
[0097] Each 2 mL emulsion tube was maintained at room temperature
for three minutes before being placed on ice for a minimum of ten
minutes. Then the tubes were centrifuged at 16,000.times.g for 5
minutes at 4.degree. C., and the upper mineral oil layer was
removed and discarded.
[0098] 200 .mu.L of cold diethyl ether was added to chemically
break the emulsion and the tubes were centrifuged at 16,000.times.g
for 2.5 minutes to pellet magnetic beads. Magnetic beads were
withdrawn using a pipette, pelleted, and resuspended in 2 mL
lysis/binding buffer (100 mM tris pH 7.5, 500 mM LiCl, 10 mM EDTA,
0.5% LiDS, 5 mM DTT). Beads were then washed and resuspended in OE
RT-PCR mixture as in Example 8. Leader peptide primers were used,
primer concentrations are given in Table 15. The OE RT-PCR mixture
bead suspension was emulsified and thermally cycled, cDNA was
extracted, and a nested PCR was performed (see Example 8). Nested
PCR product was electrophoresed to purify linked transcripts, which
were then sequenced as in Example 8 above.
[0099] After high-throughput Illumina 2.times.250 bp sequencing of
nested PCR products, 14,121 VH:VL pairs with .gtoreq.2 reads were
recovered according to the algorithm described in Example 6 (7,367
VH:VL pairs in Group 1, and 6,754 pairs in Group 2). 3,935 CDR-H3
were observed with .gtoreq.2 reads in both groups. 3,899/3,935 of
CDR-H3 observed in both groups displayed matching CDR-L3,
indicating 99.5% overall accuracy according to the formula outlined
in Example 7. The present example demonstrates the sequencing of
multiple transcripts via mRNA capture from single cells isolated
within emulsion droplets.
Example 11--Parallel Sequencing of Heavy and Light Chain cDNAs from
Single Cells
[0100] Previous examples demonstrated the use of magnetic beads to
capture mRNA and covalent linkage of desired cDNAs from a single
cell (e.g., VH and VL cDNAs) to create a single amplicon. The
single VH-VL amplicons thus generated were sequenced by high
throughput DNA sequencing to reveal the repertoire of naturally
paired VH and VL sequences.
[0101] In the example, the cDNAs captured onto beads were sequenced
directly without linking (i.e. without creating a linked VH-VL
amplicon). In this manner, the identity of the desired transcripts
from a single cell was revealed without the need for overlap
extension PCR. First, an equal mixture of three 5'-amine
functionalized primers (Table 17) was conjugated to functionalized
magnetic beads so that the immobilized oligonucleotides on each
magnetic bead were in the following proportion: 1/3 poly(T) for
mRNA capture, 1/3 primer specific for desired transcript 1 (e.g.,
the AHX89 primer of Table 1,), and 1/3 primer specific for desired
transcript 2 (Table 17). These primer-conjugated magnetic beads
served a dual purpose: first, upon lysis, poly(T) primers captured
heavy and light chain mRNA from individual cells, as in Examples
4-6; second, in the emulsion RT-PCR step, AHX89 and BRHO6 primers
caused heavy and light chain cDNA to amplify on the bead surface.
After RT-PCR, magnetic beads were used as sequencing template for
high-throughput sequencing. The process is outlined in FIG. 7.
[0102] An equal mixture of three 5'-amine oligonucleotides (Table
16) was immobilized to functionalized magnetic beads according to
manufacturer protocols (Dynal MyOne Carboxylic Acid beads, 1.0
.mu.m diameter, Invitrogen Corp.). Then, a mixture of MOPC-21 and
MOPC-315 immortalized cells were washed and suspended at 100,000
cells/mL in PBS (pH 7.4). 1.2.times.10.sup.8 functionalized
magnetic beads were added per mL of cell lysis/mRNA binding
solution, as outlined in Example 10. The cell/bead suspension was
emulsified as in Example 10, cells are lysed and mRNA anneals to
beads. Then beads were recovered by breaking the emulsion, washed
as described in Example 10, and emulsion RT-PCR was performed.
RT-PCR primer concentrations are given in Table 17. Cycling
conditions were as follows: 30 min at 55.degree. C., followed by 2
min at 94.degree. C.; four cycles of 94.degree. C. for 30 s
denature, 57.degree. C. for 1 min anneal, 72.degree. C. for 2 min
extend; 29 cycles of 94.degree. C. for 30 s denature, 59.degree. C.
for 30 s anneal, 72.degree. C. for 2 min extend; then a final
extension step for 7 min at 72.degree. C.
TABLE-US-00015 TABLE 16 Primers conjugated to the magnetic bead
surface. Conc. Primer ID 33% oligodT(25)- 5'amine 33% CHrev-AHX89-
5'amine 33% CLrev-BRH06- 5'amine
TABLE-US-00016 TABLE 17 Primers in the MOPC-21/MOPC-315 RT-PCR mix.
Conc. Primer ID 400 CHrev-AHX89 400 CLrev-BRH06 40
MOPC21-CHrev-AHX89 40 MOPC21-CLrev-BRH06 40 MOPC315-CLrev-BRH06 40
MOPC315-CHrev-AHX89 400 MOPC21-VH-OE-5'<F3> 400
MOPC21-VL-OE-5'<F5> 400 MOPC315-VH-OE-5'<F3> 400
MOPC315-VL-OE-5'<F5>
[0103] After emulsion RT-PCR, the emulsion was broken with
n-butanol according to SOLiD gene sequencing manufacturer protocols
(Applied Biosystems), and magnetic beads were submitted as direct
template for the Ion Torrent sequencing platform (Life
Technologies). Sequencing was initiated first with the <F3>
heavy chain primer to collect heavy chain cDNA sequences, followed
by sequencing with the <F5> light chain primer to collect
light chain cDNA sequences. The heavy and light chain sequences
were matched by location on the Ion Torrent sequencing platform to
obtain the native heavy and light chain pairings.
Example 12--Sequencing of Paired VH:VL Transcripts from Cells
Encoding High-Affinity Antibodies
[0104] Previous examples detailed the use of various techniques for
sequencing multiple transcripts from a variety of cell populations.
The present example describes a method for high-throughput
sequencing natively paired VH:VL antibody sequences from only cells
encoding high affinity antibodies specific to a particular antigen
of interest using antigen-dependent poly(dT) capture and subsequent
VH:VL sequencing.
[0105] Antigen-coated magnetic beads were prepared by covalently
coupling free vaccine-grade tetanus toxoid (TT) (1 mM
oligonucleotide, 40 nM TT, Statens Serum Institut, Copenhagen,
Denmark) to carboxylic acid-functionalized magnetic beads (1 .mu.m
diameter Dynal MyOne COOH beads, Life Technologies) according to
manufacturer protocols.
[0106] PBMC were collected from donated blood 14 d after
administration of tetanus toxoid (TT)/diphteria toxoid boost
vaccination (TD; 20 I.E. TT and 2 I.E. diphteria toxoid, Sanofi
Pasteur MSD GmbH, Leimen, Germany) and sorted via labeled antibody
staining and FACS sorting, as in Example 7. Memory B cells were
seeded into sterile PDMS slides as described in Example 4 along
with antigen-coated beads (approximately 40 beads/well), and cells
were sealed inside the wells using a dialysis membrane and cultured
inside the PDMS microwell slides for four days in memory B cell
stimulation media: RPMI-1640 supplemented with 10%
immunoglobulin-depleted FBS, 1.times.GlutaMAX,
1.times.non-essential amino acids, 1.times.sodium pyruvate and
1.times.penicillin/streptomycin (all from Life Technologies) along
with 10 .mu.g/mL anti-CD40 antibody (5C3, BioLegend), 500 U/ml
IL-4, and 5 ng/ml IL-5 (PeproTech, Rocky Hill, N.J., USA). During
this time, the cells were stimulated to secrete antibody
(Taubenheim et al., 2012), and any secreted antibody specific to TT
became bound to magnetic microbeads containing immobilized
antigen.
[0107] A solution of 5' streptavidin-labeled poly(dT).sub.25
oligonuclotides (Integrated DNA Technologies, USA) was mixed in an
equimolar ratio with goat anti-human IgG-biotin conjugate (B1140,
Sigma-Alrich, USA). The streptavidin and biotin associated in
solution to form anti-IgG antibodies with tethered poly(dT).sub.25
oligonucleotides for mRNA capture. After four days in culture, the
seal was broken and the slide surface was washed gently with 400
.mu.L PBS three times to wash away secreted antibodies without
disturbing cells and beads inside wells. Excess PBS was removed and
350 .mu.L of RPMI-1640 media containing 10 nM anti-IgG
antibody/poly(dT).sub.25 conjugate was added to the microwell slide
surface and the slide was incubated at room temp for 45 minutes.
Over the course of the 45 min incubation, any antigen-labeled
microbeads which had been coated by anti-TT antibodies following
the 4-day secretion phase (ie antigen-labeled microbeads
co-localized in a well with a secreting cell that encoded a
specific antibody for TT) became decorated with poly(dT).sub.25 for
mRNA capture. Subsequently the slides were gently washed three
times with 400 uL PBS to remove excess antibody/oligonucleotide
conjugate and microwells were sealed with a dialysis membrance,
cells were lysed, beads were recovered with a magnet, and emulsion
linkage RT-PCR was performed as in NEW Example 3, with the
exception that 0.1% lithium dodecyl sulfate was used in the cell
lysis buffer instead of 1% lithium dodecyl sulfate. Nested PCR was
performed and linked transcripts were sequenced using a long-read
Next Generation sequencing platform, as in NEW Example 5.
[0108] The process outlined in the present method enriched the
sequence set for high-affinity antigen-specific VH:VL pairs, as
only the antigen-labeled beads with bound IgG immunoglobulin
contained the poly(dT).sub.25 sequence required for mRNA capture
after cell lysis. Thus, the method outlined in this example
demonstrates the application of the high-throughput VH:VL pairings
technique for sequencing of a large number of antigen-specific
VH:VL pairs in a single experiment without the need for surface
expression of immunoglobulin.
Example 13--RT-PCR on Single Cells Emulsified Using a Low
Dispersity Droplet Emulsion
[0109] As in Example 6, an emulsion was formed by injecting aqueous
stream out of a nozzle into a fast-moving annular oil phase. Shear
forces generated by the carrier stream induced aqueous droplet
formation with a tightly controlled size distribution, and the
nozzle/carrier stream method generated emulsions of monodisperse
droplet sizes which reduces the incidence of multiple cells per
emulsion droplet caused by a range of droplet sizes. In this
example, a mixture of two immortalized cell lines (MOPC-21
andMOPC-315) was used to demonstrate cell encapsulation and linkage
RT-PCR directly in emulsion droplets of approximately 4 nL volume
without intermediate cell lysis or mRNA capture steps.
[0110] An equal mix of RNAse-treated and washed MOPC-21 and
MOPC-315 cells (as in Example 2) were resuspended at a
concentration of 50,000 total cells/mL in PBS, while another
aqueous phase was prepared consisting of 2.times. concentrated
RT-PCR mixture (Quanta OneStep Fast qRT-PCR) with 0.1% BSA
(Invitrogen Ultrapure BSA, 50 mg/mL), 4% SuperAse In RNAse
inhibitor (Invitrogen, USA), and 0.1% NP-40 detergent. An
emulsification apparatus was prepared as in Example 10. All needles
and needle supply tubes were pre-blocked in 1% BSA for 30 minutes
and rinsed with PBS, and cells in PBS were delivered through the
inner (26 gauge) needle while RT-PCR mixture and detergent was
delivered via the outer (19 gauge) needle, with both aqueous phases
being 500 .mu.L/min. Oil carrier phase (molecular biology grade
mineral oil with 4.5% Span-80, 0.4% Tween 80, 0.05% Triton X-100,
v/v %, oil phase reagents from Sigma Aldrich Corp.) flowed through
the outer glass tubing at a rate of 3 mL/min and samples were
collected as in Example 10. A total of 2 mL of the cell/RT-PCR
mixture mixed with 2 mL of NP-40 diluent was emulsified for
approximately 100,000 cells analyzed. Primer concentrations for the
RT-PCR mixture are given in Table 1, with the same thermal cycling
conditions being used as those in Example 11.
[0111] The cell emulsion for RT-PCR was then placed into 96-well
plates and thermally cycled, cDNA was extracted, and a nested PCR
reaction was performed (see Example 4). Nested PCR primers are
given in Table 2, and thermal cycling conditions for the PCR were
as follows: a 2 min denaturing step at 94.degree. C., followed by
thermal cycling at 94.degree. C. for 30 s denature, 62.degree. C.
for 30 s anneal, 72.degree. C. for 20 s extend, for 30 cycles.
Nested PCR product was electrophoresed to purify linked VH-VL cDNA,
which was submitted as template for NextGen sequencing.
[0112] Therefore, the present invention is well adapted to attain
the ends and advantages mentioned as well as those that are
inherent therein. The particular embodiments disclosed above are
illustrative only, as the present invention may be modified and
practiced in different but equivalent manners apparent to those
skilled in the art having the benefit of the teachings herein.
Furthermore, no limitations are intended to the details of
construction or design herein shown, other than as described in the
claims below. It is therefore evident that the particular
illustrative embodiments disclosed above may be altered or modified
and all such variations are considered within the scope and spirit
of the present invention. While compositions and methods are
described in terms of "comprising," "containing," or "including"
various components or steps, the compositions and methods can also
"consist essentially of" or "consist of" the various components and
steps. All numbers and ranges disclosed above may vary by some
amount. Whenever a numerical range with a lower limit and an upper
limit is disclosed, any number and any included range falling
within the range is specifically disclosed. In particular, every
range of values (of the form, "from about a to about b," or,
equivalently, "from approximately a to b," or, equivalently, "from
approximately a-b") disclosed herein is to be understood to set
forth every number and range encompassed within the broader range
of values. Also, the terms in the claims have their plain, ordinary
meaning unless otherwise explicitly and clearly defined by the
patentee. Moreover, the indefinite articles "a" or "an," as used in
the claims, are defined herein to mean one or more than one of the
element that it introduces. If there is any conflict in the usages
of a word or term in this specification and one or more patent or
other documents that may be incorporated herein by reference, the
definitions that are consistent with this specification should be
adopted.
[0113] All of the methods disclosed and claimed herein can be made
and executed without undue experimentation in light of the present
disclosure. While the compositions and methods of this invention
have been described in terms of preferred embodiments, it will be
apparent to those of skill in the art that variations may be
applied to the methods and in the steps or in the sequence of steps
of the method described herein without departing from the concept,
spirit and scope of the invention. More specifically, it will be
apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope and
concept of the invention as defined by the appended claims.
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