U.S. patent application number 16/992595 was filed with the patent office on 2021-01-07 for methods for adding adapters to nucleic acids and compositions for practicing the same.
The applicant listed for this patent is Takara Bio USA, Inc.. Invention is credited to Craig Betts, Nathalie Bolduc, George G. Jokhadze, Steve Oh.
Application Number | 20210002633 16/992595 |
Document ID | / |
Family ID | |
Filed Date | 2021-01-07 |
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United States Patent
Application |
20210002633 |
Kind Code |
A1 |
Betts; Craig ; et
al. |
January 7, 2021 |
Methods for Adding Adapters to Nucleic Acids and Compositions for
Practicing the Same
Abstract
Provided are methods of adding adapters to nucleic acids. The
methods include combining in a reaction mixture a template
ribonucleic acid (RNA), a template switch oligonucleotide including
a 3' hybridization domain and a sequencing platform adapter
construct, a polymerase, and dNTPs. The reaction mixture components
are combined under conditions sufficient to produce a product
nucleic acid that includes the template RNA and the template switch
oligonucleotide each hybridized to adjacent regions of a single
product nucleic acid that includes a region polymerized from the
dNTPs by the polymerase. Aspects of the invention further include
compositions and kits.
Inventors: |
Betts; Craig; (Mountain
View, CA) ; Oh; Steve; (St. Louis, MO) ;
Jokhadze; George G.; (Mountain View, CA) ; Bolduc;
Nathalie; (Mountain View, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Takara Bio USA, Inc. |
Mountain View |
CA |
US |
|
|
Appl. No.: |
16/992595 |
Filed: |
August 13, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15469364 |
Mar 24, 2017 |
10781443 |
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16992595 |
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14478978 |
Sep 5, 2014 |
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15469364 |
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61979852 |
Apr 15, 2014 |
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61892372 |
Oct 17, 2013 |
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Current U.S.
Class: |
1/1 |
International
Class: |
C12N 15/10 20060101
C12N015/10; C12Q 1/6853 20060101 C12Q001/6853 |
Claims
1-45. (canceled)
46. A method comprising: (a) providing a precursor ribonucleic acid
(RNA); (b) adding a plurality of non-templated nucleotides to an
end of the precursor RNA to produce a template RNA; (c) combining:
the template RNA; a template switch oligonucleotide (TSO)
comprising a first nucleotide sequence domain; a polymerase having
terminal transferase activity; a first strand primer comprising a
first domain that hybridizes to the non-templated nucleotides in
the template RNA and a second domain having a nucleotide sequence
that is different from the first nucleotide sequence domain present
in the TSO; and dNTPs; in a reaction mixture under conditions
sufficient to produce a product nucleic acid comprising the
template RNA and the TSO hybridized to adjacent regions of a single
product nucleic acid comprising the first strand primer and a
region polymerized from the dNTPs by the polymerase; and (d)
amplifying the product nucleic acid with a forward primer and a
reverse primer, wherein each of the forward primer and the reverse
primer comprises a sequencing platform adapter construct comprising
at least a portion of a capture sequence that is utilized by a
sequencing platform, wherein the capture sequence specifically
hybridizes to a surface-attached sequencing platform
oligonucleotide on the sequencing platform, and wherein the forward
and reverse primers are different and hybridize to different
sequences of the product nucleic acid.
47. The method according to claim 46, further comprising sequencing
the amplified product nucleic acid by the sequencing platform that
comprises the surface-attached sequencing platform oligonucleotide
that captures the at least portion of the capture sequence of the
sequencing platform adapter construct.
48. The method according to claim 46, wherein the non-templated
nucleotides are added to the 3' end of the precursor RNA.
49. The method according to claim 46, wherein the non-templated
nucleotides are added in an enzymatic reaction.
50. The method according to claim 46, wherein the non-templated
nucleotides comprise a polyadenylation (poly A) sequence.
51. The method according to claim 46, wherein the precursor RNA is
a non-polyadenylated RNA.
52. The method according to claim 51, wherein the
non-polyadenylated RNA is selected from the group consisting of a
microRNA, a siRNA, and a small RNA.
53. The method according to claim 46, wherein the polymerase is a
reverse transcriptase.
54. The method according to claim 46, wherein the template switch
oligonucleotide, the first strand primer, or both the template
switch oligonucleotide and the first strand primer comprise a
sequencing platform adapter construct that is utilized by a
sequencing platform.
55. The method according to claim 54, wherein the sequencing
platform adapter construct comprises a nucleic acid domain selected
from the group consisting of: a domain that specifically hybridizes
to the surface-attached sequencing platform oligonucleotide, a
sequencing primer binding domain, a barcode domain, a barcode
sequencing primer binding domain, a molecular identification
domain, and combinations thereof.
56. A kit, the kit comprising: a) a template switch oligonucleotide
(TSO) comprising a 3' hybridization domain; b) a first polymerase;
c) a first strand primer comprising a first domain that hybridizes
to a template RNA; d) dNTPs; and e) a second polymerase.
57. The kit according to claim 56, wherein the first polymerase
further comprises terminal transferase activity and template
switching activity
58. The kit according to claim 56, wherein the first polymerase is
a reverse transcriptase.
59. The kit according to claim 58, wherein the reverse
transcriptase is a MMLV reverse transcriptase.
60. The kit according to claim 56, wherein the second polymerase
adds dNTPs to the 3' end of a precursor RNA to produce the template
RNA.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/469,364 filed Mar. 24, 2017; which application is a
continuation of U.S. application Ser. No. 14/478,978 filed Sep. 5,
2014; which application, pursuant to 35 U.S.C. .sctn. 119 (e),
claims priority to the filing date of the U.S. Provisional Patent
Application Ser. No. 61/892,372 filed Oct. 17, 2013 and U.S.
Provisional Patent Application Ser. No. 61/979,852 filed Apr. 15,
2014; the disclosures of which applications are herein incorporated
by reference.
INTRODUCTION
[0002] Massively parallel (or "next generation") sequencing
platforms are rapidly transforming data collection and analysis in
genome, epigenome and transcriptome research. Certain sequencing
platforms, such as those marketed by Illumina.RTM., Ion
Torrent.TM., Roche.TM., and Life Technologies.TM., involve solid
phase amplification of polynucleotides of unknown sequence. Solid
phase amplification of these polynucleotides is typically performed
by first ligating known adapter sequences to each end of the
polynucleotide. The double-stranded polynucleotide is then
denatured to form a single-stranded template molecule. The adapter
sequence on the 3' end of the template is hybridized to an
extension primer that is immobilized on the solid substrate, and
amplification is performed by extending the immobilized primer. In
what is often referred to as "bridge PCR", a second immobilized
primer, identical to the 5' end of the template, serves as a
reverse primer, allowing amplification of both the forward and
reverse strands to proceed on the solid substrate, e.g., a bead or
surface of a flow cell.
[0003] A disadvantage of ligation-based approaches for sequencing
adapter addition is the number of steps involved, including the
enzymatic and wash steps that are needed to prepare the target
polynucleotide before solid phase amplification can be initiated.
As one example, after ligation of the adapter sequences, unused
adapter molecules must be separated from the ligated
polynucleotides before adding the mixture to the flow cell.
Otherwise, the unused adapter molecules can also hybridize to the
immobilized primers, preventing efficient hybridization of the
primers to the template molecules and subsequent extension.
[0004] An additional drawback of ligation-based approaches is their
lack of directionality, which makes it difficult to have different
adapters at the different ends of the nucleic acids. Moreover, the
sensitivity of such methods is low and renders them unsuitable
under circumstances where only a small amount of sample material is
available.
SUMMARY
[0005] Provided are methods of adding adapters to nucleic acids.
The methods include combining in a reaction mixture a template
ribonucleic acid (RNA), a template switch nucleic acid (e.g., a
template switch oligonucleotide) including a 3' hybridization
domain and a sequencing platform adapter construct, a polymerase,
and dNTPs. The reaction mixture components are combined under
conditions sufficient to produce a product nucleic acid that
includes the template RNA and the template switch oligonucleotide
each hybridized to adjacent regions of a single product nucleic
acid that includes a region polymerized from the dNTPs by the
polymerase. Aspects of the invention further include compositions
and kits.
BRIEF DESCRIPTION OF THE FIGURES
[0006] FIG. 1 schematically illustrates a template switch-based
method for generating a nucleic acid having adapter constructs
according to one embodiment of the present disclosure. In this
embodiment, adapter constructs having less than all nucleic acid
domains necessary for a sequencing platform of interest are
provided by a template-switch polymerization reaction. The
remaining nucleic acid domains are provided by polymerase chain
reaction (PCR) using amplification primers that include the
remaining domains.
[0007] FIG. 2 schematically illustrates a template switch-based
method for generating a nucleic acid having adapter constructs
according to one embodiment of the present disclosure. In this
embodiment, adapters that include all nucleic acid domains
necessary for a sequencing platform of interest are provided during
a template-switch polymerization reaction.
[0008] FIG. 3 schematically illustrates a template switch-based
method for generating a nucleic acid having adapter constructs
according to one embodiment of the present disclosure. In this
embodiment, non-polyadenylated RNA is used as the starting
material. The non-polyadenylated RNA is adenylated, and the
adenylated RNA serves as the donor template in a template-switch
polymerization reaction that generates a nucleic acid having
adapter constructs. From top to bottom, SEQ ID NOs:11-12.
[0009] FIG. 4 is a graph showing that a cDNA library may be
generated using the methods of the present disclosure with various
amounts of input RNA. According to this embodiment, the cDNAs that
make up the library have adapter constructs that enable sequencing
of the cDNAs by a sequencing platform of interest.
[0010] FIG. 5 shows adapter constructs according to one embodiment
of the present disclosure. In this embodiment, the constructs
include the P5, P7, Read 1, Read 2, and index domains which enable
paired-end sequencing of a cDNA corresponding to a template RNA on
an Illumina.RTM. sequencing platform. From top to bottom, SEQ ID
NOs:8-10.
[0011] FIG. 6 shows a comparison of sequencing data generated using
a method according to one embodiment of the present disclosure and
sequencing data generated using the traditional method of separate
cDNA amplification and library preparation steps.
DETAILED DESCRIPTION
[0012] Provided are methods of adding adapters to nucleic acids.
The methods include combining in a reaction mixture a template
ribonucleic acid (RNA), a template switch oligonucleotide including
a 3' hybridization domain and a sequencing platform adapter
construct, a polymerase, and dNTPs. The reaction mixture components
are combined under conditions sufficient to produce a product
nucleic acid that includes the template RNA and the template switch
oligonucleotide each hybridized to adjacent regions of a single
product nucleic acid that includes a region polymerized from the
dNTPs by the polymerase. Aspects of the invention further include
compositions and kits.
[0013] Before the methods of the present disclosure are described
in greater detail, it is to be understood that the methods are not
limited to particular embodiments described, as such may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting, since the scope of the
methods will be limited only by the appended claims.
[0014] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the methods. The
upper and lower limits of these smaller ranges may independently be
included in the smaller ranges and are also encompassed within the
methods, subject to any specifically excluded limit in the stated
range. Where the stated range includes one or both of the limits,
ranges excluding either or both of those included limits are also
included in the methods.
[0015] Certain ranges are presented herein with numerical values
being preceded by the term "about." The term "about" is used herein
to provide literal support for the exact number that it precedes,
as well as a number that is near to or approximately the number
that the term precedes. In determining whether a number is near to
or approximately a specifically recited number, the near or
approximating unrecited number may be a number which, in the
context in which it is presented, provides the substantial
equivalent of the specifically recited number.
[0016] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the methods belong. Although any
methods similar or equivalent to those described herein can also be
used in the practice or testing of the methods, representative
illustrative methods and materials are now described.
[0017] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference and are incorporated herein by reference
to disclose and describe the methods and/or materials in connection
with which the publications are cited. The citation of any
publication is for its disclosure prior to the filing date and
should not be construed as an admission that the present methods
are not entitled to antedate such publication by virtue of prior
invention. Further, the dates of publication provided may be
different from the actual publication dates which may need to be
independently confirmed.
[0018] It is noted that, as used herein and in the appended claims,
the singular forms "a", "an", and "the" include plural referents
unless the context clearly dictates otherwise. It is further noted
that the claims may be drafted to exclude any optional element. As
such, this statement is intended to serve as antecedent basis for
use of such exclusive terminology as "solely," "only" and the like
in connection with the recitation of claim elements, or use of a
"negative" limitation.
[0019] It is appreciated that certain features of the methods,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the methods, which are,
for brevity, described in the context of a single embodiment, may
also be provided separately or in any suitable sub-combination. All
combinations of the embodiments are specifically embraced by the
present invention and are disclosed herein just as if each and
every combination was individually and explicitly disclosed, to the
extent that such combinations embrace operable processes and/or
devices/systems/kits. In addition, all sub-combinations listed in
the embodiments describing such variables are also specifically
embraced by the present methods and are disclosed herein just as if
each and every such sub-combination was individually and explicitly
disclosed herein.
[0020] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present methods. Any recited method
can be carried out in the order of events recited or in any other
order which is logically possible.
Methods
[0021] Methods of adding adapters to nucleic acids are provided.
The methods utilize the ability of certain nucleic acid polymerases
to "template switch," using a first ribonucleic acid (RNA) strand
as a template for polymerization, and then switching to a second
template nucleic acid strand (which may be referred to as a
"template switch nucleic acid" or an "acceptor template") while
continuing the polymerization reaction. The result is the synthesis
of a hybrid nucleic acid strand with a 5' region complementary to
the first template nucleic acid strand and a 3' region
complementary to the template switch nucleic acid. In certain
aspects, the nucleotide sequence of all or a portion (e.g., a 5'
region) of the template switch oligonucleotide may be defined by a
practitioner of the subject methods such that the newly-synthesized
hybrid nucleic acid strand has a partial or complete sequencing
platform adapter sequence at its 3' end useful for sequencing the
hybrid nucleic acid strand using a sequencing platform of interest.
Sequencing platforms of interest include, but are not limited to,
the HiSeq.TM., MiSeq.TM. and Genome Analyzer.TM. sequencing systems
from Illumina.RTM.; the Ion PGM.TM. and Ion Proton.TM. sequencing
systems from Ion Torrent.TM.; the PACBIO RS II sequencing system
from Pacific Biosciences, the SOLiD sequencing systems from Life
Technologies.TM., the 454 GS FLX+ and GS Junior sequencing systems
from Roche, or any other sequencing platform of interest.
[0022] In certain aspects, the polymerization reaction is initiated
using a primer having a partial or complete sequencing platform
adapter sequence at its 5' end, resulting in a hybrid nucleic acid
strand having a partial or complete sequencing platform adapter
sequence at each end. The directionality of the adapters in the
hybrid nucleic acid strand may be predetermined by a practitioner
of the subject methods, e.g., by selecting the adapter sequence
present at the 5' end of the primer, and the adapter sequence
present at the 5' end of the template switch oligonucleotide. Here,
the adapter sequence present in the primer and the adapter sequence
in the template switch oligonucleotide will be present at the 5'
and 3' ends of the hybrid nucleic acid strand, respectively.
[0023] According to the methods of the present disclosure, the
reaction mixture components are combined under conditions
sufficient to produce a product nucleic acid that includes the
template RNA and the template switch oligonucleotide each
hybridized to adjacent regions of a single product nucleic acid
that includes a region polymerized from the dNTPs by the
polymerase.
[0024] By "conditions sufficient to produce a product nucleic acid"
is meant reaction conditions that permit polymerase-mediated
extension of a 3' end of a nucleic acid strand hybridized to the
template RNA, template switching of the polymerase to the template
switch oligonucleotide, and continuation of the extension reaction
using the template switch oligonucleotide as the template.
Achieving suitable reaction conditions may include selecting
reaction mixture components, concentrations thereof, and a reaction
temperature to create an environment in which the polymerase is
active and the relevant nucleic acids in the reaction interact
(e.g., hybridize) with one another in the desired manner. For
example, in addition to the template RNA, the polymerase, the
template switch oligonucleotide and dNTPs, the reaction mixture may
include buffer components that establish an appropriate pH, salt
concentration (e.g., KCl concentration), metal cofactor
concentration (e.g., Mg.sup.2+ or Mn.sup.2+ concentration), and the
like, for the extension reaction and template switching to occur.
Other components may be included, such as one or more nuclease
inhibitors (e.g., an RNase inhibitor and/or a DNase inhibitor), one
or more additives for facilitating amplification/replication of GC
rich sequences (e.g., GC-Melt.TM. reagent (Clontech Laboratories,
Inc. (Mountain View, Calif.)), betaine, DMSO, ethylene glycol,
1,2-propanediol, or combinations thereof), one or more molecular
crowding agents (e.g., polyethylene glycol, or the like), one or
more enzyme-stabilizing components (e.g., DTT present at a final
concentration ranging from 1 to 10 mM (e.g., 5 mM)), and/or any
other reaction mixture components useful for facilitating
polymerase-mediated extension reactions and template-switching.
[0025] The reaction mixture can have a pH suitable for the primer
extension reaction and template-switching. In certain embodiments,
the pH of the reaction mixture ranges from 5 to 9, such as from 7
to 9, including from 8 to 9, e.g., 8 to 8.5. In some instances, the
reaction mixture includes a pH adjusting agent. pH adjusting agents
of interest include, but are not limited to, sodium hydroxide,
hydrochloric acid, phosphoric acid buffer solution, citric acid
buffer solution, and the like. For example, the pH of the reaction
mixture can be adjusted to the desired range by adding an
appropriate amount of the pH adjusting agent.
[0026] The temperature range suitable for production of the product
nucleic acid may vary according to factors such as the particular
polymerase employed, the melting temperatures of any optional
primers employed, etc. According to one embodiment, the polymerase
is a reverse transcriptase (e.g., an MMLV reverse transcriptase)
and the reaction mixture conditions sufficient to produce the
product nucleic acid include bringing the reaction mixture to a
temperature ranging from 4.degree. C. to 72.degree. C., such as
from 16.degree. C. to 70.degree. C., e.g., 37.degree. C. to
50.degree. C., such as 40.degree. C. to 45.degree. C., including
42.degree. C.
[0027] The template ribonucleic acid (RNA) may be a polymer of any
length composed of ribonucleotides, e.g., 10 bases or longer, 20
bases or longer, 50 bases or longer, 100 bases or longer, 500 bases
or longer, 1000 bases or longer, 2000 bases or longer, 3000 bases
or longer, 4000 bases or longer, 5000 bases or longer or more
bases. In certain aspects, the template ribonucleic acid (RNA) is a
polymer composed of ribonucleotides, e.g., 10 bases or less, 20
bases or less, 50 bases or less, 100 bases or less, 500 bases or
less, 1000 bases or less, 2000 bases or less, 3000 bases or less,
4000 bases or less, or 5000 bases or less. The template RNA may be
any type of RNA (or sub-type thereof) including, but not limited
to, a messenger RNA (mRNA), a microRNA (miRNA), a small interfering
RNA (siRNA), a transacting small interfering RNA (ta-siRNA), a
natural small interfering RNA (nat-siRNA), a ribosomal RNA (rRNA),
a transfer RNA (tRNA), a small nucleolar RNA (snoRNA), a small
nuclear RNA (snRNA), a long non-coding RNA (lncRNA), a non-coding
RNA (ncRNA), a transfer-messenger RNA (tmRNA), a precursor
messenger RNA (pre-mRNA), a small Cajal body-specific RNA (scaRNA),
a piwi-interacting RNA (pi RNA), an endoribonuclease-prepared si
RNA (esiRNA), a small temporal RNA (stRNA), a signal recognition
RNA, a telomere RNA, a ribozyme, or any combination of RNA types
thereof or subtypes thereof.
[0028] The RNA sample that includes the template RNA may be
combined into the reaction mixture in an amount sufficient for
producing the product nucleic acid. According to one embodiment,
the RNA sample is combined into the reaction mixture such that the
final concentration of RNA in the reaction mixture is from 1
fg/.mu.L to 10 .mu.g/.mu.L, such as from 1 pg/.mu.L to 5
.mu.g/.mu.L, such as from 0.001 .mu.g/.mu.L to 2.5 .mu.g/.mu.L,
such as from 0.005 .mu.g/.mu.L to 1 .mu.g/.mu.L, such as from 0.01
.mu.g/.mu.L to 0.5 .mu.g/.mu.L, including from 0.1 .mu.g/.mu.L to
0.25 .mu.g/.mu.L. In certain aspects, the RNA sample that includes
the template RNA is isolated from a single cell. In other aspects,
the RNA sample that includes the template RNA is isolated from 2,
3, 4, 5, 6, 7, 8, 9, 10 or more, 20 or more, 50 or more, 100 or
more, or 500 or more cells. According to certain embodiments, the
RNA sample that includes the template RNA is isolated from 500 or
less, 100 or less, 50 or less, 20 or less, 10 or less, 9, 8, 7, 6,
5, 4, 3, or 2 cells.
[0029] The template RNA may be present in any nucleic acid sample
of interest, including but not limited to, a nucleic acid sample
isolated from a single cell, a plurality of cells (e.g., cultured
cells), a tissue, an organ, or an organism (e.g., bacteria, yeast,
or the like). In certain aspects, the nucleic acid sample is
isolated from a cell(s), tissue, organ, and/or the like of a mammal
(e.g., a human, a rodent (e.g., a mouse), or any other mammal of
interest). In other aspects, the nucleic acid sample is isolated
from a source other than a mammal, such as bacteria, yeast, insects
(e.g., drosophila), amphibians (e.g., frogs (e.g., Xenopus)),
viruses, plants, or any other non-mammalian nucleic acid sample
source.
[0030] Approaches, reagents and kits for isolating RNA from such
sources are known in the art. For example, kits for isolating RNA
from a source of interest--such as the NucleoSpin.RTM.,
NucleoMag.RTM. and NucleoBond.RTM. RNA isolation kits by Clontech
Laboratories, Inc. (Mountain View, Calif.)--are commercially
available. In certain aspects, the RNA is isolated from a fixed
biological sample, e.g., formalin-fixed, paraffin-embedded (FFPE)
tissue. RNA from FFPE tissue may be isolated using commercially
available kits--such as the NucleoSpin.RTM. FFPE RNA kits by
Clontech Laboratories, Inc. (Mountain View, Calif.).
[0031] In certain aspects, the subject methods include producing
the template RNA from a precursor RNA. For example, when it is
desirable to control the size of the template RNA that is combined
into the reaction mixture, an RNA sample isolated from a source of
interest may be subjected to shearing/fragmentation, e.g., to
generate a template RNA that is shorter in length as compared to a
precursor non-sheared RNA (e.g., a full-length mRNA) in the
original sample. The template RNA may be generated by a
shearing/fragmentation strategy including, but not limited to,
passing the sample one or more times through a micropipette tip or
fine-gauge needle, nebulizing the sample, sonicating the sample
(e.g., using a focused-ultrasonicator by Covaris, Inc. (Woburn,
Mass.)), bead-mediated shearing, enzymatic shearing (e.g., using
one or more RNA-shearing enzymes), chemical based fragmentation,
e.g., using divalent cations, fragmentation buffer (which may be
used in combination with heat) or any other suitable approach for
shearing/fragmenting a precursor RNA to generate a shorter template
RNA. In certain aspects, the template RNA generated by
shearing/fragmentation of a starting nucleic acid sample has a
length of from 10 to 20 nucleotides, from 20 to 30 nucleotides,
from 30 to 40 nucleotides, from 40 to 50 nucleotides, from 50 to 60
nucleotides, from 60 to 70 nucleotides, from 70 to 80 nucleotides,
from 80 to 90 nucleotides, from 90 to 100 nucleotides, from 100 to
150 nucleotides, from 150 to 200, from 200 to 250 nucleotides in
length, or from 200 to 1000 nucleotides or even from 1000 to 10,000
nucleotides, for example, as appropriate for the sequencing
platform chosen.
[0032] Additional strategies for producing the template RNA from a
precursor RNA may be employed. For example, producing the template
RNA may include adding nucleotides to an end of the precursor RNA.
In certain aspects, the precursor RNA is a non-polyadenylated RNA
(e.g., a microRNA, small RNA, or the like), and producing the
template RNA includes adenylating (e.g., polyadenylating) the
precursor RNA. Adenylating the precursor RNA may be performed using
any convenient approach. According to certain embodiments, the
adenylation is performed enzymatically, e.g., using Poly(A)
polymerase or any other enzyme suitable for catalyzing the
incorporation of adenine residues at the 3' terminus of the
precursor RNA. Reaction mixtures for carrying out the adenylation
reaction may include any useful components, including but not
limited to, a polymerase, a buffer (e.g., a Tris-HCL buffer), one
or more metal cations (e.g., MgCl.sub.2, MnCl.sub.2, or
combinations thereof), a salt (e.g., NaCl), one or more
enzyme-stabilizing components (e.g., DTT), ATP, and any other
reaction components useful for facilitating the adenylation of a
precursor RNA. The adenylation reaction may be carried out at a
temperature (e.g., 30.degree. C.-50.degree. C., such as 37.degree.
C.) and pH (e.g., pH 7-pH 8.5, such as pH 7.9) compatible with the
polymerase being employed, e.g., polyA polymerase. Other approaches
for adding nucleotides to a precursor RNA include ligation-based
strategies, where an RNA ligase (e.g., T4 RNA ligase) catalyzes the
covalent joining of a defined sequence to an end (e.g., the 3' end)
of the precursor RNA to produce the template RNA.
[0033] The methods of the present disclosure include combining a
polymerase into the reaction mixture. A variety of polymerases may
be employed when practicing the subject methods. The polymerase
combined into the reaction mixture is capable of template
switching, where the polymerase uses a first nucleic acid strand as
a template for polymerization, and then switches to the 3' end of a
second "acceptor" template nucleic acid strand to continue the same
polymerization reaction. In certain aspects, the polymerase
combined into the reaction mixture is a reverse transcriptase (RT).
Reverse transcriptases capable of template-switching that find use
in practicing the methods include, but are not limited to,
retroviral reverse transcriptase, retrotransposon reverse
transcriptase, retroplasmid reverse transcriptases, retron reverse
transcriptases, bacterial reverse transcriptases, group II
intron-derived reverse transcriptase, and mutants, variants
derivatives, or functional fragments thereof. For example, the
reverse transcriptase may be a Moloney Murine Leukemia Virus
reverse transcriptase (MMLV RT) or a Bombyx mori reverse
transcriptase (e.g., Bombyx mori R2 non-LTR element reverse
transcriptase). Polymerases capable of template switching that find
use in practicing the subject methods are commercially available
and include SMARTScribe.TM. reverse transcriptase available from
Clontech Laboratories, Inc. (Mountain View, Calif.). In certain
aspects, a mix of two or more different polymerases is added to the
reaction mixture, e.g., for improved processivity, proof-reading,
and/or the like. In some instances, the polymer is one that is
heterologous relative to the template, or source thereof.
[0034] The polymerase is combined into the reaction mixture such
that the final concentration of the polymerase is sufficient to
produce a desired amount of the product nucleic acid. In certain
aspects, the polymerase (e.g., a reverse transcriptase such as an
MMLV RT or a Bombyx mori RT) is present in the reaction mixture at
a final concentration of from 0.1 to 200 units/.mu.L (U/.mu.L),
such as from 0.5 to 100 U/.mu.L, such as from 1 to 50 U/.mu.L,
including from 5 to 25 U/.mu.L, e.g., 20 U/.mu.L.
[0035] In addition to a template switching capability, the
polymerase combined into the reaction mixture may include other
useful functionalities to facilitate production of the product
nucleic acid. For example, the polymerase may have terminal
transferase activity, where the polymerase is capable of catalyzing
template-independent addition of deoxyribonucleotides to the 3'
hydroxyl terminus of a DNA molecule. In certain aspects, when the
polymerase reaches the 5' end of the template RNA, the polymerase
is capable of incorporating one or more additional nucleotides at
the 3' end of the nascent strand not encoded by the template. For
example, when the polymerase has terminal transferase activity, the
polymerase may be capable of incorporating 1, 2, 3, 4, 5, 6, 7, 8,
9, 10 or more additional nucleotides at the 3' end of the nascent
DNA strand. In certain aspects, a polymerase having terminal
transferase activity incorporates 10 or less, such as 5 or less
(e.g., 3) additional nucleotides at the 3' end of the nascent DNA
strand. All of the nucleotides may be the same (e.g., creating a
homonucleotide stretch at the 3' end of the nascent strand) or at
least one of the nucleotides may be different from the other(s). In
certain aspects, the terminal transferase activity of the
polymerase results in the addition of a homonucleotide stretch of
2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the same nucleotides (e.g.,
all dCTP, all dGTP, all dATP, or all dTTP). According to certain
embodiments, the terminal transferase activity of the polymerase
results in the addition of a homonucleotide stretch of 10 or less,
such as 9, 8, 7, 6, 5, 4, 3, or 2 (e.g., 3) of the same
nucleotides. For example, according to one embodiment, the
polymerase is an MMLV reverse transcriptase (MMLV RT). MMLV RT
incorporates additional nucleotides (predominantly dCTP, e.g.,
three dCTPs) at the 3' end of the nascent DNA strand. As described
in greater detail elsewhere herein, these additional nucleotides
may be useful for enabling hybridization between the 3' end of the
template switch oligonucleotide and the 3' end of the nascent DNA
strand, e.g., to facilitate template switching by the polymerase
from the template RNA to the template switch oligonucleotide.
[0036] As set forth above, the subject methods include combining a
template switch nucleic acid into the reaction mixture. In certain
aspects, the template switch nucleic acid is a template switch
oligonucleotide. By "template switch oligonucleotide" is meant an
oligonucleotide template to which a polymerase switches from an
initial template (e.g., the template RNA in the subject methods)
during a nucleic acid polymerization reaction. In this regard, the
template RNA may be referred to as a "donor template" and the
template switch oligonucleotide may be referred to as an "acceptor
template." As used herein, an "oligonucleotide" is a
single-stranded multimer of nucleotides from 2 to 500 nucleotides,
e.g., 2 to 200 nucleotides. Oligonucleotides may be synthetic or
may be made enzymatically, and, in some embodiments, are 10 to 50
nucleotides in length. Oligonucleotides may contain ribonucleotide
monomers (i.e., may be oligoribonucleotides or "RNA
oligonucleotides") or deoxyribonucleotide monomers (i.e., may be
oligodeoxyribonucleotides or "DNA oligonucleotides").
Oligonucleotides may be 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51
to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150 or 150 to 200, up
to 500 or more nucleotides in length, for example.
[0037] The reaction mixture includes the template switch
oligonucleotide at a concentration sufficient to permit template
switching of the polymerase from the template RNA to the template
switch oligonucleotide. For example, the template switch
oligonucleotide may be added to the reaction mixture at a final
concentration of from 0.01 to 100 .mu.M, such as from 0.1 to 10
.mu.M, such as from 0.5 to 5 .mu.M, including 1 to 2 .mu.M (e.g.,
1.2 .mu.M).
[0038] The template switch oligonucleotide may include one or more
nucleotides (or analogs thereof) that are modified or otherwise
non-naturally occurring. For example, the template switch
oligonucleotide may include one or more nucleotide analogs (e.g.,
LNA, FANA, 2'-O-Me RNA, 2'-fluoro RNA, or the like), linkage
modifications (e.g., phosphorothioates, 3'-3' and 5'-5' reversed
linkages), 5' and/or 3' end modifications (e.g., 5' and/or 3'
amino, biotin, DIG, phosphate, thiol, dyes, quenchers, etc.), one
or more fluorescently labeled nucleotides, or any other feature
that provides a desired functionality to the template switch
oligonucleotide.
[0039] The template switch oligonucleotide includes a 3'
hybridization domain and a sequencing platform adapter construct.
The 3' hybridization domain may vary in length, and in some
instances ranges from 2 to 10 nts in length, such as 3 to 7 nts in
length. The sequence of the 3' hybridization may be any convenient
sequence, e.g., an arbitrary sequence, a heterpolymeric sequence
(e.g., a hetero-trinucleotide) or homopolymeric sequence (e.g., a
homo-trinucleotide, such as G-G-G), or the like. Examples of 3'
hybridization domains and template switch oligonucleotides are
further described in U.S. Pat. No. 5,962,272, the disclosure of
which is herein incorporated by reference. In addition to a 3'
hybridization domain, the template switch oligonucleotide includes
a sequencing platform adapter construct. By "sequencing platform
adapter construct" is meant a nucleic acid construct that includes
at least a portion of a nucleic acid domain (e.g., a sequencing
platform adapter nucleic acid sequence) utilized by a sequencing
platform of interest, such as a sequencing platform provided by
Illumina.RTM. (e.g., the HiSeq.TM., MiSeq.TM. and/or Genome
Analyzer.TM. sequencing systems); Ion Torrent.TM. (e.g., the Ion
PGM.TM. and/or Ion Proton.TM. sequencing systems); Pacific
Biosciences (e.g., the PACBIO RS II sequencing system); Life
Technologies.TM. (e.g., a SOLiD sequencing system); Roche (e.g.,
the 454 GS FLX+ and/or GS Junior sequencing systems); or any other
sequencing platform of interest.
[0040] In certain aspects, the sequencing platform adapter
construct includes a nucleic acid domain selected from: a domain
(e.g., a "capture site" or "capture sequence") that specifically
binds to a surface-attached sequencing platform oligonucleotide
(e.g., the P5 or P7 oligonucleotides attached to the surface of a
flow cell in an Illumina.RTM. sequencing system); a sequencing
primer binding domain (e.g., a domain to which the Read 1 or Read 2
primers of the Illumina.RTM. platform may bind); a barcode domain
(e.g., a domain that uniquely identifies the sample source of the
nucleic acid being sequenced to enable sample multiplexing by
marking every molecule from a given sample with a specific barcode
or "tag"); a barcode sequencing primer binding domain (a domain to
which a primer used for sequencing a barcode binds); a molecular
identification domain (e.g., a molecular index tag, such as a
randomized tag of 4, 6, or other number of nucleotides) for
uniquely marking molecules of interest to determine expression
levels based on the number of instances a unique tag is sequenced;
or any combination of such domains. In certain aspects, a barcode
domain (e.g., sample index tag) and a molecular identification
domain (e.g., a molecular index tag) may be included in the same
nucleic acid.
[0041] The sequencing platform adapter constructs may include
nucleic acid domains (e.g., "sequencing adapters") of any length
and sequence suitable for the sequencing platform of interest. In
certain aspects, the nucleic acid domains are from 4 to 200
nucleotides in length. For example, the nucleic acid domains may be
from 4 to 100 nucleotides in length, such as from 6 to 75, from 8
to 50, or from 10 to 40 nucleotides in length. According to certain
embodiments, the sequencing platform adapter construct includes a
nucleic acid domain that is from 2 to 8 nucleotides in length, such
as from 9 to 15, from 16-22, from 23-29, or from 30-36 nucleotides
in length.
[0042] The nucleic acid domains may have a length and sequence that
enables a polynucleotide (e.g., an oligonucleotide) employed by the
sequencing platform of interest to specifically bind to the nucleic
acid domain, e.g., for solid phase amplification and/or sequencing
by synthesis of the cDNA insert flanked by the nucleic acid
domains. Example nucleic acid domains include the P5
(5'-AATGATACGGCGACCACCGA-3') (SEQ ID NO:01), P7
(5'-CAAGCAGAAGACGGCATACGAGAT-3') (SEQ ID NO:02), Read 1 primer
(5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3') (SEQ ID NO:03) and Read 2
primer (5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3') (SEQ ID NO:04)
domains employed on the Illumina.RTM.-based sequencing platforms.
Other example nucleic acid domains include the A adapter
(5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3') (SEQ ID NO:05) and P1
adapter (5'-CCTCTCTATGGGCAGTCGGTGAT-3') (SEQ ID NO:06) domains
employed on the Ion Torrent.TM.-based sequencing platforms.
[0043] The nucleotide sequences of nucleic acid domains useful for
sequencing on a sequencing platform of interest may vary and/or
change over time. Adapter sequences are typically provided by the
manufacturer of the sequencing platform (e.g., in technical
documents provided with the sequencing system and/or available on
the manufacturer's website). Based on such information, the
sequence of the sequencing platform adapter construct of the
template switch oligonucleotide (and optionally, a first strand
synthesis primer, amplification primers, and/or the like) may be
designed to include all or a portion of one or more nucleic acid
domains in a configuration that enables sequencing the nucleic acid
insert (corresponding to the template RNA) on the platform of
interest.
[0044] According to certain embodiments, the template switch
oligonucleotide includes a modification that prevents the
polymerase from switching from the template switch oligonucleotide
to a different template nucleic acid after synthesizing the
compliment of the 5' end of the template switch oligonucleotide
(e.g., a 5' adapter sequence of the template switch
oligonucleotide). Useful modifications include, but are not limited
to, an abasic lesion (e.g., a tetrahydrofuran derivative), a
nucleotide adduct, an iso-nucleotide base (e.g., isocytosine,
isoguanine, and/or the like), and any combination thereof.
[0045] The template switch oligonucleotide may include a sequence
(e.g., a defined nucleotide sequence 5' of the 3' hybridization
domain of the template switch oligonucleotide), that enables second
strand synthesis and/or PCR amplification of the single product
nucleic acid. For example, the template switch oligonucleotide may
include a sequence, where subsequent to generating the single
product nucleic acid, second strand synthesis is performed using a
primer that has that sequence. The second strand synthesis produces
a second strand DNA complementary to the single product nucleic
acid. Alternatively, or additionally, the single product nucleic
acid may be amplified using a primer pair in which one of the
primers has that sequence. Accordingly, in certain aspects, the
methods of the present disclosure may further include producing the
product nucleic acid and contacting a 3' region of the single
product nucleic acid complementary to the template switch
oligonucleotide with a second strand primer configured to bind
thereto under hybridization conditions. Following contacting the 3'
region of the single product nucleic acid complementary to the
template switch oligonucleotide with the second strand primer, the
methods may further include subjecting the reaction mixture to
nucleic acid polymerization conditions.
[0046] The term "complementary" as used herein refers to a
nucleotide sequence that base-pairs by non-covalent bonds to all or
a region of a target nucleic acid (e.g., a region of the product
nucleic acid). In the canonical Watson-Crick base pairing, adenine
(A) forms a base pair with thymine (T), as does guanine (G) with
cytosine (C) in DNA. In RNA, thymine is replaced by uracil (U). As
such, A is complementary to T and G is complementary to C. In RNA,
A is complementary to U and vice versa. Typically, "complementary"
refers to a nucleotide sequence that is at least partially
complementary. The term "complementary" may also encompass duplexes
that are fully complementary such that every nucleotide in one
strand is complementary to every nucleotide in the other strand in
corresponding positions. In certain cases, a nucleotide sequence
may be partially complementary to a target, in which not all
nucleotides are complementary to every nucleotide in the target
nucleic acid in all the corresponding positions. For example, a
primer may be perfectly (i.e., 100%) complementary to the target
nucleic acid, or the primer and the target nucleic acid may share
some degree of complementarity which is less than perfect (e.g.,
70%, 75%, 85%, 90%, 95%, 99%). The percent identity of two
nucleotide sequences can be determined by aligning the sequences
for optimal comparison purposes (e.g., gaps can be introduced in
the sequence of a first sequence for optimal alignment). The
nucleotides at corresponding positions are then compared, and the
percent identity between the two sequences is a function of the
number of identical positions shared by the sequences (i.e., %
identity=# of identical positions/total # of positions.times.100).
When a position in one sequence is occupied by the same nucleotide
as the corresponding position in the other sequence, then the
molecules are identical at that position. A non-limiting example of
such a mathematical algorithm is described in Karlin et al., Proc.
Natl. Acad. Sci. USA 90:5873-5877 (1993). Such an algorithm is
incorporated into the NBLAST and XBLAST programs (version 2.0) as
described in Altschul et al., Nucleic Acids Res. 25:389-3402
(1997). When utilizing BLAST and Gapped BLAST programs, the default
parameters of the respective programs (e.g., NBLAST) can be used.
In one aspect, parameters for sequence comparison can be set at
score=100, wordlength=12, or can be varied (e.g., wordlength=5 or
wordlength=20).
[0047] As used herein, the term "hybridization conditions" means
conditions in which a primer specifically hybridizes to a region of
the target nucleic acid (e.g., the template RNA, the single product
nucleic acid, etc.). Whether a primer specifically hybridizes to a
target nucleic acid is determined by such factors as the degree of
complementarity between the polymer and the target nucleic acid and
the temperature at which the hybridization occurs, which may be
informed by the melting temperature (T.sub.M) of the primer. The
melting temperature refers to the temperature at which half of the
primer-target nucleic acid duplexes remain hybridized and half of
the duplexes dissociate into single strands. The T.sub.m of a
duplex may be experimentally determined or predicted using the
following formula T.sub.m=81.5+16.6(log.sub.10[Na.sup.+])+0.41
(fraction G+C)-(60/N), where N is the chain length and [Na.sup.+]
is less than 1 M. See Sambrook and Russell (2001; Molecular
Cloning: A Laboratory Manual, 3.sup.rd ed., Cold Spring Harbor
Press, Cold Spring Harbor N.Y., Ch. 10). Other more advanced models
that depend on various parameters may also be used to predict
T.sub.m of primer/target duplexes depending on various
hybridization conditions. Approaches for achieving specific nucleic
acid hybridization may be found in, e.g., Tijssen, Laboratory
Techniques in Biochemistry and Molecular Biology-Hybridization with
Nucleic Acid Probes, part I, chapter 2, "Overview of principles of
hybridization and the strategy of nucleic acid probe assays,"
Elsevier (1993).
[0048] As described above, the subject methods include combining
dNTPs into the reaction mixture. In certain aspects, each of the
four naturally-occurring dNTPs (dATP, dGTP, dCTP and dTTP) are
added to the reaction mixture. For example, dATP, dGTP, dCTP and
dTTP may be added to the reaction mixture such that the final
concentration of each dNTP is from 0.01 to 100 mM, such as from 0.1
to 10 mM, including 0.5 to 5 mM (e.g., 1 mM). According to one
embodiment, at least one type of nucleotide added to the reaction
mixture is a non-naturally occurring nucleotide, e.g., a modified
nucleotide having a binding or other moiety (e.g., a fluorescent
moiety) attached thereto, a nucleotide analog, or any other type of
non-naturally occurring nucleotide that finds use in the subject
methods or a downstream application of interest.
[0049] The addition of a primer to the reaction mixture is not
necessary when the template RNA provides a suitable substrate for
initiation of first-strand synthesis. For example, when the
template RNA has double-stranded regions and an overhang at one or
both of its ends, the "non-overhanging" strand of the dsRNA can
prime a first-strand synthesis reaction in which the overhanging
strand serves as the template. In this manner, the polymerase may
be used to "fill in" the overhang, switch to the template switch
oligonucleotide, and complete the first strand synthesis using the
template switch oligonucleotide as an acceptor template to produce
the product nucleic acid (where a terminal transferase reaction by
the polymerase optionally precedes the template switch as described
elsewhere herein). Accordingly, the addition of a primer is
obviated when the template RNA includes, e.g., an overhang at one
or both of its ends.
[0050] In certain circumstances, however, it may be desirable to
add a primer to the reaction mixture to prime the synthesis of the
single product nucleic acid. For example, if the template RNA is
single-stranded, a primer may be useful for purposes of initiating
first-strand synthesis. In addition, use of a primer can give a
practitioner of the subject methods more control over which RNA(s)
in an RNA sample will serve as the template RNA(s) for production
of the product nucleic acid, e.g., where it is desirable to produce
product nucleic acids corresponding to a template RNA of interest
(e.g., polyadenylated RNA, for which an oligo dT-based primer that
hybridizes to the polyA tail of the RNA may be used to prime the
first strand synthesis).
[0051] Accordingly, in certain aspects, the subject methods further
include contacting the template RNA with a first primer that primes
the synthesis of the single product nucleic acid. The contacting is
performed under conditions sufficient for the primer to hybridize
to the template RNA, which conditions are described elsewhere
herein. According to one embodiment, the entire sequence of the
primer is arbitrary, e.g., the primer may be a random hexamer or
any other random primer of suitable length (or mixtures thereof).
In other aspects, the primer has a defined sequence, e.g., the
primer sequence may be designed by one practicing the subject
methods to specifically hybridize to a known complementary sequence
in a template RNA of interest (e.g., a polyA tail of the template
RNA).
[0052] According to certain embodiments, the primer includes two or
more domains. For example, the primer may include a first (e.g.,
3') domain that hybridizes to the template RNA and a second (e.g.,
5') domain that does not hybridize to the template RNA. The
sequence of the first and second domains may be independently
defined or arbitrary. In certain aspects, the first domain has a
defined sequence and the sequence of the second domain is defined
or arbitrary. In other aspects, the first domain has an arbitrary
sequence (e.g., a random sequence, such as a random hexamer
sequence) and the sequence of the second domain is defined or
arbitrary. According to one embodiment, the second domain includes
a nucleotide sequence that is the same as, or different from, a
nucleotide sequence present in the template switch
oligonucleotide.
[0053] In some embodiments, the second domain of the primer
includes a sequencing platform adapter construct. The sequencing
platform adapter construct of the second domain may include a
nucleic acid domain selected from a domain (e.g., a "capture site"
or "capture sequence") that specifically binds to a
surface-attached sequencing platform oligonucleotide (e.g., the P5
or P7 oligonucleotides attached to the surface of a flow cell in an
Illumina.RTM. sequencing system), a sequencing primer binding
domain (e.g., a domain to which the Read 1 or Read 2 primers of the
Illumina.RTM. platform may bind), a barcode domain (e.g., a domain
that uniquely identifies the sample source of the nucleic acid
being sequenced to enable sample multiplexing by marking every
molecule from a given sample with a specific barcode or "tag"), a
barcode sequencing primer binding domain (a domain to which a
primer used for sequencing a barcode binds), a molecular
identification domain, or any combination of such domains.
[0054] In certain aspects, the sequencing platform adapter
construct of the second domain of the primer is different from the
sequencing platform adapter construct of the template switch
oligonucleotide. Such embodiments find use, e.g., where one wishes
to produce a single product nucleic acid (e.g., a cDNA or library
thereof) with one end having one or more sequencing platform
adapter sequences and the second end having one or more sequencing
platform adapter sequences different from the first end. Having
ends with different adapter sequences is useful, e.g., for
subsequent solid phase amplification (e.g., cluster generation
using the surface-attached P5 and P7 primers in an
Illumina.RTM.-based sequencing system), DNA sequencing (e.g., using
the Read 1 and Read 2 primers in an Illumina.RTM.-based sequencing
system), and any other steps performed by a sequencing platform
requiring different adapter sequences at opposing ends of the
nucleic acid to be sequenced. Having different ends is also useful
in providing strand specific information, since the directionality
of the sequenced strand is defined by the different ends. Current
methods in the art for doing this require multiple steps and
degradation of the undesired strand--e.g., using UDG and
incorporation of dU into the undesired strand. The current method
is far more streamlined and requires less steps, generating
strand-specific information directly.
[0055] When the methods include contacting the template RNA with a
primer that primes the synthesis of the single product nucleic
acid, the primer may include one or more nucleotides (or analogs
thereof) that are modified or otherwise non-naturally occurring.
For example, the primer may include one or more nucleotide analogs
(e.g., LNA, FANA, 2'-O-Me RNA, 2'-fluoro RNA, or the like), linkage
modifications (e.g., phosphorothioates, 3'-3' and 5'-5' reversed
linkages), 5' and/or 3' end modifications (e.g., 5' and/or 3'
amino, biotin, DIG, phosphate, thiol, dyes, quenchers, etc.), one
or more fluorescently labeled nucleotides, or any other feature
that provides a desired functionality to the primer that primes the
synthesis of the single product nucleic acid.
[0056] In certain aspects, when the methods include contacting the
template RNA with a primer that primes the synthesis of the single
product nucleic acid, it may be desirable to prevent any subsequent
extension reactions which use the single product nucleic acid as a
template from extending beyond a particular position in the region
of the single product nucleic acid corresponding to the primer. For
example, according to certain embodiments, the primer that primes
the synthesis of the single product nucleic acid includes a
modification that prevents a polymerase using the region
corresponding to the primer as a template from polymerizing a
nascent strand beyond the modification. Useful modifications
include, but are not limited to, an abasic lesion (e.g., a
tetrahydrofuran derivative), a nucleotide adduct, an iso-nucleotide
base (e.g., isocytosine, isoguanine, and/or the like), and any
combination thereof.
[0057] Any nucleic acids that find use in practicing the methods of
the present disclosure (e.g., the template switch oligonucleotide,
a primer that primes the synthesis of the single product nucleic
acid, a second strand synthesis primer, one or more primers for
amplifying the product nucleic acid, and/or the like) may include
any useful nucleotide analogues and/or modifications, including any
of the nucleotide analogues and/or modifications described
herein.
[0058] Once the product nucleic acid is produced, the methods may
include inputting the product nucleic acid directly into a
downstream application of interest (e.g., a sequencing application,
etc.). In other aspects, the methods may include using the product
nucleic acid as a template for second-strand synthesis and/or PCR
amplification (e.g., for subsequent sequencing of the amplicons).
According to one embodiment, the methods of the present disclosure
further include subjecting the product nucleic acid to nucleic acid
amplification conditions. Such conditions may include the addition
of forward and reverse primers configured to amplify all or a
desired portion of the product nucleic acid, dNTPs, and a
polymerase suitable for effecting the amplification (e.g., a
thermostable polymerase). The single product nucleic acid may have
an amplification sequence at its 5' end and an amplification
sequence at its 3' end, and be subjected to PCR amplification
conditions with primers complementary to the 5' and 3'
amplification sequences. The amplification sequences may be (or
overlap with) a nucleic acid domain in a sequencing platform
adapter construct, or may be outside of the sequencing platform
adapter construct. An initial step in carrying out the
amplification may include denaturing the product nucleic acid to
dissociate the template RNA and template switch oligonucleotide
from the single product nucleic acid, thereby making the single
product nucleic acid available for primer binding.
[0059] In certain aspects, when the single product nucleic acid is
amplified following its production, the amplification may be
carried out using a primer pair in which one or both of the primers
include a sequencing platform adapter construct. The sequencing
platform adapter construct(s) may include any of the nucleic acid
domains described elsewhere herein (e.g., a domain that
specifically binds to a surface-attached sequencing platform
oligonucleotide, a sequencing primer binding domain, a barcode
domain, a barcode sequencing primer binding domain, a molecular
identification domain, or any combination thereof). Such
embodiments finds use, e.g., where the single product nucleic does
not include all of the adapter domains useful or necessary for
sequencing in a sequencing platform of interest, and the remaining
adapter domains are provided by the primers used for the
amplification of the single product nucleic acid. An example method
according to this embodiment is shown in FIG. 1. As shown, template
RNA 102, polymerase 104, template switch oligonucleotide 106, and
dNTPs (not shown) are combined into reaction mixture 100 under
conditions sufficient to produce the product nucleic acid. Template
switch oligonucleotide 106 includes sequencing platform adapter
construct B. Although optional, the embodiment shown in FIG. 1
employs a first primer, primer 108, which is extended by the
polymerase for first strand synthesis. Primer 108 includes first
(3') domain 110 that hybridizes to the template RNA and second (5')
domain 112 that does not hybridize to the template RNA. The second
domain includes sequencing platform adapter construct A. The
nucleotide sequence of first domain 110 may be arbitrary (e.g., a
random sequence, such as a random hexamer sequence) or the sequence
of the first domain may be defined (e.g., a sequence specifically
selected to hybridize to a particular region of a particular
template RNA of interest). In this example, first domain 110 of
primer 108 is complementary to sequence 114 within template RNA
102, and second domain 112 includes sequencing platform adapter
construct A having one or more sequencing platform nucleic acid
domains (e.g., a domain that specifically binds to a
surface-attached sequencing platform oligonucleotide, a sequencing
primer binding domain, a barcode domain, a barcode sequencing
primer binding domain, a molecular identification domain, and
combinations thereof).
[0060] Upon hybridization of primer 108 to template RNA 102, first
strand synthesis proceeds when polymerase 104 extends primer 108
along template RNA 102. In this example, the polymerase has
terminal transferase activity, such that when the extension
reaction reaches the 5' end of the template RNA, the polymerase
adds an arbitrary sequence that can be homodimeric or
heterodimeric, and may range in length of nucleotides (e.g., 2 to
10 nts, such as 2 to 5 nts) such as a homonucleotide stretch (e.g.,
a homo-trinucleotide shown here as NNN) to the extension product.
According to this embodiment, template switch oligonucleotide has a
3' hybridization domain that includes a homonucleotide stretch
(shown here as a homo-trinucleotide stretch, NNN) complementary to
the homonucleotide stretch at the 3' end of the extension product.
This complementarity promotes hybridization of the 3' hybridization
domain of the template switch oligonucleotide to the 3' end of the
extension product. Hybridization brings the acceptor template
region of the template switch oligonucleotide (located 5' of the 3'
hybridization domain) within sufficient proximity of the polymerase
such that the polymerase can template switch to the acceptor
template region and continue the extension reaction to the 5'
terminal nucleotide of the template switch oligonucleotide, thereby
producing the product nucleic acid that includes the template RNA
and the template switch oligonucleotide each hybridized to adjacent
regions of the single product nucleic acid.
[0061] In this example, the template switch oligonucleotide
includes sequencing platform adapter construct B having one or more
sequencing platform nucleic acid domains (e.g., a domain that
specifically binds to a surface-attached sequencing platform
oligonucleotide, a sequencing primer binding domain, a barcode
domain, a barcode sequencing primer binding domain, a molecular
identification domain, and combinations thereof), such that the
single product nucleic acid includes sequencing platform adapter
construct A at its 5' end and sequencing platform adapter construct
B' at its 3' end. According to this embodiment, the method further
includes a second strand synthesis step, where a primer
complementary to a 3' region of the single product nucleic acid
hybridizes to the 3' region of the single product nucleic acid and
is extended by a polymerase--using the single product nucleic acid
as a template--to the 5' end of the single product nucleic acid.
The result of this second strand synthesis step is a
double-stranded DNA that includes the single product nucleic acid
and its complementary strand.
[0062] In the example shown in FIG. 1, adapter constructs A/A' and
B/B' do not include all of the sequencing platform nucleic acid
domains useful or necessary for downstream sequencing of the
nucleic acid. To add the remaining sequencing platform nucleic acid
domains, the nucleic acid is amplified using primers having adapter
constructs C and D (e.g., present in a non-hybridizing 5' region of
the primers) which provide the remaining sequencing platform
nucleic acid domains. The amplicons include adapter constructs A/A'
and C/C' at one end and adapter constructs B/B' and D/D' at the
opposite end. One practicing the subject methods may select the
sequences of the sequencing platform adapter construct of the first
strand synthesis primer, the template switch oligonucleotide, and
the amplification primers, to provide all of the necessary domains
in a suitable configuration for sequencing on a sequencing platform
of interest. As just one example, constructs A/A' and B/B' may
include sequencing primer binding domains (e.g., primer binding
domains for the Read 1 and Read 2 sequencing primers employed in
Illumina.RTM.-based sequencing platforms), while constructs C/C'
and D/D' include a domain that specifically binds to a
surface-attached sequencing platform oligonucleotide (e.g., domains
that specifically bind to the surface-attached P5 and P7 primers of
an Illumina.RTM. sequencing system). Any of adapter constructs
A/A'-D/D' may include any additional sequence elements useful or
necessary for sequencing on a sequencing platform of interest.
[0063] As summarize above, a primer having a sequencing platform
adapter construct may be used to prime the synthesis of the single
product nucleic acid, so that the single product nucleic acid has a
sequencing platform adapter construct at its 5' and 3' ends. In
certain aspects, the sequencing platform adapter constructs of the
single product nucleic acid include all of the useful or necessary
domains for sequencing the nucleic acid on a sequencing platform of
interest. As shown in FIG. 2, a product nucleic acid is produced
using an approach similar to that shown in FIG. 1. However, in the
embodiment shown in FIG. 2, sequencing adapter constructs A/A' and
B/B' include all of the sequencing platform nucleic acid domains
useful or necessary for sequencing the single product nucleic acid
on a sequencing platform of interest (e.g., a domain that
specifically binds to a surface-attached sequencing platform
oligonucleotide, a sequencing primer binding domain, a barcode
domain, a barcode sequencing primer binding domain, a molecular
identification domain, and combinations thereof). According to
certain embodiments, the single product nucleic acid is PCR
amplified prior to sequencing on the sequencing platform. In other
embodiments, the single product nucleic acid is not amplified prior
to sequencing.
[0064] A method according to an additional embodiment of the
present disclosure is shown in FIG. 3. In this example,
non-polyadenylated precursor RNA 302 undergoes 3' polyadenylation
to produce template RNA 303. In this example, first strand
synthesis is primed using an oligo(dT) primer having a sequencing
platform adapter construct (A) at its 5' end, so that the single
product nucleic acid has sequencing platform adapter constructs A
and B' at its 5' and 3' ends, respectively. The sequencing platform
adapter constructs may include less than all of the useful or
necessary domains for sequencing on a sequencing platform of
interest (e.g., similar to the embodiment shown in FIG. 1) or may
include all useful or necessary domains (e.g., similar to the
embodiment shown in FIG. 2). Embodiments such as the one shown in
FIG. 3 find use, e.g., in generating a sequencing-ready library of
cDNAs which correspond to non-polyadenylated RNAs (e.g., microRNAs,
small RNAs, siRNAs, or the like) present in a biological sample of
interest.
[0065] In certain embodiments, the subject methods may be used to
generate a cDNA library corresponding to mRNAs for downstream
sequencing on a sequencing platform of interest (e.g., a sequencing
platform provided by Illumina.RTM., Ion Torrent.TM., Pacific
Biosciences, Life Technologies.TM., Roche, or the like). In one
embodiment, mRNAs are sheared to a length of approximately 200 bp,
or any other appropriate length as defined by the sequencing
platform being used (e.g. 400-800 bp), and then used as templates
in a template switch polymerization reaction as described elsewhere
herein. The first strand synthesis is primed using a primer having
a sequencing primer binding domain (e.g., an Illumina.RTM. Read 2
N6 primer binding domain), and the template switch oligonucleotide
includes a second sequencing primer binding domain of the
sequencing platform (e.g., an Illumina.RTM. Read 1 primer binding
domain). In certain aspects, the first strand synthesis is primed
using a random primer. The resulting library may then optionally be
PCR amplified with primers that add nucleic acid domains that bind
to surface-attached sequencing platform oligonucleotides (e.g., the
P5 and P7 oligonucleotides attached to the flow cell in an
Illumina.RTM. sequencing system). The library may be mixed 50:50
with a control library (e.g., Illumina.RTM.'s PhiX control library)
and sequenced on the sequencing platform (e.g., an Illumina.RTM.
sequencing system). The control library sequences may be removed
and the remaining sequences mapped to the transcriptome of the
source of the mRNAs (e.g., human, mouse, or any other mRNA
source).
[0066] According to certain embodiments, the subject methods may be
used to generate a cDNA library corresponding to non-polyadenylated
RNAs for downstream sequencing on an Illumina.RTM.-based sequencing
system. In one embodiment, microRNAs are polyadenylated and then
used as templates in a template switch polymerization reaction as
described elsewhere herein. The first strand synthesis is primed
using an Illumina.RTM. dT primer, and the template switch
oligonucleotide included an Illumina.RTM. Read 1 primer binding
domain.
[0067] FIG. 5 shows example sequences that may be added to nucleic
acids according to one embodiment of the present disclosure. In
this example, a template switch oligonucleotide (top) includes a 3'
hybridization domain (GGG) and a sequencing platform adapter
construct that includes a binding site for a surface-attached
sequencing platform oligonucleotide (in this example, the
surface-attached P5 primer of an Illumina.RTM. system) and a
sequencing primer binding site (in this example, a binding site for
the Read 1 sequencing primer of an Illumina.RTM. system) to
facilitate sequencing on a sequencing platform of interest. A
sequencing platform adapter construct (bottom) which may be
included in the nucleic acid at an end opposite the template switch
oligonucleotide includes a binding site for a second
surface-attached sequencing platform oligonucleotide (in this
example, the surface-attached P7 primer of an Illumina.RTM.
system), an index barcode, and a second sequencing primer binding
site (in this example, the binding site for a Read 2 sequencing
primer of an Illumina.RTM. system) to facilitate sequencing on a
sequencing platform of interest.
[0068] The subject methods may further include combining a
thermostable polymerase (e.g., a Taq, Pfu, Tfl, Tth, Tli, and/or
other thermostable polymerase)--in addition to the template
switching polymerase--into the reaction mixture. Alternatively, the
template switching polymerase may be a thermostable polymerase.
Either of these embodiments find use, e.g., when it is desirable to
achieve sequencing platform adapter construct addition and
amplification (e.g., amplification with or without further adapter
addition) of the product nucleic acid in a single tube. For
example, the contents of the single tube may be placed under
conditions suitable for the template switch polymerization reaction
to occur (as described elsewhere herein), followed by placing the
reaction contents under thermocycling conditions (e.g.,
denaturation, primer annealing, and polymerization conditions) in
which the first-strand synthesis product is PCR amplified using
amplification primers and the thermostable polymerase present in
the single tube. Due to its thermostability, the thermostable
polymerase will retain its activity even when present during the
PCR phase of this embodiment.
Compositions
[0069] Also provided by the present disclosure are compositions.
The subject compositions may include, e.g., one or more of any of
the reaction mixture components described above with respect to the
subject methods. For example, the compositions may include one or
more of a template ribonucleic acid (RNA), a polymerase (e.g., a
polymerase capable of template-switching, a thermostable
polymerase, combinations thereof, or the like), a template switch
oligonucleotide, dNTPs, a salt, a metal cofactor, one or more
nuclease inhibitors (e.g., an RNase inhibitor), one or more
enzyme-stabilizing components (e.g., DTT), or any other desired
reaction mixture component(s).
[0070] In certain aspects, the subject compositions include a
template ribonucleic acid (RNA) and a template switch
oligonucleotide each hybridized to adjacent regions of a nucleic
acid strand, where the template switch oligonucleotide includes a
3' hybridization domain and a sequencing platform adapter
construct. The sequencing platform adapter construct may include
any sequencing platform nucleic acid domain of interest, including
any of the domains described above with respect to the subject
methods (e.g., a domain that specifically binds to a
surface-attached sequencing platform oligonucleotide, a sequencing
primer binding domain, a barcode domain, a barcode sequencing
primer binding domain, a molecular identification domain, or any
combination thereof). Approaches for isolating RNA samples from a
nucleic acid source of interest, as well as strategies for
generating template RNAs from precursor RNAs, are described
elsewhere herein.
[0071] In certain aspects, the 3' hybridization domain of the
template switch oligonucleotide includes an arbitrary sequence,
e.g., as described above.
[0072] The subject compositions may be present in any suitable
environment. According to one embodiment, the composition is
present in a reaction tube (e.g., a 0.2 mL tube, a 0.6 mL tube, a
1.5 mL tube, or the like) or a well. In certain aspects, the
composition is present in two or more (e.g., a plurality of)
reaction tubes or wells (e.g., a plate, such as a 96-well plate).
The tubes and/or plates may be made of any suitable material, e.g.,
polypropylene, or the like. In certain aspects, the tubes and/or
plates in which the composition is present provide for efficient
heat transfer to the composition (e.g., when placed in a heat
block, water bath, thermocycler, and/or the like), so that the
temperature of the composition may be altered within a short period
of time, e.g., as necessary for a particular enzymatic reaction to
occur. According to certain embodiments, the composition is present
in a thin-walled polypropylene tube, or a plate having thin-walled
polypropylene wells. In certain embodiments it may be convenient
for the reaction to take place on a solid surface or a bead, in
such case, the template switch oligonucleotide or one or more of
the primers may be attached to the solid support or bead by methods
known in the art--such as biotin linkage or by covalent linkage)
and reaction allowed to proceed on the support.
[0073] Other suitable environments for the subject compositions
include, e.g., a microfluidic chip (e.g., a "lab-on-a-chip
device"). The composition may be present in an instrument
configured to bring the composition to a desired temperature, e.g.,
a temperature-controlled water bath, heat block, or the like. The
instrument configured to bring the composition to a desired
temperature may be configured to bring the composition to a series
of different desired temperatures, each for a suitable period of
time (e.g., the instrument may be a thermocycler).
Kits
[0074] Aspects of the present disclosure also include kits. The
kits may include, e.g., one or more of any of the reaction mixture
components described above with respect to the subject methods. For
example, the kits may include one or more of a template ribonucleic
acid (RNA), components for producing a template RNA from a
precursor RNA (e.g., a poly(A) polymerase and associated reagents
for polyadenylating a non-polyadenylated precursor RNA), a
polymerase (e.g., a polymerase capable of template-switching, a
thermostable polymerase, combinations thereof, or the like), a
template switch oligonucleotide, dNTPs, a salt, a metal cofactor,
one or more nuclease inhibitors (e.g., an RNase inhibitor and/or a
DNase inhibitor), one or more molecular crowding agents (e.g.,
polyethylene glycol, or the like), one or more enzyme-stabilizing
components (e.g., DTT), or any other desired kit component(s), such
as solid supports, e.g., tubes, beads, microfluidic chips, etc.
[0075] According to one embodiment, the subject kits include a
template switch oligonucleotide comprising a 3' hybridization
domain and a sequencing platform adapter construct, and a template
switching polymerase. The sequencing platform adapter construct may
include any sequencing platform nucleic acid domain of interest,
including any of the domains described above with respect to the
subject methods and compositions (e.g., a domain that specifically
binds to a surface-attached sequencing platform oligonucleotide, a
sequencing primer binding domain, a barcode domain, a barcode
sequencing primer binding domain, a molecular identification
domain, or any combination thereof).
[0076] Kits of the present disclosure may include a first-strand
synthesis primer that includes a first domain that hybridizes to a
template RNA and a second domain that does not hybridize to the
template RNA. The first domain may have a defined or arbitrary
sequence. The second domain of such primers may include, e.g., a
sequencing platform adapter construct that includes a nucleic acid
domain selected from a domain that specifically binds to a
surface-attached sequencing platform oligonucleotide, a sequencing
primer binding domain, a barcode domain, a barcode sequencing
primer binding domain, a molecular identification domain, and any
combination thereof.
[0077] In certain embodiments, the kits include reagents for
isolating RNA from a source of RNA. The reagents may be suitable
for isolating nucleic acid samples from a variety of RNA sources
including single cells, cultured cells, tissues, organs, or
organisms. The subject kits may include reagents for isolating a
nucleic acid sample from a fixed cell, tissue or organ, e.g.,
formalin-fixed, paraffin-embedded (FFPE) tissue. Such kits may
include one or more deparaffinization agents, one or more agents
suitable to de-crosslink nucleic acids, and/or the like.
[0078] Components of the kits may be present in separate
containers, or multiple components may be present in a single
container. For example, the template switch oligonucleotide and the
template switching polymerase may be provided in the same tube, or
may be provided in different tubes. In certain embodiments, it may
be convenient to provide the components in a lyophilized form, so
that they are ready to use and can be stored conveniently at room
temperature.
[0079] In addition to the above-mentioned components, a subject kit
may further include instructions for using the components of the
kit, e.g., to practice the subject method. The instructions are
generally recorded on a suitable recording medium. For example, the
instructions may be printed on a substrate, such as paper or
plastic, etc. As such, the instructions may be present in the kits
as a package insert, in the labeling of the container of the kit or
components thereof (i.e., associated with the packaging or
subpackaging) etc. In other embodiments, the instructions are
present as an electronic storage data file present on a suitable
computer readable storage medium, e.g. CD-ROM, diskette, Hard Disk
Drive (HDD) etc. In yet other embodiments, the actual instructions
are not present in the kit, but means for obtaining the
instructions from a remote source, e.g. via the internet, are
provided. An example of this embodiment is a kit that includes a
web address where the instructions can be viewed and/or from which
the instructions can be downloaded. As with the instructions, this
means for obtaining the instructions is recorded on a suitable
substrate.
Utility
[0080] The subject methods find use in a variety of applications,
including those that require the presence of particular nucleotide
sequences at one or both ends of nucleic acids of interest. Such
applications exist in the areas of basic research and diagnostics
(e.g., clinical diagnostics) and include, but are not limited to,
the generation of sequencing-ready cDNA libraries. Such libraries
may include adapter sequences that enable sequencing of the library
members using any convenient sequencing platform, including: the
HiSeq.TM., MiSeq.TM. and Genome Analyzer.TM. sequencing systems
from Illumina.RTM.; the Ion PGM.TM. and Ion Proton.TM. sequencing
systems from Ion Torrent.TM.; the PACBIO RS II sequencing system
from Pacific Biosciences, the SOLiD sequencing systems from Life
Technologies.TM., the 454 GS FLX+ and GS Junior sequencing systems
from Roche, or any other convenient sequencing platform. The
methods of the present disclosure find use in generating sequencing
ready cDNA libraries corresponding to any RNA starting material of
interest (e.g., mRNA) and are not limited to polyadenylated RNAs.
For example, the subject methods may be used to generate
sequencing-ready cDNA libraries from non-polyadenylated RNAs,
including microRNAs, small RNAs, siRNAs, and/or any other type
non-polyadenylated RNAs of interest. The methods also find use in
generating strand-specific information, which can be helpful in
determining allele-specific expression or in distinguishing
overlapping transcripts in the genome.
[0081] An aspect of the subject methods is that--utilizing a
template RNA--a cDNA species having sequencing platform adapter
sequences at one or both of its ends is generated in a single step,
e.g., without the added steps associated with traditional
ligation-based approaches for generating hybrid nucleic acid
molecules for downstream sequencing applications. Such steps
include a ligation step (which may require a prior restriction
digest), washing steps, and any other necessary steps associated
with traditional ligation-based approaches. Accordingly, the
methods of the present disclosure are more efficient,
cost-effective, and provide more flexibility than the traditional
approaches.
[0082] The following examples are offered by way of illustration
and not by way of limitation.
Experimental
I. Library Construction
[0083] 1 .mu.g of Human Brain PolyA RNA (Clontech) was fragmented
with addition of 5.times. fragmentation Buffer (200 mM Tris
acetate, pH 8.2, 500 mM potassium acetate, and 150 mM magnesium
acetate) and heating at 94.degree. C. for 2 min 30 s. Fragmented
RNA was purified using a Nucleospin RNA XS spin column (Macharey
Nagel).
[0084] Fragmented RNA was diluted to either 1 ng/.mu.l of 5
ng/.mu.l in RNase free water. 1 .mu.l of fragmented RNA or water
was combined with 1 .mu.l 120 first strand primer and 2.5 .mu.l of
RNase free water. Samples were heated to 72.degree. C. for 3
minutes and then placed on ice. To these samples were added 2 .mu.l
5.times. first strand buffer (Clontech), 0.25 .mu.l 100 mM DTT,
0.25 .mu.l recombinant RNase inhibitor (Takara), 10 mM dNTP mix
(Clontech), 1 .mu.l 120 template switch oligo and 1 .mu.l
SMARTScribe RT (Clontech). Samples were incubated at 42.degree. C.
for 90 minutes followed by 70.degree. C. for 10 minutes.
[0085] First strand cDNA reactions were purified by addition of 15
.mu.l water and 25 .mu.l Ampure XP beads (Beckman Coulter). Samples
were well mixed and incubated at room temperature for 8 minutes.
Samples were bound to a magnetic stand for 5 minutes, and the beads
were washed twice with 200 .mu.l 80% ethanol and allowed to air dry
for 5 minutes.
[0086] cDNA on the beads was eluted by addition of 50 .mu.l PCR
Mastermix (5 .mu.l 10 Advantage2 buffer, 5 .mu.l GC Melt reagent, 1
.mu.l 10 mM dNTPs, 1 .mu.l Advantage2 polymerase (Clontech), 240 nM
forward PCR primer, 240 nM reverse PCR primer, and 36.8 .mu.l
water). Samples were thermocycled for 12 PCR cycles with the
settings 95.degree. C. 1 minute, 12.times. (95.degree. C. 15
seconds, 65.degree. C. 30 seconds, 68.degree. C. 1 minute). PCR
products were purified with 50 .mu.l Ampure XP beads and eluted in
40 .mu.l TE buffer.
Samples were diluted and run on an Agilent Bioanalyzer using the
high sensitivity DNA assay. The results are provided in FIG. 4.
II. Construction of Illumina Sequenced Libraries
A. Library Construction
[0087] 1 .mu.g of Mouse Brain PolyA RNA (Clontech) was fragmented
addition of 5.times. fragmentation Buffer (200 mM Tris acetate, pH
8.2, 500 mM potassium acetate, and 150 mM magnesium acetate) and
heating at 94.degree. C. for 2 min 30 s. Fragmented RNA was
purified using a Nucleospin RNA XS spin column (Macharey
Nagel).
[0088] 10 ng of fragmented RNA in 3.5 .mu.l was combined with 1
.mu.l 120 first strand primer. Samples were heated to 72.degree. C.
for 3 minutes and then placed on ice. To these samples were added 2
.mu.l 5.times. first strand buffer (Clontech), 0.25 .mu.l 100 mM
DTT, 0.25 .mu.l recombinant RNase inhibitor (Takara), 10 mM dNTP
mix (Clontech), 1 .mu.l 120 template switch oligo, and 1 .mu.l
SMARTScribe RT (Clontech). Samples were incubated at 42.degree. C.
for 90 minutes followed by 70.degree. C. for 10 minutes.
[0089] First strand cDNA reactions were purified by addition of 15
.mu.l water and 25 .mu.l Ampure XP beads (Beckman Coulter). Samples
were well mixed and incubated at room temperature for 8 minutes.
Samples were bound to a magnetic stand for 5 minutes, and the beads
were washed twice with 200 .mu.l 80% ethanol and allowed to air dry
for 5 minutes.
[0090] cDNA on the beads was eluted by addition of 50 .mu.l PCR
Mastermix (5 .mu.l 10 Advantage2 buffer, 5 .mu.l GC Melt reagent, 1
.mu.l 10 mM dNTPs, 1 .mu.l Advantage2 polymerase (All Clontech),
240 nM forward PCR primer, 240 nM reverse PCR primer, and 36.8
.mu.l water). Samples were thermocycled for 12 PCR cycles with the
settings 95.degree. C. 1 minute, 12.times. (95.degree. C. 15
seconds, 65.degree. C. 30 seconds, 68.degree. C. 1 minute). PCR
products were purified with 50 .mu.l Ampure XP beads and eluted in
40 .mu.l TE buffer. Samples were diluted and run on an Agilent
Bioanalyzer using the high sensitivity DNA assay.
B. Sequencing
[0091] The above sequencing library was diluted to 2 nM and
combined with an equal amount of PhiX Control Library (Illumina).
Samples were loaded onto an Illumina MiSeq instrument with a final
loading concentration of 8 pM and sequenced as a single 66 bp
read.
C. Analysis Summary
[0092] All Analysis was performed on a linux workstation. Sequences
were trimmed of the first three nucleotides, and PhiX sequences
were bioinformatically removed by mapping all sequences to the PhiX
genome with the Bowtie2 software package and retaining all unmapped
reads.
[0093] Remaining sequencing reads were mapped to the mouse
transcriptome (build MM10) using the tophat2 software package. Gene
expression values were calculated using the Cufflinks software
using the genome annotation as a guide.
[0094] Gene expression values were compared to a previously
sequenced library generated with the SMARTer Universal kit
(Clontech) from ribosomally depleted Mouse Brain Total RNA
(Clontech).
[0095] Gene expression comparisons and plotting were done In R
using the CummeRbund analysis package.
[0096] Gene body coverage and strand specificity were calculated
using geneBody_coverage.py and infer_experiment.py scripts
respectively from the RSeQC software collection.
III. miRNA Library Construction
[0097] 1 .mu.l of 5 .mu.M synthetic miR-22
(AAGCUGCCAGUUGAAGAACUGUA) (RNA) (SEQ ID NO:07) was combined with 2
.mu.l 5.times. First Strand Buffer (Clontech), 0.25 .mu.l 100 mM
DTT, 0.25 .mu.l Recombinant RNase inhibitor (Takara), 0.25 .mu.l
Poly(A) polymerase (Takara), 1 .mu.l 10 mM ATP, 5.25 .mu.l RNase
free water. Samples were incubated at 37.degree. C. for 10 minutes
followed by 65.degree. C. for 20 minutes.
[0098] Reactions were diluted with 10 .mu.l RNase free water. 3.5
.mu.l diluted polyadenylated miRNA was combined with 1 .mu.l 12
.mu.M first strand primer. Samples were heated to 72.degree. C. for
3 minutes and then placed on ice. To these samples were added 2
.mu.l 5.times. first strand buffer (Clontech), 0.25 .mu.l 100 mM
DTT, 0.25 .mu.l recombinant RNase inhibitor (Takara), 10 mM dNTP
mix (Clontech), 1 .mu.l 120 template switch oligo, and 1 .mu.l
SMARTScribe RT (Clontech). Samples were incubated at 42.degree. C.
for 60 minutes followed by 70.degree. C. for 15 minutes.
[0099] First strand reactions were diluted with 40 .mu.l TE buffer.
5 .mu.l diluted cDNA was combined with 45 .mu.l PCR Mastermix (5
.mu.l 10 Advantage2 buffer, 1 .mu.l 10 mM dNTPs, 1 .mu.l Advantage2
polymerase (All Clontech), 240 nM forward PCR primer, 240 nM
reverse PCR primer (and 36 .mu.l water). Samples were thermocycled
for 20 PCR cycles with the settings 95.degree. C. 1 minute,
20.times. (95.degree. C. 15 seconds, 65.degree. C. 30 seconds). 5
.mu.l PCR products were resolved on a 1% agarose gel.
[0100] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is readily apparent to those of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing
from the spirit or scope of the appended claims.
[0101] Accordingly, the preceding merely illustrates the principles
of the invention. It will be appreciated that those skilled in the
art will be able to devise various arrangements which, although not
explicitly described or shown herein, embody the principles of the
invention and are included within its spirit and scope.
Furthermore, all examples and conditional language recited herein
are principally intended to aid the reader in understanding the
principles of the invention and the concepts contributed by the
inventors to furthering the art, and are to be construed as being
without limitation to such specifically recited examples and
conditions. Moreover, all statements herein reciting principles,
aspects, and embodiments of the invention as well as specific
examples thereof, are intended to encompass both structural and
functional equivalents thereof. Additionally, it is intended that
such equivalents include both currently known equivalents and
equivalents developed in the future, i.e., any elements developed
that perform the same function, regardless of structure. The scope
of the present invention, therefore, is not intended to be limited
to the exemplary embodiments shown and described herein. Rather,
the scope and spirit of present invention is embodied by the
appended claims.
Sequence CWU 1
1
12120DNAArtificial sequenceSynthetic oligonucleotide 1aatgatacgg
cgaccaccga 20224DNAArtificial sequenceSynthetic oligonucleotide
2caagcagaag acggcatacg agat 24333DNAArtificial sequenceSynthetic
oligonucleotide 3acactctttc cctacacgac gctcttccga tct
33434DNAArtificial sequenceSynthetic oligonucleotide 4gtgactggag
ttcagacgtg tgctcttccg atct 34530DNAArtificial sequenceSynthetic
oligonucleotide 5ccatctcatc cctgcgtgtc tccgactcag
30623DNAArtificial sequenceSynthetic oligonucleotide 6cctctctatg
ggcagtcggt gat 23723RNAArtificial sequenceSynthetic oligonucleotide
7aagcugccag uugaagaacu gua 23861DNAArtificial sequenceSynthetic
oligonucleotide 8aatgatacgg cgaccaccga gatctacact ctttccctac
acgacgctct tccgatctgg 60g 619258DNAArtificial sequenceSynthetic
oligonucleotidemisc_feature(35)..(234)n at positions 35-234 may be
any nucleotide and between 4 and 200 of the nucleotides may be
present. 9agatcggaag agcacacgtc tgaactccag tcacnnnnnn nnnnnnnnnn
nnnnnnnnnn 60nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 120nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnatctcg 240tatgccgtct tctgcttg
25810258DNAArtificial sequenceSynthetic
oligonucleotidemisc_feature(25)..(224)n at positions 25-224 may be
any nucleotide and between 4 and 200 of the nucleotides may be
present. 10caagcagaag acggcatacg agatnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 60nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 120nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnngtgact ggagttcaga 240cgtgtgctct tccgatct
2581114DNAArtificial Sequencepoly-adenosine
sequencemisc_feature(14)..(14)the nucleotide at this position may
be repeated n times 11aaaaaaaaaa aaaa 141213DNAArtificial
Sequencepoly-thymidine sequence 12tttttttttt ttt 13
* * * * *