U.S. patent application number 16/122176 was filed with the patent office on 2019-01-03 for acoustic energy mediation of genetic fragmentation.
This patent application is currently assigned to Covaris, Inc. The applicant listed for this patent is Covaris, Inc.. Invention is credited to Hamid KHOJA, James A. Laugharn, JR..
Application Number | 20190002944 16/122176 |
Document ID | / |
Family ID | 54542497 |
Filed Date | 2019-01-03 |
United States Patent
Application |
20190002944 |
Kind Code |
A1 |
Laugharn, JR.; James A. ; et
al. |
January 3, 2019 |
ACOUSTIC ENERGY MEDIATION OF GENETIC FRAGMENTATION
Abstract
Method and apparatus for controlling acoustic treatment of a
sample to mediate a tagmentation process used on double stranded
DNA.
Inventors: |
Laugharn, JR.; James A.;
(Boston, MA) ; KHOJA; Hamid; (Rancho Santa
Margarita, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Covaris, Inc. |
Woburn |
MA |
US |
|
|
Assignee: |
Covaris, Inc
Woburn
MA
|
Family ID: |
54542497 |
Appl. No.: |
16/122176 |
Filed: |
September 5, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14881632 |
Oct 13, 2015 |
10093955 |
|
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16122176 |
|
|
|
|
62063683 |
Oct 14, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 3/5082 20130101;
C12P 19/34 20130101; B01F 11/0283 20130101; B01F 13/0052 20130101;
G01N 1/286 20130101; C12Q 1/6806 20130101; C12Q 1/6806 20130101;
C12Q 2523/301 20130101; C12Q 2525/301 20130101; C12Q 2535/122
20130101 |
International
Class: |
C12P 19/34 20060101
C12P019/34; G01N 1/28 20060101 G01N001/28; B01F 13/00 20060101
B01F013/00; B01F 11/02 20060101 B01F011/02; C12Q 1/6806 20060101
C12Q001/6806 |
Claims
1. A method for processing a sample containing genomic material,
comprising: providing a sample in a vessel, the sample including
double stranded DNA material with segments having a starting base
pair length, the sample having a starting viscosity; subjecting the
sample to acoustic energy to reduce a viscosity of the sample to a
reduced viscosity that is less than the starting viscosity and to
cause random shearing of the segments of double stranded DNA
material in the sample to form fragments of the double stranded DNA
material having a base pair length of less than the starting base
pair length; and providing a hyperactive mutant of Tn5 transposase
and oligonucleotide material with the fragments of double stranded
DNA material to cut the fragments of double stranded DNA material
with the Tn5 transposase and insert oligonucleotide material into
the fragments of double stranded DNA material at areas cut by the
Tn5 transposase.
2. The method of claim 1, wherein the step of providing a
hyperactive mutant of Tn5 transposase and oligonucleotide material
with the fragments of double stranded DNA material occurs before
the step of subjecting the sample to acoustic energy.
3. The method of claim 1, further comprising: subjecting the sample
to acoustic energy to mix the hyperactive mutant of Tn5
transposase, oligonucleotide material, and the fragments of double
stranded DNA material after the step of subjecting the sample to
acoustic energy to cause shearing.
4. The method of claim 1, wherein the step of providing a
hyperactive mutant of Tn5 transposase and oligonucleotide material
with the fragments of double stranded DNA material occurs after the
step of subjecting the sample to acoustic energy.
5. The method of claim 4, further comprising: subjecting the sample
to acoustic energy to mix the hyperactive mutant of Tn5
transposase, oligonucleotide material, and the fragments of double
stranded DNA material after the step of subjecting the sample to
acoustic energy to cause shearing.
6. The method of claim 1, wherein the oligonucleotide material
includes synthetic oligonucleotides.
7. The method of claim 1, wherein the sample has a volume of about
15 microliters.
8. The method of claim 1, wherein the vessel has a volume of about
100 microliters.
9. The method of claim 1, wherein the starting base pair length is
in excess of 10000 bp.
10. The method of claim 1, wherein the fragments of the double
stranded DNA material have a base pair length less than 3000
bp.
11. The method of claim 1, wherein the fragments of the double
stranded DNA material have a base pair length between 1000 bp and
1500 bp.
12. The method of claim 1, wherein the step of subjecting the
sample to acoustic energy reduces a viscosity of the sample so as
to enhance enzyme interaction that occurs during the step of
providing a hyperactive mutant of Tn5 transposase and
oligonucleotide material.
13. The method of claim 1, wherein the subjecting step is performed
over a time period of 30-200 seconds.
14. The method of claim 1, wherein the step of providing a
hyperactive mutant of Tn5 transposase and oligonucleotide material
with the fragments of double stranded DNA material occurs after the
step of subjecting the sample to acoustic energy.
15. The method of claim 14, further comprising: subjecting the
sample to acoustic energy to mix the hyperactive mutant of Tn5
transposase, oligonucleotide material, and the fragments of double
stranded DNA material after the step of subjecting the sample to
acoustic energy to cause shearing.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. application Ser.
No. 14/881,632, filed Oct. 13, 2015, which claims the benefit of
U.S. Provisional Application No. 62/063,683, filed Oct. 14, 2014,
which applications are hereby incorporated by reference in their
entirety.
BACKGROUND
1. Field of the Invention
[0002] Systems and methods for processing of samples with acoustic
energy are generally disclosed.
2. Related Art
[0003] Acoustic energy-based sample processing devices, such as
Adaptive Focused Acoustic apparatuses made by Covaris of Woburn,
Mass., are effective for homogenization and disruption of
biological tissues, cells and other sample material. With such
devices, a controlled acoustic field enables repeatable processes
to be developed which often result in higher recovery of target
molecules. Such target molecules may be, for example, DNA, RNA,
proteins, and the like. Target molecules or other materials may be
contained as samples within a vessel.
SUMMARY OF INVENTION
[0004] Tagmentation is a process in which a hyperactive mutant of
the Tn5 transposase is used to incorporate a synthetic
oligonucleotide into double stranded DNA, essentially carrying out
a cut/paste procedure in which the double stranded DNA is cut, and
synthetic sequence is inserted. Its utility in generating libraries
for next generation sequencing (NGS) systems was first described in
a paper by Andrew Adey et al. in 2010 (Adey A, Morrison H G, Asan,
Xun X, Kitzman J O, Turner E H, Stackhouse B, MacKenzie A P,
Caruccio N C, Zhang X, et al. Rapid, low-input, low-bias
construction of shotgun fragment libraries by high-density in vitro
transposition. Genome Biol 11: R119, 2010.) In commercially
available products such as Nextera from Illumina, and MuSeek from
Thermo Scientific, the transposase inserts NGS system-specific
adaptor oligos in the double stranded DNA sample, and subsequent
limited PCR is used to enrich fragments containing the desired
adaptors and barcode indices on either end of the DNA
fragments.
[0005] The inventors have appreciated that the known tagmentation
process has some well-known limitations, including: [0006] G+C bias
inherent in transposase-mediated fragmentation; [0007] Insertion
bias towards AT rich region containing similar insertion nucleic
acid sequences described for TN5 transposases; [0008] The process
is highly sensitive to the DNA input concentration, requiring
precise quantification upstream; [0009] DNA fragments distribution
after tagmentation is wide, thus limiting library yield after size
selection.
[0010] To address at least some of these limitations, the inventors
have developed a tagmentation process that is mediated by acoustic
energy. In at least some embodiments, acoustic energy can be used
to fragment double stranded DNA having a relatively long base pair
length, e.g., in excess of 3000 bp, e.g., 10000 bp or more. This
fragmentation can have one or more benefits, such as reducing
viscosity of the DNA sample which can enhance enzymatic activity of
the tagmentation process. The acoustic energy can also have other
benefits, such as shearing double stranded DNA to a smaller
fragment size under 3000 bp, e.g., 1000 bp to 1500 bp. This may
help create a narrower DNA fragmentation size after the
tagmentation process, thereby enhancing the library yield. In other
embodiments, the acoustic energy treatment may randomly shear
segments of double stranded DNA material in GC and AT rich regions,
and in regions having long repeat portions, so as to form fragments
of the double stranded DNA material having a base pair length less
than 3000 bp. This may help reduce the G+C bias inherent in
transposase-mediated fragmentation and/or insertion bias towards AT
rich regions containing similar insertion nucleic acid sequences
described for TN5 transposases. In yet other embodiments, acoustic
energy treatment may reduce a transposase concentration required to
successfully perform the tagmentation process s, e.g., because the
DNA fragmentation caused by the acoustic treatment and/or mixing
caused by the acoustic treatment may enhance enzymatic activity of
the transposase.
[0011] In one aspect of the invention, a method for processing a
sample containing genomic material includes providing a sample in a
vessel, the sample including double stranded DNA material with
segments having a base pair length in excess of a starting base
pair length. In some cases, the starting base pair length may be
greater than 3000 bp or more, e.g., more than 10000 bp or 48000 bp.
The sample may be subjected to acoustic energy to cause shearing of
the segments of double stranded DNA material in the sample to form
fragments of the double stranded DNA material having a final base
pair length, the starting base pair length being two or more times
the final base pair length. For example, the fragments of DNA
material created by acoustic energy treatment may have a base pair
length of less than or about 3000 bp, e.g., 1000 to 1500 bp. A
hyperactive mutant of Tn5 transposase and oligonucleotide material
may be provided with the fragments of double stranded DNA material
to cut the fragments of double stranded DNA material with the Tn5
transposase and insert oligonucleotide material into the fragments
of double stranded DNA material at areas cut by the Tn5
transposase. The transposase and oligonucleotide material may be
provided with the sample before or after subjecting the sample to
acoustic energy to shear the DNA into fragments. In some
embodiments, the sample may be subjected to acoustic energy to mix
the hyperactive mutant of Tn5 transposase, oligonucleotide
material, and the fragments of double stranded DNA material after
the step of subjecting the sample to acoustic energy to cause
shearing. This may help enhance the enzymatic and oligonucleotide
insertion process.
[0012] In another aspect of the invention, a method for processing
a sample containing genomic material includes providing a sample in
a vessel, the sample including double stranded DNA material with
segments having a starting base pair length, and the sample having
a starting viscosity. The sample may be subjected to acoustic
energy to reduce a viscosity of the sample to a reduced viscosity
that is less than the starting viscosity. The acoustic energy
treatment may also cause shearing of the segments of double
stranded DNA material in the sample to form fragments of the double
stranded DNA material having a base pair length of less than the
starting base pair length. A hyperactive mutant of Tn5 transposase
and oligonucleotide material may be provided with the fragments of
double stranded DNA material to cut the fragments of double
stranded DNA material with the Tn5 transposase and insert
oligonucleotide material into the fragments of double stranded DNA
material at areas cut by the Tn5 transposase. The reduction of
viscosity of the sample caused by acoustic energy treatment may
enhance the enzymatic activity of the transposase, e.g., because
steric hindrance presence in higher viscosity solutions may be
reduced with reduced sample viscosity. As a result, the
tagmentation process may be performed more efficiently. In some
embodiments, a concentration of transposase needed to perform the
tagmentation process may be significantly reduced as compared to
tagmentation processes performed without the use of acoustic
energy. The hyperactive mutant of Tn5 transposase and
oligonucleotide material may be provided with the fragments of
double stranded DNA material before or after the step of subjecting
the sample to acoustic energy to shear the DNA segments, and in
some cases, acoustic energy treatment may be used to mix the
hyperactive mutant of Tn5 transposase, oligonucleotide material,
and the fragments of double stranded DNA material after the step of
subjecting the sample to acoustic energy to cause shearing. The
acoustic energy used to mix may be provided at a lower power than
the acoustic energy used to shear the DNA segments.
[0013] In another aspect of the invention, a method for processing
a sample containing genomic material includes providing a sample in
a vessel, the sample including double stranded DNA material with
segments having a base pair length in excess of 3000 bp. The
segments of double stranded DNA material may be randomly sheared in
GC and AT rich regions, and in regions having long repeat portions,
to form fragments of the double stranded DNA material having a base
pair length less than 3000 bp. For example, in some embodiments,
acoustic energy may be employed to randomly shear the DNA segments.
A hyperactive mutant of Tn5 transposase and oligonucleotide
material may be provided with the fragments of double stranded DNA
material to cut the fragments of double stranded DNA material with
the Tn5 transposase and insert oligonucleotide material into the
fragments of double stranded DNA material at areas cut by the Tn5
transposase.
[0014] Generally speaking, shearing of DNA and other genomic
fragments using acoustic treatment is known from U.S. Patent
Publication 2009/0317884. For example, U.S. Patent Publication
2009/0317884 discloses placing DNA fragments having a base pair
length of 10 kbp and up into a 50-100 microliter vessel along with
an energy director in the form of a polymer rod or bead, and
acoustically treating the DNA fragments so as to shear the DNA
fragments into smaller fragment sizes of about 3 kbp. Other
acoustic energy treatment protocols have been provided by Covaris,
Inc. of Woburn, Ma. For example, DNA shearing to base pair sizes
between about 150 to 1500 bp may be performed using a Covaris S220
system by employing a peak incident power (PIP) between 140 and 175
Watts, a 2 to 10% duty factor, 200 cycles per burst and a treatment
time of 15 to 430 seconds. Sample volume may be 50 or 130
microliters and may be held in a Covaris microTUBE. (The energy
applied to a sample via acoustic energy is measured in Joules and
given by the product of peak incident power (PIP in watts) by the
duty cycle of the applied energy (DC in percentage terms) by the
total processing time (T in seconds) or E=PIP*DC*T).) As will be
understood, the acoustic energy employed in such shearing
operations is focused acoustic energy such that a focal zone is
present at the sample being treated.
[0015] Other advantages and novel features of the invention will
become apparent from the following detailed description of various
non-limiting embodiments when considered in conjunction with the
accompanying figures and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Aspects of the invention are described with reference to the
following drawings in which numerals reference like elements, and
wherein:
[0017] FIG. 1 shows a schematic block diagram of an acoustic
treatment system that incorporates one or more aspects of the
invention; and
[0018] FIG. 2 shows a cross sectional view of a vessel containing a
sample that may be used with aspects of the invention;
[0019] FIG. 3 illustrates schematic steps of a method for
performing tagmentation in an illustrative embodiment; and
[0020] FIG. 4 shows a vessel used to hold a sample in Example One
below.
DETAILED DESCRIPTION
[0021] Aspects of the invention are not limited in application to
the details of construction and the arrangement of components set
forth in the following description or illustrated in the drawings.
Other embodiments may be employed and aspects of the inventions may
be practiced or be carried out in various ways. Also, the
phraseology and terminology used herein is for the purpose of
description and should not be regarded as limiting.
[0022] Acoustic treatment systems can be useful for the
homogenization and disruption of biological tissues, cells and
other sample material, with the end goal of recovering target
molecules from the sample material, such as DNA, RNA, proteins, and
the like. In addition, such systems may be used along with aspects
of the invention for DNA and/or other genomic fragment shearing,
e.g., to reduce the base pair length of DNA fragments from 1,000s
or 10,000s of base pairs to lengths of 200-1000 base pairs. As
described in more detail below, acoustic energy, and specifically
focused acoustics, can be useful in mediating a tagmentation
process. Generally speaking the inventors have found the following
features and benefits to employing focused acoustics with a
tagmentation process: [0023] Utilizing acoustic energy to randomly
shear DNA to an average size of around 1-1.5 kb may: [0024] a.
Increase the complexity of the resulting library by randomly
shearing the genomic DNA in GC and AT rich regions, as well as long
stretches of repeats, leading to reduced G+C bias of the library.
[0025] b. The 1-1.5 kb fragments will render the fragments more
accessible to the transposases since long stretches of low
concentration genomic DNA are converted into higher concentration,
partially fragmented DNA. The resultant transposase-processed DNA
should have a tighter size distribution as compared to genomic DNA
processed by a transposase alone. [0026] Carrying out the
tagmentation reaction in the presence of acoustic energy, e.g., of
relatively low power as compared to acoustic energy used for
shearing, will provide efficient gentle mixing of the reaction
components. As a result, the tagmentation reaction may experience:
[0027] a. An increase in the rate of sample/enzyme interaction
leading to a more efficient tagmentation reaction. This can reduce
the concentration of transposase required per reaction further
reducing the cost of library preparation. [0028] b. Further
reduction in the distribution of DNA fragments generated by
tagmentation as a result of efficient mixing of the components
during the tagmentation reaction. [0029] c. Better control over the
tagmentation fragment size. This will allow for greater utility of
tagmentation in applications requiring longer fragment inserts.
[0030] d. Reduce tagmentation sensitivity to input DNA mass and
concentration.
[0031] FIG. 1 shows a schematic block diagram of an acoustic
treatment system 100 that incorporates one or more aspects of the
invention and/or can be employed with one or more aspects of the
invention. It should be understood that although embodiments
described herein may include most or all aspects of the invention,
aspects of the invention may be used alone or in any suitable
combination with other aspects of the invention. In this
illustrative embodiment, the acoustic treatment system 100 includes
an acoustic transducer 14 (e.g., including one or more
piezoelectric elements) that is capable of generating an acoustic
field (e.g., at a focal zone 17) suitable to cause mixing, e.g.,
caused by cavitation, and/or other affects in a sample 1 contained
in a vessel 4. The acoustic transducer 14 may produce acoustic
energy within a frequency range of between about 100 kilohertz and
about 100 megahertz such that the focal zone 17 has a width of
about 2 centimeters or less. The focal zone 17 of the acoustic
energy may be any suitable shape, such as spherical, ellipsoidal,
rod-shaped, or column-shaped, for example, and be positioned at the
sample 1. The focal zone 17 may be larger than the sample volume,
or may be smaller than the sample volume, as shown in FIG. 1. U.S.
Pat. Nos. 6,948,843 and 6,719,449 are incorporated by reference
herein for details regarding the construction and operation of an
acoustic transducer and its control.
[0032] The vessel 4 may have any suitable size or other
arrangement, e.g., may be a glass tube, a plastic container, a well
in a microtiter plate, a vial, or other, and may be supported at a
location by a vessel holder 12. Although a vessel holder 12 is not
necessarily required, the vessel holder 12 may serve to interface
with the acoustic processing device so that the vessel 4 and the
sample in the vessel is positioned in a known location relative to
an acoustic field, for example, at least partially within a focal
zone of acoustic energy. In this embodiment, the vessel 4 is a 130
microliter borosilicate glass tube, but it should be understood
that the vessel 4 may have other suitable shapes, sizes, materials,
or other feature, as discussed more below. For example, the vessel
4 may be a cylindrical tube with a flat bottom and a threaded top
end to receive a cap, may include a cylindrical collar with a
depending flexible bag-like portion to hold a sample, may be a
single well in a multiwell plate, may be a cube-shaped vessel, or
may be of any other suitable arrangement. The vessel 4 may be
formed of glass, plastic, metal, composites, and/or any suitable
combinations of materials, and formed by any suitable process, such
as molding, machining, stamping, and/or a combination of
processes.
[0033] The acoustic treatment system 100 may also include a
coupling medium container 15 that is capable of holding a medium 16
(such as water or other liquid, gas, gel, solid, semi-solid, and/or
a combination of such components) which transmits acoustic energy
from the transducer 14 to the vessel 4. In some embodiments, the
acoustic field may be controlled, the acoustic transducer 14 may be
moved, and/or the vessel 4 may be moved (e.g., by way of moving a
holder 12, such as a rack, tray, platform, etc., that supports the
vessel 4) so that the sample is positioned in a desired location
relative to the focal zone 17. Also, the holder 12 is not limited
to a device like that shown in FIG. 1, and instead may include a
rack, slot, tray, gripper element, clamp, box or any other suitable
arrangement for holding the vessel, or multiple vessels, in a
desired location. For example, the holder 12 may include one or
more multi-vessel supports and a rack. Each support may hold a
plurality of vessels 4, e.g., a plurality of vessels may be held in
a linear array. Each support may include an identifier, such as a
barcode, RFID chip, or other component that may be read so as to
identify the support and/or vessels 4 associated with the support.
The rack may hold multiple supports with vessels and make it easier
to physically manipulate or otherwise handle multiple vessels 4,
e.g., in an automated processing environment in which one or more
robotic devices manipulate vessels for acoustic or other
processing. For example, the support may include a strip of
material with holes into which each vessel is inserted. The rack
may be arranged in the form of a multiwell plate such that vessel
bottoms extending below the support may be received into a
corresponding opening or well of the plate. The rack may also
include an identifier so that the rack and/or supports on the rack
can be identified in a automated way, e.g., by a laser scanner,
optical camera, RFID tag reader or other arrangement.
[0034] To control the acoustic transducer 14, the acoustic
treatment system 100 may include a system control circuit 10 that
controls various functions of the system 100 including operation of
the acoustic transducer 14. For example, the system control circuit
10 may provide control signals to a load current control circuit,
which controls a load current in a winding of a transformer. Based
on the load current, the transformer may output a drive signal to a
matching network, which is coupled to the acoustic transducer 14
and provides suitable signals for the transducer 14 to produce
desired acoustic energy. As discussed in more detail below, the
system control circuit 10 may control various other acoustic
treatment system 100 functions, such as positioning of the vessel 4
and/or acoustic transducer 14, receiving operator input (such as
commands for system operation), outputting information (e.g., to a
visible display screen, indicator lights, sample treatment status
information in electronic data form, and so on), and others.
[0035] In this illustrative embodiment, the sample 1 includes DNA
segments 2 and a liquid 3, e.g., 15 to 130 microliters of liquid
containing 20-30 nanograms of DNA fragments per microliter.
(Although the DNA segments 2 are shown schematically as a single
block of material, this is for purposes of illustration only. The
DNA segments 2 may be dispersed in the liquid 3 and generally will
not form a solid mass.) The DNA segments 2 may have a starting base
pair length of 3 kbp, 5 kbp, 10 bkp, 48 kbp or more, e.g., such
that 90% or more of the DNA fragments have a base pair length over
3 kbp, 5 kbp, etc. Of course, those of skill in the art will
appreciate that the sample 1 is not limited to including a liquid
3, as the sample 1 may take any suitable form, such as a solid only
form, a gel, a semi-solid, etc.
[0036] In at least some embodiments, the sample volume may be less
than the volume of the vessel, and thus an interface 5 will
separate the sample 1 from a headspace 6 in the vessel, i.e., a
gaseous region immediately above the sample 1. This arrangement may
cause portions of the sample 1 to be splashed or otherwise ejected
from the interface 5 in some conditions, e.g., to adhere to the
vessel 4 sidewalls above the interface 5. However, the presence of
one or more beads 8 in the sample 1 may reduce splashing or other
sample 1 ejection from the interface 5. The beads 8 may function as
a nucleation site for cavitation induced by the acoustic energy and
cause shear forces created during cavitation bubble collapse to be
directed to the surface of the bead, instead of to other portions
in the sample. By arranging all portions of the sample within a
maximum distance, e.g., 2 mm or less, of a bead surface, all
portions of the sample may be positioned suitably near the bead
surface (e.g., due to mixing during acoustic treatment) to
experience shear or other forces that cause shearing of genomic
material.
[0037] Thus, in some embodiments, the presence of the bead(s) 8 in
the sample 1 having a volume of 1-30 microliters may enable
shearing of DNA to occur under lower power or energy conditions
than would otherwise be possible. In addition, DNA or other genomic
segments may be sheared to lengths much shorter than previously
possible under relatively low power or energy conditions. DNA
segments having a base pair length of 3 kbp, 5 kbp, 10 bkp, 48 kbp
or more may be sheared by acoustic energy having a PIP of 20 watts
or less and a duty cycle of 10-20% in 30-200 seconds such that the
90%, 95% or more of the fragments end up with a base pair length of
200-1500 bp. This is a significant and surprising improvement over
prior processes.
[0038] The bead(s) may have a diameter of about 1-3 mm, with a
diameter of 1.57 mm being found particularly effective in genomic
shearing with sample volumes around 15 microliters. In some
embodiments, three beads 8 having a 1.57 mm diameter have been
found particularly useful. Beads made of PTFE have been found
effective, although other polymer materials are expected to work as
well. Beads having a coarse surface finish, as opposed to a
polished surface finish, have been found to be effective in many
applications. Generally, the beads are non-buoyant so as to remain
immersed in the sample during acoustic processing, but the beads
could be formed as part of a vessel wall, be attached to a vessel
wall, or be neutrally buoyant. Also, although beads 8 in the
illustrated embodiment are shown as spherical in shape, the beads
may have a variety of shapes, e.g., like jewelry elements commonly
referred to as "beads" have a variety of different shapes and
sizes. However, it should be understood that the use of beads 8 is
not required, and instead, DNA shearing may be performed in the
absence of any beads 8 or other elements in the vessel 4.
[0039] The embodiment shown in FIG. 1 also includes a cap 9 that
may be used to close the open end of the vessel 4. By capping the
open end of the vessel 4, an operator may be able to prevent flow
out of/into the vessel 4 and/or prevent contamination of the sample
1 by the outside environment. The cap 9 may engage the vessel 4 in
any way, such as by screw thread, interference fit, frictional
engagement to the inner or outer surface of the vessel sidewall,
etc. The use of a cap 9 is optional, and not required.
[0040] In one aspect of the invention, a vessel in which genomic
material is sheared may have a conically shaped bottom arranged
such that the conical walls diverge upwardly from each other at an
aperture angle of about 12-20 degrees. For example, FIG. 2 shows an
arrangement in which the sidewalls 41 of the vessel 4 diverge from
each other at an aperture angle .alpha. of about 16 degrees. The
sidewalls 41 may be relatively thin, e.g., about 0.25 mm in
thickness, so as to reduce interference with acoustic energy and/or
enhance heat exchange with the coupling medium 15. The extreme
bottom 42 of the vessel may have a partial spherical shape with a
radius of about 1-2 mm. The bottom 42 may be thicker than the
sidewalls 41 as shown, or may have the same or smaller thickness.
The partial spherical bottom may help with recovery of sample after
processing, since sample may tend to collect at the bottom,
allowing pipetting from the vessel. The sample 1 may have a height
h in the vessel 4 of about 3-4 mm as measured from the inner bottom
of the vessel to the interface 5.
[0041] FIG. 3 outlines steps in a method of performing a
tagmentation process with genomic material in accordance with
aspects of the invention. A vessel 4 is provided having a vessel
volume, which may be 50-150 microliters or more (or less). The
vessel 4 may be made of glass or a polymer or other material, if
suitable. Glass materials may aid in heat transfer to a coupling
medium, and some polymer materials have been found to aid in
genomic material shearing. The vessel 4 may have a conically-shaped
bottom with sidewalls upwardly diverging at an angle of about 12-20
degrees, e.g., as shown in FIG. 2. The extreme bottom of the vessel
4 may have a partial spherical shape. This arrangement has been
found particularly useful in shearing DNA in a sample volume of
about 15 microliters. Alternately, the vessel 4 may be a
cylindrical tube with vertical sidewalls and a flat or spherical
bottom, as shown.
[0042] Next, a sample 1 containing DNA segments 2 and liquid 3 may
be placed in the vessel 4. The sample 1 may have a volume of about
1 to 130 microliters, with a volume of 15 microliters having been
found in some examples to be particularly suitable for effective
DNA shearing. The sample 1 may have a height in the vessel 4 of
about 3-4 mm above the vessel inner bottom. Genomic fragments in
the sample may be provided at a concentration of about 20-30
nanograms/microliter, and may have fragment lengths of more than 3
kbp, 5 kbp, 10 kbp, 48 kbp or more. Optionally, one to three
polymer beads, e.g., made of PTFE having a diameter of 1-3 mm may
be provided in the sample 1 so the beads are immersed in the
sample. Providing three spherical PTFE beads having a diameter of
about 1.57 mm in a 15 microliter sample has been found particularly
suitable for shearing DNA.
[0043] Thereafter, the sample 1 may be treated with acoustic energy
to shear the DNA segments in the sample 1. The acoustic energy may
have a frequency range of between about 100 kilohertz and about 100
megahertz and have a focal zone 17 with a width of about 2
centimeters or less. The focal zone 17 may be positioned so that
the entire sample 1 is located in the focal zone 17, or so that a
portion of the sample is in the focal zone 17. In some embodiments,
the sample may move through the focal zone 17, whether by moving
the focal zone 17 relative to the vessel 4 or moving the vessel 4
relative to the focal zone 17. The acoustic energy may have a peak
incident power (PIP) of 20 watts or less and a duty cycle of
10-20%. The sample 1 may be treated with the acoustic energy over a
time period of 30-200 seconds. As a result, 90%, 95%, 99% or more
of the genomic material having a relatively long starting base pair
length may be sheared to fragments having a resulting base pair
length that is at least one-half or smaller than the starting base
pair length, e.g., 1000 to 1500 bp. In some embodiments, 99% or
more of the initial genomic material may be sheared to have a base
pair length of 1000-1500 bp.
[0044] With acoustic energy shearing complete, a hyperactive mutant
of Tn5 transposase and oligonucleotide material may be provided
with the fragments of double stranded DNA material to cut the
fragments of double stranded DNA material with the Tn5 transposase
and insert oligonucleotide material into the fragments of double
stranded DNA material at areas cut by the Tn5 transposase. The
transposase and oligonucleotide material may be provided with the
sample by pipette 11 or in other ways, and may be provided before
or after subjecting the sample to acoustic energy to shear the DNA
into fragments. That is, the DNA segments may be sheared by
acoustic energy while in the presence of the transposase and
olionucleotide material. In some embodiments, the sample may be
subjected to acoustic energy to mix the hyperactive mutant of Tn5
transposase, oligonucleotide material, and the fragments of double
stranded DNA material after the sample is exposed to acoustic
energy to cause shearing. This may help enhance the enzymatic and
oligonucleotide insertion process. In other embodiments, once
acoustic energy shearing and tagmentation is complete, other
processes may be performed on the sample while in the vessel, such
as PCR amplification, stirring, catalyzing, heating, disruption of
molecular bonds, or any other appropriate process. Such processes
may be performed using acoustic energy, or not, e.g., PCR
processing may be performed by a standard thermocycler machine.
[0045] A few illustrative examples of DNA shearing and tagmentation
processes using methods and systems according to the invention are
described below.
Example One
[0046] A vessel having a volume of 130 microliters was provided
with a 15 microliter sample containing lambda DNA (i.e., DNA
fragments having a base pair length of 48 kbp or more) at a
concentration of about 28 nanograms/microliter, but may be lower,
e.g., down to 7 nanograms/microliter. Three 1.57 mm PTFE beads were
provided in the sample as well, and the borosilicate glass vessel
having a spherical bottom was closed by a split septum, as shown in
FIG. 4. (Dimensions in FIG. 4 are in millimeters.) The sample was
acoustically treated using a Covaris S220 ultrasonicator set to
provide a PIP of 18 watts, a 20% duty cycle and 50 cycles per burst
for 60 seconds. Some splashing of the sample was observed during
acoustic treatment. More than 95% of the lambda DNA fragments were
sheared to DNA fragments having a length under 1000 bp, with an
average base pair length of the sheared DNA being about 336 bp.
More than 75% of the sheared DNA had a base pair length of 100-500
bp. Also of note is that more than 96% of the sample was recovered
from the vessel after acoustic treatment, and more than 93% of the
DNA material initially provided in the vessel was recovered by
pipetting. The experiment was repeated 12 times, and a coefficient
of variation of less than 4% was determined, i.e., the results of
DNA shearing were found to be highly repeatable and consistent. The
total energy of about 216 Joules to shear the lambda DNA is far
less than expected, and is thought to be due at least in part to
the relatively small sample size and the presence of three beads in
the sample.
Example Two
[0047] Example One was repeated, except that the samples were
processed with two 1.57 mm beads and with one 1.57 mm beads.
(Processing time for the single bead experiment was increased from
60 seconds to 120 seconds.) In both experiments, more than 90% of
the lambda DNA fragments were sheared to DNA fragments having a
length under 1000 bp, with an average base pair length of the
sheared DNA being about 332 bp for two beads, and about 412 bp for
one bead. The coefficient of variation for two beads was about
4.0%, and for one bead was about 7.2% based on 12 repeat
experiments for each.
Example Three
[0048] Example One was repeated, except that the samples were
processed using one 2.36 mm bead, i.e., a larger bead than those
used in Examples One and Two. The results are shown in FIG. 7. More
than 95% of the lambda DNA fragments were sheared to DNA fragments
having a length under 1000 bp, with an average base pair length of
the sheared DNA being about 337 bp. The coefficient of variation
was about 10.5% based on 12 repeat experiments, i.e., a significant
drop in repeatability versus the smaller bead sizes. Recovery of
the sample volume was about 80%, also significantly less than that
found in the three bead example above.
Example Four
[0049] Double stranded DNA having segments with a starting base
pair size of about 5000 bp is sheared using a Covaris S220 system
and employing a Covaris protocol to shear DNA to a resulting base
pair size of about 1500 bp. For example, 2 micrograms of E. coli
double stranded DNA is provided in a 130 microliter sample
including water in a Covaris microTUBE having a 130 microliter
volume. A Tris EDTA buffer having a pH of 8.0 is included along
with a Covaris microTUBE fiber. The sample is treated with focused
acoustic energy from the S220 system employing a peak incident
power (PIP) of 140 Watts, a 2% duty factor, 200 cycles per burst
and a treatment time of 15 seconds. This treatment shears the
double stranded DNA to create fragments having a resulting base
pair size of about 1500 bp. Prior to acoustic energy treatment,
Dnase I enzyme is used to nick the input DNA sample material using
an enzyme concentration of 0.1 to 0.001 units and an incubation
time of 5 to 60 minutes. The Dnase I enzyme activity is stopped by
adding EDTA in a final concentration of 10 mM to the sample and
heating to 70 degrees C. for 15 minutes. After acoustic treatment
to shear the DNA, the Nextera XT protocol is followed in which a
hyperactive mutant of Tn5 transposase and oligonucleotide material
is provided with the fragments of double stranded DNA material to
cut the fragments of double stranded DNA material with the Tn5
transposase and insert oligonucleotide material into the fragments
of double stranded DNA material at areas cut by the Tn5
transposase. An Agilent Bioanalyzer and associated high sensitivity
DNA assay is used to analyze aliquots of sample material for
fragment size distribution. A Kapa library quantification kit is
used to measure library efficiency.
[0050] As described above, the system control circuit 10 may
include any suitable components to perform desired control,
communication and/or other functions. For example, the system
control circuit 10 may include one or more general purpose
computers, a network of computers, one or more microprocessors,
etc. for performing data processing functions, one or more memories
for storing data and/or operating instructions (e.g., including
volatile and/or non-volatile memories such as optical disks and
disk drives, semiconductor memory, magnetic tape or disk memories,
and so on), communication buses or other communication devices for
wired or wireless communication (e.g., including various wires,
switches, connectors, Ethernet communication devices, WLAN
communication devices, and so on), software or other
computer-executable instructions (e.g., including instructions for
carrying out functions related to controlling the load current
control circuit as described above and other components), a power
supply or other power source (such as a plug for mating with an
electrical outlet, batteries, transformers, etc.), relays and/or
other switching devices, mechanical linkages, one or more sensors
or data input devices (such as a sensor to detect a temperature
and/or presence of the medium 16, a video camera or other imaging
device to capture and analyze image information regarding the
vessel 4 or other components, position sensors to indicate
positions of the acoustic transducer 14 and/or the vessel 4, and so
on), user data input devices (such as buttons, dials, knobs, a
keyboard, a touch screen or other), information display devices
(such as an LCD display, indicator lights, a printer, etc.), and/or
other components for providing desired input/output and control
functions.
[0051] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0052] The phrase "and/or," as used herein in the specification and
in the claims, should be understood to mean "either or both" of the
elements so conjoined, i.e., elements that are conjunctively
present in some cases and disjunctively present in other cases.
Multiple elements listed with "and/or" should be construed in the
same fashion, i.e., "one or more" of the elements so conjoined.
Other elements may optionally be present other than the elements
specifically identified by the "and/or" clause, whether related or
unrelated to those elements specifically identified.
[0053] The use of "including," "comprising," "having,"
"containing," "involving," and/or variations thereof herein, is
meant to encompass the items listed thereafter and equivalents
thereof as well as additional items.
[0054] It should also be understood that, unless clearly indicated
to the contrary, in any methods claimed herein that include more
than one step or act, the order of the steps or acts of the method
is not necessarily limited to the order in which the steps or acts
of the method are recited.
[0055] While aspects of the invention have been described with
reference to various illustrative embodiments, such aspects are not
limited to the embodiments described. Thus, it is evident that many
alternatives, modifications, and variations of the embodiments
described will be apparent to those skilled in the art.
Accordingly, embodiments as set forth herein are intended to be
illustrative, not limiting. Various changes may be made without
departing from the spirit of aspects of the invention.
* * * * *