U.S. patent application number 11/407567 was filed with the patent office on 2006-08-24 for reading chemical arrays.
Invention is credited to Herbert F. Cattell.
Application Number | 20060190193 11/407567 |
Document ID | / |
Family ID | 25103516 |
Filed Date | 2006-08-24 |
United States Patent
Application |
20060190193 |
Kind Code |
A1 |
Cattell; Herbert F. |
August 24, 2006 |
Reading chemical arrays
Abstract
Methods of using an addressable array of biopolymers which has
been exposed to a sample, and apparatus and computer program
products for use of the arrays. In one embodiment the method
includes detecting signals from the exposed array to obtain a
signal image of the array. The array signal image is saved in a
memory. Based on the detected signals, a shape of each region in
one or more sets of multiple regions on the array signal image is
established. A definition of the shapes of the established regions
of each set is saved in a memory. Each region of each set is
processed according to a corresponding routine for that set.
Inventors: |
Cattell; Herbert F.;
(Mountain View, CA) |
Correspondence
Address: |
AGILENT TECHNOLOGIES, INC.;Legal Department DL429
Intellectual Property Administration
P.O. Box 7599
Loveland
CO
80537-0599
US
|
Family ID: |
25103516 |
Appl. No.: |
11/407567 |
Filed: |
April 19, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09775163 |
Jan 31, 2001 |
7027930 |
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11407567 |
Apr 19, 2006 |
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Current U.S.
Class: |
702/20 |
Current CPC
Class: |
G16B 25/00 20190201 |
Class at
Publication: |
702/020 |
International
Class: |
G06F 19/00 20060101
G06F019/00 |
Claims
1-42. (canceled)
43. A method of reading an addressable array of biopolymers on a
substrate, comprising: (a) detecting signals from the exposed array
to obtain a signal image of the array; (b) establishing, based on
the detected signals, a shape of each region in one or more sets of
multiple regions on the array signal image, wherein the shape of
regions of a set include an identified shape of any of an array
feature, a background region, and an identified inlier or outlier
region within an array feature or background region; and (c)
processing each region of each set of a region processing
routine.
44. The method of claim 43, further comprising displaying the
established shapes.
45. The method of claim 44, wherein the displayed shapes are
saved.
46. The method of claim 43, wherein processed results from step (c)
are saved.
47. The method of claim 44, wherein the displayed shapes are saved
in a first file and processed results from step (c) are saved in a
second file.
48. The method of claim 43, wherein the biopolymers comprise
nucleic acids.
49. The method of claim 48, wherein the biopolymers comprise
DNA.
50. The method of claim 47, wherein the first file is smaller than
the second file.
51. The method of claim 47 additionally comprising, subsequent to
processing of each region, retrieving the saved shapes of the
established regions and displaying images of them.
52. The method of claim 43 additionally comprising, saving the
array signal image, and subsequent to processing each region,
retrieving the array signal image and saved shapes of the
established regions and displaying overlaid images of them.
53. The method of claim 52, wherein each displayed region is linked
to processed results for that region.
54. The method of claim 53, additionally comprising evaluating the
results from the processing based on the displayed retrieved
shapes.
55. The method of claim 54, additionally comprising, based on the
displayed retrieved shapes, altering a parameter used in the
processing and re-processing a region using the altered
parameter.
56. The method of claim 53 additionally comprising re-processing a
region in response to an alteration of one of the shapes of a set
using the altered shape.
57. The method of claim 52, wherein the shape of regions of a first
set are established which comprise regions of signal strength
higher than a predetermined value representing detected array
features, surrounded by regions of lower signal strength
representing detected background.
58. The method of claim 57, wherein the regions of the first set
are processed by a first routine comprising a summation of signal
strength within sub-regions of each first set region to provide a
total signal strength for each first set region.
59. The method of claim 47, wherein the shape of regions of a
second set are established each of which comprises a local
background region of lower signal strength than a corresponding
detected feature about which the background region is
positioned.
60. The method of claim 58, wherein the first routine comprises
subtracting local background region signal strength from the
corresponding detected feature signal strength.
61. An apparatus for reading an addressable array of biopolymers on
a substrate which array has been exposed to a sample, comprising:
(a) a detector to detect signals from the exposed array to obtain a
signal image of the array; (b) a memory; and (c) a processor which:
establishes, based on the detected signals, a shape of each region
in one or more sets of multiple regions on the array signal image,
wherein the shape of regions of a set include an identified shape
of any of an array feature, a background region, and an identified
inlier or outlier region within an array feature or background
region; saves a definition of the shapes of the established regions
of each set in a memory; and processes each region of each set of a
region processing routine.
62. The apparatus of claim 61, wherein the shapes of the
established regions are saved in a first file and results of region
processing are saved in a second file of the memory.
63. The apparatus of claim 61, further comprising a display for
displaying the established shapes.
64. The apparatus of claim 63, wherein each displayed region is
linked to processed results for that region.
65. A computer program product, comprising: a computer readable
storage medium having a computer program stored thereon for
performing, when loaded into a computer, the steps of: (a)
receiving signals from a biopolymer array, as a signal image of the
array; (b) saving the array signal image in a memory; (c)
establishing, based on the detected signals, a shape of each region
in one or more sets of multiple regions on the array signal image,
wherein the shape of regions of a set include an identified shape
of any of an array feature, a background region, and an identified
inlier or outlier region within an array feature or background
region; (d) saving a definition of the shapes of the established
regions of each set in a memory; and (e) processing each region of
each set of a region processing routine.
66. A method of reading an addressable array of biopolymers on a
substrate which array has been exposed to a sample, comprising: (a)
detecting signals from the exposed array to obtain a signal image
of the array; (b) establishing, based on the detected signals, a
shape of each region in one or more sets of multiple regions on the
array signal image, wherein the shape of regions of a set include
an identified shape of an inlier or outlier region within an array
feature; and (c) processing each region of each set of a region
processing routine.
67. The method of claim 66, further comprising displaying the
established shapes.
68. The method of claim 67, further comprising saving the displayed
shapes in a first file and the processed results in a second
file.
69. The method of claim 66, wherein the biopolymers comprise
nucleic acids.
70. The method of claim 66, wherein the biopolymers comprise
DNA.
71. The method of claim 68 wherein the first file is smaller than
the second file.
72. The method of claim 66 additionally comprising, subsequent to
processing of each region, retrieving saved shapes of the
established regions and displaying images of them.
73. The method of claim 66 additionally comprising, saving the
array signal image, and subsequent to processing each region,
retrieving the array signal image and saved shapes of the
established regions and displaying overlaid images of them.
74. The method of claim 67 wherein each displayed region is linked
to processed results for that region.
75. The method of claim 74, additionally comprising evaluating the
results from the processing based on the displayed retrieved
shapes.
76. The method of claim 75 additionally comprising, based on the
displayed retrieved shapes, altering a parameter used in the
processing and re-processing a region using the altered
parameter.
77. The method of claim 73 additionally comprising re-processing a
region in response to an alteration of one of the shapes of a set
using the altered shape.
78. An apparatus for reading an addressable array of biopolymers on
a substrate which array has been exposed to a sample, comprising:
(a) a detector to detect signals from the exposed array to obtain a
signal image of the array; (b) a memory; and (c) a processor which:
establishes, based on the detected signals, a shape of each region
in one or more sets of multiple regions on the array signal image,
wherein the shape of regions of a set include an identified shape
of an identified inlier or outlier region within an array feature;
saves a definition of the shapes of the established regions of each
set in a memory; and processes each region of each set of a region
processing routine.
79. The apparatus of claim 78, wherein the established shapes are
saved in a first file and processed results are saved in a second
file of the memory.
80. The apparatus of claim 78, further comprising a display for
displaying the established shapes.
81. A computer program product, comprising: a computer readable
storage medium having a computer program stored thereon for
performing, when loaded into a computer, the steps of: (a)
receiving signals from a biopolymer array, as a signal image of the
array; (b) saving the array signal image in a memory; (c)
establishing, based on the detected signals, a shape of each region
in one or more sets of multiple regions on the array signal image,
wherein the shape of regions of a set include an identified shape
of an identified inlier or outlier region within an array feature;
(d) saving a definition of the shapes of the established regions of
each set in a memory; and (e) processing each region of each set of
a region processing routine.
Description
FIELD OF THE INVENTION
[0001] This invention relates to arrays, particularly biopolymer
arrays (such polynucleotide arrays, and particularly DNA arrays)
which are useful in diagnostic, screening, gene expression
analysis, and other applications.
BACKGROUND OF THE INVENTION
[0002] Arrays of biopolymers, such as arrays of peptides or
polynucleotides (such as DNA or RNA), are known and are used, for
example, as diagnostic or screening tools. Such arrays include
regions (sometimes referenced as features or spots) of usually
different sequence biopolymers arranged in a predetermined
configuration on a substrate. The arrays, when exposed to a sample,
will exhibit a pattern of binding which is indicative of the
presence and/or concentration of one or more components of the
sample, such as an antigen in the case of a peptide array or a
polynucleotide of particular sequence in the case of a
polynucleotide array. The binding pattern can be detected by
reading the array, for example, by observing a fluorescence pattern
on the array following exposure to a fluid sample in which all
potential targets (for example, DNA) in the sample have been
labeled with a suitable fluorescent label.
[0003] Methods of fabricating biopolymer arrays can be fabricated
using light directed methods, in situ synthesis methods or
deposition of the previously obtained biopolymers. In known light
directed synthesis methods the aim is to form an array of
oligonucleotides on a surface by removing a photoremovable group
from a surface, coupling a monomer to the exposed region of the
surface, and repeating the process. The in situ synthesis methods
include those described in U.S. Pat. No. 5,449,754 for synthesizing
peptide arrays, as well as WO 98/41531 and the references cited
therein for synthesizing polynucleotides (specifically, DNA). Such
in situ synthesis methods can be basically regarded as iterating
the sequence of depositing droplets of: (a) a protected monomer
onto predetermined locations on a substrate to link with either a
suitably activated substrate surface (or with a previously
deposited deprotected monomer); (b) deprotecting the deposited
monomer so that it can now react with a subsequently deposited
protected monomer; and (c) depositing another protected monomer for
linking. Different monomers may be deposited at different regions
on the substrate during any one iteration so that the different
regions of the completed array will have different desired
biopolymer sequences. One or more intermediate further steps may be
required in each iteration, such as oxidation and washing
steps.
[0004] The "deposition method" basically involve depositing
previously obtained biopolymers at predetermined locations on a
substrate which are suitably activated such that the biopolymers
can link thereto. The deposited biopolymers may, for example, be
obtained from synthetic or biological sources. Biopolymers of
different sequence may be deposited at different regions of the
substrate to yield the completed array. Washing or other additional
steps may also be used. Typical procedures known in the art for
deposition of polynucleotides, particularly DNA such as whole
oligomers or cDNA, are to load a small volume of DNA in solution in
one or more drop dispensers such as the tip of a pin or in an open
capillary and, touch the pin or capillary to the surface of the
substrate. Such a procedure is described in U.S. Pat. No.
5,807,522. When the fluid touches the surface, some of the fluid is
transferred. The pin or capillary must be washed prior to picking
up the next type of DNA for spotting onto the array. This process
is repeated for many different sequences and, eventually, the
desired array is formed. Alternatively, the DNA can be loaded into
a drop dispenser in the form of an inkjet head and fired onto the
substrate. Such a technique has been described, for example, in PCT
publications WO 95/25116 and WO 98/41531, and elsewhere. This
method has the advantage of non-contact deposition. Still other
methods include pipetting and positive displacement pumps such as
the Biodot equipment (available from Bio-Dot Inc., Irvine Calif.,
USA).
[0005] In array fabrication, the quantities of DNA available for
the array are usually very small and expensive. Sample quantities
available for testing are usually also very small and it is
therefore desirable to simultaneously test the same sample against
a large number of different probes on an array. These conditions
require use of arrays with large numbers of very small, closely
spaced spots (features). For example, typical arrays may contain
more than a thousand or ten thousand features in an area no larger
than about 2 to 4 cm.sup.2. During array fabrication however, the
resulting fabricated array may not have an actual layout which is
precisely the same as a target layout. For example, some features
actually deposited may have errors in position, dimensions, or
presence (and may not be present at all due to intermittent errors
in the drop deposition apparatus).
[0006] Thus, during reading of an array by a user following
exposure to a sample, a determination should be made as to the
exact shape of regions that represent actual features. Incorrectly
designating regions as features or otherwise incorrectly
calculating a total signal value for a feature in a determination
of results from the exposed array, can read to serious errors in
result interpretation. Determination of array features can be done
manually by an operator viewing a display of a signal image
acquired from a sample exposed array, and manually designating
(with a mouse or other graphical computer pointer device) features
based on a subjective evaluation of the boundaries of strong signal
regions. The results for such manually designated feature regions
can then be processed by known routines (such as by adding the
signal values of each pixel within designated features of a digital
signal image to obtain a total feature signal value). However, with
thousands of features on each array, such a manual procedure is
very slow, and does not lend itself to reproducible results.
Therefore, computer implemented routines which automatically
determine feature (or other region) boundaries are preferred
particularly where many sample exposed arrays are to be read and
analyzed in an efficient high-throughput manner. These routines and
subsequent processing of features to obtain processed results for
each (such as final signal values for each feature), are referred
to as feature extraction.
[0007] However, the present invention recognizes that even
sophisticated feature extraction routines will not be able to
account for every possible error in designating feature shapes. For
example, a routine which establishes a feature boundary based only
on regions of highest signal strength, may fail to recognize a
region where features have been smeared due to a scratch or other
contact with the array. The present invention recognizes then that
while automated feature extraction methods are needed for
high-throughput reading and processing of array data, it would be
desirable to have some convenient and rapid method by which an
operator or some other means can review what shapes (for example,
feature boundaries) were assigned. The present invention further
recognizes that while this can be reproduced by running the same
feature extraction program on the raw signal data acquired during
array reading such a method is computationally intensive and
therefore time consuming. Thus, the present invention realizes that
it would be desirable, particularly where many arrays are subject
to automated feature extraction, to have some method by which an
individual or other means can readily review the shapes assigned to
feature or other regions even after feature extraction.
SUMMARY OF THE INVENTION
[0008] The present invention then, provides in one aspect, a method
of using an addressable array of biopolymers (for example,
peptides, or polynucleotides such as DNA or RNA) on a substrate
which array has been exposed to a sample. The method includes
detecting signals from the exposed array to obtain a signal image
of the array and optionally saving the array signal image in a
memory. Based on the detected signals, a shape of each region in
one or more sets of multiple regions on the array signal image, is
established. Such sets may include, for example, regions which
represent detected array features or local background regions
(discussed further below). The shapes of the established regions of
each set is saved in a memory, and each region of each set
processed according to a corresponding routine for that set. The
displayed shapes may optionally be saved in a first file and at
least some, or the majority, of the processed results may be saved
in a second file. These files may, for example, both be saved in a
non-volatile memory. It will be appreciated that where the detected
signals have been previously detected and saved a method of the
present invention need not necessarily include the detecting and
saving of the signal image.
[0009] In another aspect, the method may include retrieving the
saved shapes of the established regions from the memory and
displaying images of them. This retrieval may particularly be
performed prior to retrieving from the memory the majority (that
is, greater than 50%, and optionally greater than 60% or 80%) or
any of other processed data for each region of each set of the
retrieved shapes, which data was processed according to a
corresponding routine for that set. Optionally, the array signal
image may also be retrieved and the retrieved region shapes and
array signal images displayed as overlaid images. In one
embodiment, each displayed region may be linked to processed data
for that region. This may be accomplished using a graphical user
interface in which an individual viewing the displayed region may
retrieve saved data (such as the processed results for the
corresponding region) simply by selecting any desired region of
interest (for example, by pointing and clicking on any region of
interest). The method may further optionally include altering a
result obtained from the processing of a region based on the
displayed shapes of the established regions, for example in
response to an alteration of the shape of the displayed region by a
user. The present invention further provides a method in which a
sub-set of a set of processed data from an addressable array of
biopolymers which was exposed to a sample, is retrieved from a
memory (such as a non-volatile memory). Optionally, the retrieved
data may be reviewed and additional processed data of the set
retrieved from the non-volatile memory based on the results of the
review. The sub-set retrieved may, for example, be from regions of
any one or more sets of regions-as discussed herein. For example,
an operator (or a suitably programmed processor) may review an
initially retrieved sub-set (such as shapes of regions of one or
more sets) on a display and, if such data appears questionable
(that is, some part or all of it appears to be outside
predetermined limits), may then retrieve further data to confirm or
dismiss an error (such as in the data or the exposed array). Such a
method can, for example, be operated to facilitate rapid review of
processed data from multiple different arrays, by allowing the
operator to initially review for each only a predetermined sub-set
expected to most likely reveal an error. If no error is detected
during a sub-set review for an array, review of further processed
data for the same array may be avoided thereby saving time during a
review of the data for the multiple arrays while still retaining a
relatively high level of confidence that an error for that array is
unlikely.
[0010] Different sets of regions may be established according to
any desired feature extraction routine. For example, the shape of
regions of a first set are established which comprise regions of
signal strength higher than a predetermined value and which
represent detected array features surrounded by regions of lower
signal strength representing detected background. Similarly, the
shape of regions of a second set may be established each of which
comprises a local background region of lower signal strength than a
corresponding detected feature about which the background region is
positioned.
[0011] Various region processing routines may be used as desired by
a feature extraction procedure. For example, a first routine for
processing a set of regions (such as detected features) may include
summing signal strength within sub-regions of each first set region
to provide a total signal strength for each first set region. The
sub-regions may, for example, be pixels or pixel blocks or groups
where the signal image is a digital image. Other options include
subtracting local background region signal strength from the
corresponding detected feature signal strength (for example, by
subtracting average local background pixel values from each pixel
of the corresponding detected feature).
[0012] The present invention further provides an apparatus for
using an addressable array of biopolymers on a substrate which
array has been exposed to a sample. The apparatus may include a
processor, and optionally may also include a detector to detect
signals from the exposed array to obtain a signal image of the
array and also optionally a memory. The processor is capable of
executing any steps of a method of the present invention (other
than detecting the actual signals from the array, which is
performed by the detector). For example, the processor may save the
array signal image in the memory and establish, based on the
detected signals, a shape of each region in one or more sets of
multiple regions on the array signal image. The processor may
further save the definition of the shapes of the established
regions of each set in a memory, and process each region of each
set according to a corresponding routine for that set. It will be
appreciated that the detected signals from the array may have been
previously detected and saved and thus, an apparatus of the present
invention need not necessarily include a detector and the processor
need not necessarily perform the signal image saving.
[0013] The present invention further provides a computer program
product, comprising: a computer readable storage medium having a
computer program stored thereon. The computer program may perform
any one or more of the steps required by a method of the present
invention (other than the array signal detection), in any different
combinations. In another aspect, the computer program performs,
when loaded into a computer, the steps of: receiving signals from a
biopolymer array which has been exposed to a sample, as a signal
image of the array; saving the array signal image in a memory;
establishing, based on the detected signals, a shape of each region
in one or more sets of multiple regions on the array signal image;
saving a definition of the shapes of the established regions of
each set in a memory; and processing each region of each set
according to a corresponding routine for that set. The saving of
the signal image into memory may be omitted where this has been
previously done and the memory made accessible to the program. In
another aspect, the computer program may perform the steps of
retrieving the signal image and shape definitions and displaying
them as overlaid images, without necessarily performing other
steps.
[0014] One or more of the various aspects of the present invention
may provide one or more of the following, or other, useful
benefits. For example, region shape definitions for one or more
arrays can be quickly reviewed to facilitate detection of a
potential problem in the application of an automatic feature
extraction routine to a particular array in a large batch of
automatically extracted arrays.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Embodiments of the invention will now be described with
reference to the drawings in which:
[0016] FIG. 1 illustrates a substrate carrying multiple arrays,
such as may be used in methods and apparatus of the present
invention;
[0017] FIG. 2 is an enlarged view of a portion of FIG. 1 showing
multiple ideal spots or features;
[0018] FIG. 3 is an enlarged illustration of a portion of the
substrate in FIG. 2;
[0019] FIG. 4 is a schematic diagram of an apparatus at a user site
which can execute a method of the present invention;
[0020] FIG. 5 illustrates the display of image and region shapes
for a read array; and
[0021] FIG. 6 is a flowchart illustrating a method of the present
invention.
[0022] To facilitate understanding, identical reference numerals
have been used, where practical, to designate identical elements
that are common to the figures.
DETAILED DESCRIPTION OF THE INVENTION
[0023] In the present application, unless a contrary intention
appears, the following terms refer to the indicated
characteristics. A "biopolymer" is a polymer of one or more types
of repeating units. Biopolymers are typically found in biological
systems and particularly include polysaccharides (such as
carbohydrates), and peptides (including polypeptides and proteins)
and polynucleotides as well as such compounds composed of or
containing amino acid analogs or non-amino acid groups, or
nucleotide analogs or non-nucleotide groups. This includes
polynucleotides in which the conventional backbone has been
replaced with a non-naturally occurring or synthetic backbone, and
nucleic acids (or synthetic or naturally occurring analogs) in
which one or more of the conventional bases has been replaced with
a group (natural or synthetic) capable of participating in
Watson-Crick type hydrogen bonding interactions. Polynucleotides
include single or multiple stranded configurations, where one or
more of the strands may or may not be completely aligned with
another. A "nucleotide" refers to a sub-unit of a nucleic acid and
has a phosphate group, a 5 carbon sugar and a nitrogen containing
base, as well as functional analogs (whether synthetic or naturally
occurring) of such sub-units which in the polymer form (as a
polynucleotide) can hybridize with naturally occurring
polynucleotides in a sequence specific manner analogous to that of
two naturally occurring polynucleotides. For example, a
"biopolymer" includes DNA (including cDNA), RNA, oligonucleotides,
and PNA and other polynucleotides as described in U.S. Pat. No.
5,948,902 and references cited therein (all of which are
incorporated herein by reference), regardless of the source. An
"oligonucleotide" generally refers to a nucleotide multimer of
about 10 to 100 nucleotides in length, while a "polynucleotide"
includes a nucleotide multimer having any number of
nucleotides.
[0024] An "array", unless a contrary intention appears, includes
any one, two or three dimensional arrangement of addressable
regions bearing a particular chemical moiety or moieties (for
example, biopolymers such as polynucleotide sequences) associated
with that region. An array is "addressable" in that it has multiple
regions of different moieties (for example, different
polynucleotide sequences) such that a region (a "feature" or "spot"
of the array) at a particular predetermined location (an "address")
on the array will detect a particular target or class of targets
(although a feature may incidentally detect non-targets of that
feature). Array features are typically, but need not be, separated
by intervening spaces. In the case of an array, the "target" will
be referenced as a moiety in a mobile phase (typically fluid), to
be detected by probes ("target probes") which are bound to the
substrate at the various regions. However, either of the "target"
or "target probes" may be the one which is to be evaluated by the
other (thus, either one could be an unknown mixture of
polynucleotides to be evaluated by binding with the other). An
"array layout" refers collectively to one or more characteristics
of the features, such as feature positioning, one or more feature
dimensions, errors, or some indication of a moiety at a given
location. "Hybridizing" and "binding", with respect to
polynucleotides, are used interchangeably.
[0025] When one item is indicated as being "remote" from another,
this is referenced that the two items are at least in different
buildings, and may be at least one mile, ten miles, or at least one
hundred miles apart. "Communicating" information references
transmitting the data representing that information as electric or
electromagnetic (including light) signals over a suitable
communication circuit or channel. It will also be appreciated that
throughout the present application, that words such as "top",
"upper", and "lower" are used in a relative sense only. "Fluid" is
used herein to reference a liquid. A "set" or a "sub-set" may have
one or more members but a set of multiple members has more than
one. A "processor" includes any one or more electrical and/or
optical processors which can execute all the steps required of it,
or any hardware or software combination which will perform those or
equivalent steps, such as one or more general purpose digital
microprocessors suitably programmed from a computer readable medium
carrying necessary program code. Any "memory" includes any suitable
device or combination of devices in which a processor can store
and/or retrieve data as required, and includes: non-volatile memory
which remains without a source of power, such as magnetic, optical,
or certain solid state storage devices (for example, magnetic or
optical disks or tape or non-volatile memory chips) as well as
volatile memory (for example, volatile memory chips). Any memory
may be either fixed or portable. Steps recited in a particular
order in relation to any method can be carried out in the recited
order or changed in order where this is logically possible and not
otherwise indicated. Reference to a singular item, includes the
possibility that there are plural of the same items present. All
patents, patent applications, and other cited references are
incorporated into this application by reference except to the
extent that anything in them conflicts with the present
application.
[0026] Referring first to FIGS. 1-3, methods and apparatus of the
present invention may be used with a contiguous planar substrate 10
carrying one or more arrays 12 disposed across a front surface 11a
of substrate 10 and separated by inter-array areas 13. A back
surface 11b of substrate 10 does not carry any arrays 12. The
arrays on substrate 10 can be designed for testing against any type
of sample, whether a trial sample, reference sample, a combination
of them, or a known mixture of polynucleotides (in which latter
case the arrays may be composed of features carrying unknown
sequences to be evaluated). While ten arrays 12 are shown in FIG. 1
and the different embodiments described below may use a substrate
with only one array 12 on it, it will be understood that substrate
10 and the embodiments to be used with it may have any number of
desired arrays 12. Similarly, substrate 10 may be of any shape, and
any apparatus used with it adapted accordingly. Depending upon
intended use, any or all of arrays 12 may be the same or different
from one another and each will contain multiple spots or features
16 of biopolymers such as polynucleotides. A typical array may
contain from more than ten, more than one hundred, more than one
thousand or ten thousand features, or even more than from one
hundred thousand features. All of the features 16 may be different,
or some or all could be the same. In the embodiment illustrated,
there are interfeature areas 17 between features, which do not
carry any polynucleotide. It will be appreciated though, that the
interfeature areas 17 could be of various sizes and configurations.
It will be appreciated that there need not be any space separating
arrays 12 from one another, nor features 16 within an array from
one another. However, in the case where arrays 12 are formed by the
deposition method as described above, such inter-array and
inter-feature areas 17 will typically be present. Each feature
carries a predetermined polynucleotide (which includes the
possibility of mixtures of polynucleotides). As per usual, A, C, G,
T represent the usual nucleotides. It will be understood that there
may be a linker molecule (not shown) of any known types between the
front surface 11a and the first nucleotide.
[0027] FIGS. 2 and 3 are enlarged views illustrating portions of
ideal features on an array where the actual features formed are the
same as the desired features (sometimes referenced as the "target"
or "aim" features), with each feature 16 being uniform in shape,
size and composition, and the features being regularly spaced. In
practice though, such an ideal result is not obtained with there
being some error in one or more feature characteristics.
[0028] Arrays such as arrays 12 may be fabricated at a fabrication
station using in situ or deposition techniques, as more fully
described, for example, in U.S. patent application Ser. No.
09/302898 for "Polynucleotide Array Fabrication" filed Apr. 30,
1999 by Caren et al.
[0029] FIG. 4 illustrates an apparatus of the present invention at
which an addressable array 12 may be used, in particular a single
"user station" which may or may not be remote from the fabrication
station (usually at the location of the customer which ordered a
received array 12). The user station includes a processor 162, a
non-volatile memory 184, user input device 194 (such as a keyboard
and/or mouse or touch screen), monitor 192, an array reader in the
form of a scanner 160 to read an array following exposure to a
sample, data writer/reader 186 (which may be capable of
writing/reading to the same type of media as writer/reader 320),
and a communication module 164 which also has access to a
communication channel 180. Processor 162 can be programmed from any
computer readable medium carrying a suitable computer program which
may be read locally (such as by reader/writer 186) or from a remote
location through communication channel 180. Scanner 160 may include
a holder 161 which receives and holds an array unit 15, as well as
a source of illumination (such as a laser) and a detector in the
form of light sensor 165 to read fluorescent light signals from
respective features on the array. Communication module 164 may be
any type of suitable communication module, such as telephone modem,
WAN card, or satellite modem card. Scanner 160 can be any suitable
apparatus for reading an array, such as one which can read the
location and intensity of fluorescence at each feature of an array
following exposure to a fluorescently labeled sample. For example,
such a scanner may be similar to the GENEARRAY scanner available
from Hewlett-Packard, Palo Alto, Calif. The scanning components of
scanner 160, holder 161, and reader 163 may all be contained within
the same housing of a single same apparatus.
[0030] The operation of the user station of FIG. 4 will now be
described with reference to FIGS. 4-6. Steps and data in FIG. 6 are
referenced by numerals in parentheses. In FIG. 4, an array in the
form of an array unit 15 is received from the remote fabrication
station. Unit 15 includes one or more arrays 12 on substrate 10 and
optionally in a housing (which may include a closed chamber
accessible through one or more ports normally closed by septa). A
sample, for example a test sample, is exposed to the array 12 on
the array unit 15. Following hybridization and washing in a known
manner, the array unit 15 is then inserted into holder 161 in
scanner 160 for reading of the array (such as information
representing the fluorescence pattern on the array 12) under
control of processor 162. During reading signals from light sensor
165 are obtained (400) at processor 162 in the form of a digital
signal image. The digital signal image is merely a spatial
representation of the detected pixels each associated with its
corresponding signal value. The signal value may, for example,
simply be a level on a digital scale (for example, an 8 or 10 bit
scale for any single color) and may be displayed on a monitor 192
as varying brightness. Detector 165 may, for example, provide an
image signal with a pixel resolution of about 2 to 4 .mu.m where
each feature has an approximate diameter of about 10 to 30 .mu.m.
Shapes for detected features and local background regions around
respective features, are established (402) by processor 162 based
on a predetermined algorithm. For example, pixels in a group which
have a signal strength higher than a predetermined value, may be
established as the shape of a detected feature. Pixels around and
within a certain number of pixels of an outer boundary of a
detected feature, and which have signal strength lower than any
pixels of a corresponding detected feature may be established as a
local background region for that feature. In this manner, detected
features (which form a first set of multiple regions) with their
surrounding local backgrounds (forming a second set of multiple
regions) may be established. Other sets of multiple regions may
include inlier or outlier regions within an array feature or
background feature. The definition of the shapes of these sets of
regions, when displayed, results in an image such as that of FIG. 5
in which local backgrounds 3102-3110 surround respective detected
features 3112-3120. The digital signal image and the definition of
the shapes are saved (404) in memory 184 as data signal image and
region shape data (408). For example, the shape definitions and
optionally the digital signal image may be saved in any known image
format such as JPEG, TTFF, uncompressed bitmapped, vector mapped,
or the like. The regions of each set may then be processed
according to the routine for that set. For example, pixels (which
are sub-regions) of a detected feature may have their digital
signal strength values summed to provide a total signal strength
for each feature. Prior to such summation a background value such
as an average of a local background pixel value for a feature, may
be subtracted from each pixel of the feature surrounded by that
local background region. Aspects and different routines used during
feature extraction are further discussed in U.S. patent application
Ser. No. 09/659415 filed Sep. 11, 2000 for "Method And System For
Extracting Data From Surface Array Deposited Features". Note that
the shapes of the sets of regions may be saved in memory 184 in a
first file, while the processed results are saved in a second file
(each file having its own identifier or name). This permits fast
retrieval and display of the shapes before the retrieval of the
processed results. Some of the processed results can also be saved
in the first file (such as less than 5%, 10% 20%, 40%, or less than
50% of the total bits of the processed results). However,
regardless of the format of the saved shapes and processed
information, the saved shapes may be retrieved and displayed by
processor 162 from memory 184 prior to retrieving the majority of
other processed data for the retrieved and displayed shapes.
[0031] Many arrays can be read and processed rapidly and
automatically by scanner 160 under control of processor 162. At any
suitable time subsequent to processing each established region of
an array (such as the detected features and local backgrounds) the
saved signal image and region shape data (408) can be retrieved and
displayed (416) as overlaid images. These have the appearance of
FIG. 5 but additionally including the image signal pixels with
signal strength displayed as increased brightness. Note that the
established shapes can be displayed as solid shapes or as
boundaries, such as just the outer boundaries of the detected
features and local background regions. A user may evaluate the
results from the processing based on the display of the shapes at
any time (for example, a detected feature shape which deviates more
than a predetermined amount from an expected circular shape may
indicate a processed result from that feature to be unreliable).
The retrieval and display can be done either continuously during
scanning and reading for each array of a batch of arrays, or at any
time subsequently. Processor 162 may link regions such as detected
features with processed data from that region, such that a user can
access that data with an appropriate command to select the
corresponding region. For example, with an appropriate graphical
user interface a user can point and click on a displayed detected
feature using user input device 194 and the processed data (for
example, total signal strength for that feature) is retrieved and
displayed in a window on monitor 192. This facilitates a user
evaluating whether the established shape may be in error.
Additionally, the graphical user interface allows the user to use
input device 194 to alter a parameter used in the processing such
as by altering the shape of region on display 192. This can be
done, for example, by dragging a boundary of a detected feature or
local background region. Processor 162 then re-processes the
altered region according to the same routine previously used (such
as the pixel signal addition routine described above in connection
with detected features) using the altered shape.
[0032] Results from the array reading can include the processed
results discussed above. The results of the reading (processed or
not) can be forwarded (such as by communication) along with data
(408) to be received at a remote location for further evaluation
and/or processing, or use, using communication channel 180 or
reader/writer 186 and medium 190. This data may be transmitted by
others as required to reach the remote location, or re-transmitted
to elsewhere as desired.
[0033] Modifications in the particular embodiments described above
are, of course, possible. For example, where a pattern of arrays is
desired, any of a variety of geometries may be constructed other
than the organized rows and columns of arrays 12 of FIG. 1. For
example, arrays 12 can be arranged in a series of curvilinear rows
across the substrate surface (for example, a series of concentric
circles or semi-circles of spots), and the like. Similarly, the
pattern of regions 16 may be varied from the organized rows and
columns of spots in FIG. 2 to include, for example, a series of
curvilinear rows across the substrate surface(for example, a series
of concentric circles or semi-circles of spots), and the like. Even
irregular arrangements of the arrays or the regions within them can
be used provided the locations of features of identified
biopolymers are known.
[0034] The present methods and apparatus may be used to deposit
biopolymers or other moieties on surfaces of any of a variety of
different substrates, including both flexible and rigid substrates.
Thus, in any of the above described methods "biopolymer" or
"biopolymers" could more broadly be replaced with "moiety" or
"moieties". Preferred materials for the substrate provide physical
support for the deposited material and endure the conditions of the
deposition process and of any subsequent treatment or handling or
processing that may be encountered in the use of the particular
array. The array substrate may take any of a variety of
configurations ranging from simple to complex. Thus, the substrate
could have generally planar form, as for example a slide or plate
configuration, such as a rectangular or square or disc. In many
embodiments, the substrate will be shaped generally as a
rectangular solid, having a length in the range about 4 mm to 200
mm, usually about 4 mm to 150 mm, more usually about 4 mm to 125
mm; a width in the range about 4 mm to 200 mm, usually about 4 mm
to 120 mm and more usually about 4 mm to 80 mm; and a thickness in
the range about 0.01 mm to 5.0 mm, usually from about 0.1 mm to 2
mm and more usually from about 0.2 to 1 mm. However, larger
substrates can be used, particularly when such are cut after
fabrication into smaller size substrates carrying a smaller total
number of arrays 12. Substrates of other configurations and
equivalent areas can be chosen. The configuration of the array may
be selected according to manufacturing, handling, and use
considerations.
[0035] The substrates may be fabricated from any of a variety of
materials. In certain embodiments, such as for example where
production of binding pair arrays for use in research and related
applications is desired, the materials from which the substrate may
be fabricated should ideally exhibit a low level of non-specific
binding during hybridization events. In many situations, it will
also be preferable to employ a material that is transparent to
visible and/or UV light. For flexible substrates, materials of
interest include: nylon, both modified and unmodified,
nitrocellulose, polypropylene, and the like, where a nylon
membrane, as well as derivatives thereof, may be particularly
useful in this embodiment. For rigid substrates, specific materials
of interest include: glass; fused silica, silicon, plastics (for
example, polytetrafluoroethylene, polypropylene, polystyrene,
polycarbonate, and blends thereof, and the like); metals (for
example, gold, platinum, and the like).
[0036] The substrate surface onto which the polynucleotide
compositions or other moieties is deposited may be porous or
non-porous, smooth or substantially planar, or have irregularities,
such as depressions or elevations. The surface may be modified with
one or more different layers of compounds that serve to modify the
properties of the surface in a desirable manner. Such modification
layers, when present, will generally range in thickness from a
monomolecular thickness to about 1 mm, usually from a monomolecular
thickness to about 0.1 mm and more usually from a monomolecular
thickness to about 0.001 mm. Modification layers of interest
include: inorganic and organic layers such as metals, metal oxides,
polymers, small organic molecules and the like. Polymeric layers of
interest include layers of: peptides, proteins, polynucleic acids
or rimetics thereof (for example, peptide nucleic acids and the
like); polysaccharides, phospholipids, polyurethanes, polyesters,
polycarbonates, polyureas, polyamides, polyethyleneamines,
polyarylene sulfides, polysiloxanes, polyimides, polyacetates, and
the like, where the polymers may be hetero- or homopolymeric, and
may or may not have separate functional moieties attached thereto
(for example, conjugated),
[0037] Various further modifications to the particular embodiments
described above are, of course, possible. Accordingly, the present
invention is not limited to the particular embodiments described in
detail above.
* * * * *