U.S. patent application number 10/648819 was filed with the patent office on 2005-02-17 for scanned image alignment systems and methods.
This patent application is currently assigned to AFFYMETRIX, INC., a California corporation. Invention is credited to Bartell, Dan M., Fiekowsky, Peter.
Application Number | 20050037367 10/648819 |
Document ID | / |
Family ID | 26749615 |
Filed Date | 2005-02-17 |
United States Patent
Application |
20050037367 |
Kind Code |
A9 |
Fiekowsky, Peter ; et
al. |
February 17, 2005 |
Scanned image alignment systems and methods
Abstract
Systems and methods for aligning scanned images are provided. A
pattern is included in the scanned image so that when the image is
convolved with a filter, a recognizable pattern is generated in the
convolved image. The scanned image may then be aligned according to
the position of the recognizable pattern in the convolved image.
The filter may also act to remove the portions of the scanned image
that do not correspond to the pattern in the scanned image.
Inventors: |
Fiekowsky, Peter; (Los
Altos, CA) ; Bartell, Dan M.; (San Carlos,
US) |
Correspondence
Address: |
AFFYMETRIX, INC
ATTN: CHIEF IP COUNSEL, LEGAL DEPT.
3380 CENTRAL EXPRESSWAY
SANTA CLARA
CA
95051
US
|
Assignee: |
AFFYMETRIX, INC., a California
corporation
Santa Clara
CA
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 0096883 A1 |
May 20, 2004 |
|
|
Family ID: |
26749615 |
Appl. No.: |
10/648819 |
Filed: |
August 25, 2003 |
Related U.S. Patent Documents
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Application
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Patent Number |
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10648819 |
Aug 25, 2003 |
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09542151 |
Apr 4, 2000 |
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6611767 |
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09542151 |
Apr 4, 2000 |
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08996737 |
Dec 23, 1997 |
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6090555 |
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10648819 |
Aug 25, 2003 |
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09699852 |
Oct 30, 2000 |
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6741344 |
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09699852 |
Oct 30, 2000 |
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08823824 |
Mar 25, 1997 |
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6141096 |
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08823824 |
Mar 25, 1997 |
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08195889 |
Feb 10, 1994 |
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5631734 |
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60069032 |
Dec 11, 1997 |
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Current U.S.
Class: |
435/6.18 ;
382/128; 435/6.1; 702/20 |
Current CPC
Class: |
G06T 5/20 20130101; G06T
2207/20068 20130101; G06T 3/60 20130101; G16B 25/00 20190201; G06K
9/3216 20130101; G06T 7/0012 20130101; G06T 7/70 20170101; G06T
2207/30072 20130101 |
Class at
Publication: |
435/006 ;
382/128; 702/020 |
International
Class: |
C12Q 001/68; G06F
019/00; G01N 033/48; G01N 033/50; G06K 009/00 |
Claims
What is claimed is:
1. In a computer system, a method of aligning scanned images,
comprising: convolving a scanned image with a filter, the scanned
image including a first pattern that the filter will convolve into
a second pattern in a convolved image; and aligning the scanned
image according to a position of the second pattern in the
convolved image.
2. The method of claim 1, wherein convolving a scanned image with a
filter comprises setting a convolved pixel to a difference between
a selected odd pixel and a selected even pixel of the first
pattern.
3. The method of claim 2, wherein the selected odd pixel has the
lowest intensity of the odd pixels and the selected even pixel has
the highest intensity of the even pixels, if the average intensity
of the odd pixels is greater than the average intensity of the even
pixels.
4. The method of claim 2, wherein the selected odd pixel has the
highest intensity of the odd pixels and the selected even pixel has
the lowest intensity of the even pixels, if the average intensity
of the odd pixels is not greater than the average intensity of the
even pixels.
5. The method of claim 1, wherein the first pattern is a
checkerboard pattern.
6. The method of claim 1, wherein the second pattern is a grid
pattern.
7. The method of claim 1, wherein aligning the scanned image
comprises aligning a grid over the scanned image.
8. The method of claim 7, further comprising adjusting the position
of the grid to minimize a sum of the intensities of pixels along a
direction in the grid.
9. The method of claim 1, wherein the scanned image includes
multiple copies of the first pattern.
10. The method of claim 9, wherein the scanned image is a rectangle
with a copy of the first pattern near each corner.
11. A computer program product that aligns scanned images,
comprising: computer code that convolves a scanned image with a
filter, the scanned image including a first pattern that the filter
will convolve into a second pattern in a convolved image; computer
code that aligns the scanned image according to a position of the
second pattern in the convolved image; and a computer readable
medium that stores the computer codes.
12. A method of aligning scanned images, comprising: synthesizing a
chip having attached nucleic acid sequences, the chip including a
first pattern of nucleic acid sequences; hybridizing labeled
nucleic acid sequences to nucleic acid sequences on the chip;
scanning the hybridized chip to produce a scanned image; convolving
the scanned image with a filter, the filter convolving the first
pattern into a second pattern in a convolved image; and aligning
the scanned image according to a position of the second pattern in
the convolved image.
13. The method of claim 12, wherein convolving the scanned image
with a filter comprises setting a convolved pixel to a difference
between a selected odd pixel and a selected even pixel of the first
pattern.
14. The method of claim 13, wherein the selected odd pixel has the
lowest intensity of the odd pixels and the selected even pixel has
the highest intensity of the even pixels, if the average intensity
of the odd pixels is greater than the average intensity of the even
pixels.
15. The method of claim 13, wherein the selected odd pixel has the
highest intensity of the odd pixels and the selected even pixel has
the lowest intensity of the even pixels, if the average intensity
of the odd pixels is not greater than the average intensity of the
even pixels.
16. The method of claim 12, wherein the first pattern is a
checkerboard pattern.
17. The method of claim 16, wherein the labeled nucleic acid
sequences include control nucleic acid sequences that hybridize to
alternating squares in the checkerboard pattern.
18. The method of claim 12, wherein the second pattern is a grid
pattern.
19. The method of claim 12, wherein aligning the scanned image
comprises aligning a grid over the scanned image.
20. The method of claim 19, further comprising adjusting the
position of the grid to minimize a sum of the intensities of pixels
along a direction in the grid.
21. The method of claim 12, wherein the scanned image includes
multiple copies of the first pattern.
22. The method of claim 21, wherein the scanned image is a
rectangle with a copy of the first pattern near each corner.
23. A computer program product that aligns scanned images,
comprising: computer code that receives as input a scanned image of
a chip having attached nucleic acid sequences to which labeled
nucleic acid sequences are hybridized, the chip including a first
pattern of nucleic acid sequences; computer code that convolves the
scanned image with a filter, the filter convolving the first
pattern into a second pattern in a convolved image; computer code
that aligns the scanned image according to a position of the second
pattern in the convolved image; and a computer readable medium that
stores the computer codes.
Description
COPYRIGHT NOTICE
[0001] A portion of the disclosure of this patent document contains
material that is subject to copyright protection. The copyright
owner has no objection to the xerographic reproduction by anyone of
the patent document or the patent disclosure in exactly the form it
appears in the Patent and Trademark Office patent file or records,
but otherwise reserves all copyright rights whatsoever.
SOFTWARE APPENDICES
[0002] A Software Appendix of source code for an embodiment of the
invention including two (2) sheets is included herewith.
BACKGROUND OF THE INVENTION
[0003] The present invention relates to the field of image
processing. More specifically, the present invention relates to
computer systems for aligning grids on a scanned image of a chip
including hybridized nucleic acid sequences.
[0004] Devices and computer systems for forming and using arrays of
materials on a chip or substrate are known. For example, PCT
applications WO92/10588 and 95/11995, both incorporated herein by
reference for all purposes, describe techniques for sequencing or
sequence checking nucleic acids and other materials. Arrays for
performing these operations may be formed in arrays according to
the methods of, for example, the pioneering techniques disclosed in
U.S. Pat. Nos. 5,445,934, 5,384,261 and 5,571,639, each
incorporated herein by reference for all purposes.
[0005] According to one aspect of the techniques described therein,
an array of nucleic acid probes is fabricated at known locations on
a chip. A labeled nucleic acid is then brought into contact with
the chip and a scanner generates an image file (also called a cell
file) indicating the locations where the labeled nucleic acids are
bound to the chip. Based upon the image file and identities of the
probes at specific locations, it becomes possible to extract
information such as the nucleotide or monomer sequence of DNA or
RNA. Such systems have been used to form, for example, arrays of
DNA that may be used to study and detect mutations relevant to
genetic diseases, cancers, infectious diseases, HIV, and other
genetic characteristics.
[0006] The VLSIPS.TM. technology provides methods of making very
large arrays of oligonucleotide probes on very small chips. See
U.S. Pat. No. 5,143,854 and PCT patent publication Nos. WO 90/15070
and 92/10092, each of which is incorporated by reference for all
purposes. The oligonucleotide probes on the DNA probe array are
used to detect complementary nucleic acid sequences in a sample
nucleic acid of interest (the "target" nucleic acid).
[0007] For sequence checking applications, the chip may be tiled
for a specific target nucleic acid sequence. As an example, the
chip may contain probes that are perfectly complementary to the
target sequence and probes that differ from the target sequence by
a single base mismatch. For de novo sequencing applications, the
chip may include all the possible probes of a specific length. The
probes are tiled on a chip in rows and columns of cells, where each
cell includes multiple copies of a particular probe. Additionally,
"blank" cells may be present on the chip which do not include any
probes. As the blank cells contain no probes, labeled targets
should not bind specifically to the chip in this area. Thus, a
blank cell provides a measure of the background intensity.
[0008] In the scanned image file, a cell is typically represented
by multiple pixels. Although a visual inspection of the scanned
image file may be performed to identify the individual cells in the
scanned image file. It would be desirable to utilize
computer-implemented image processing techniques to align the
scanned image file.
SUMMARY OF THE INVENTION
[0009] Embodiments of the present invention provide innovative
techniques for aligning scanned images. A pattern is included in
the scanned image so that when the image is convolved with a
filter, a recognizable pattern is generated in the convolved image.
The scanned image may then be aligned according to the position of
the recognizable pattern in the convolved image. The filter may
also act to remove or "filter out" the portions of the scanned
image that do not correspond to the pattern in the scanned image.
Several embodiments of the invention are described below.
[0010] In one embodiment, the invention provides a
computer-implemented method of aligning scanned images. The scanned
image is convolved with a filter. The scanned image includes a
first pattern that the filter will convolve into a second pattern
in the convolved image. The scanned image is then aligned according
to the position of the second pattern in the convolved image. In a
preferred embodiment, the first pattern may be a checkerboard
pattern that is convolved into a grid pattern in the convolved
image.
[0011] In another embodiment, the invention provides a method of
aligning scanned images of chips with hybridized nucleic sequences.
A chip having attached nucleic acid sequences (probes) is
synthesized, with the chip including a first pattern of nucleic
acid sequences. Labeled nucleic acid sequences are hybridized to
nucleic acid sequences on the chip and the hybridized chip is
scanned to produce a scanned image. The scanned image is convolved
with a filter that will convolve the first pattern into a second
pattern in the convolved image. The scanned image is then aligned
according to the position of the second pattern in the convolved
image. In a preferred embodiment, the first pattern may be a
checkerboard pattern that is generated by control nucleic acid
sequences that hybridize to alternating squares in the checkerboard
pattern.
[0012] Other features and advantages of the invention will become
readily apparent upon review of the following detailed description
in association with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 illustrates an example of a computer system that may
be utilized to execute the software of an embodiment of the
invention.
[0014] FIG. 2 illustrates a system block diagram of the computer
system of FIG. 1.
[0015] FIG. 3 illustrates an overall system for forming and
analyzing arrays of biological materials such as DNA or RNA.
[0016] FIG. 4 is a high level flowchart of a process of
synthesizing a chip.
[0017] FIG. 5 illustrates conceptually the binding of probes on
chips.
[0018] FIG. 6 illustrates a flowchart of how a chip is hybridized
and analyzed to produce experimental results.
[0019] FIG. 7A shows a checkerboard pattern in a scanned image and
FIG. 7B shows a grid that has been aligned over the scanned image
to show the individual cells on the chip.
[0020] FIG. 8 illustrates a flowchart of a process of image
alignment.
[0021] FIG. 9A shows a checkerboard pattern in a scanned image and
FIG. 9B shows a convolved image of FIG. 9A with a grid pattern that
was generated by the checkerboard pattern.
[0022] FIG. 10 illustrates a flowchart of a process of convolving
the scanned image.
[0023] FIG. 11 shows neighbor pixels that may be analyzed to
produce a convolved pixel in the convolved image.
[0024] FIGS. 12A-12D show how the filter may be moved over the
scanned image to produce the convolved image.
[0025] FIG. 13 illustrates a flowchart of a process of refining the
grid alignment over the scanned image.
[0026] FIG. 14 shows the grid lines in the scanned image that may
be analyzed to refine the grid alignment.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0027] Overview
[0028] In the description that follows, the present invention will
be described in reference to preferred embodiments that utilize
VLSIPS.TM. technology for making very large arrays of
oligonucleotide probes on chips. However, the invention is not
limited to images produced in this fashion and may be
advantageously applied other hybridization technologies or images
in other technology areas. Therefore, the description of the
embodiments that follows for purposes of illustration and not
limitation.
[0029] FIG. 1 illustrates an example of a computer system that may
be used to execute the software of an embodiment of the invention.
FIG. 1 shows a computer system 1 that includes a display 3, screen
5, cabinet 7, keyboard 9, and mouse 11. Mouse 11 may have one or
more buttons for interacting with a graphical user interface.
Cabinet 7 houses a CD-ROM drive 13, system memory and a hard drive
(see FIG. 2) which may be utilized to store and retrieve software
programs incorporating computer code that implements the invention,
data for use with the invention, and the like. Although a CD-ROM 15
is shown as an exemplary computer readable storage medium, other
computer readable storage media including floppy disk, tape, flash
memory, system memory, and hard drive may be utilized.
Additionally, a data signal embodied in a carrier wave (e.g., in a
network including the Internet) may be the computer readable
storage medium.
[0030] FIG. 2 shows a system block diagram of computer system 1
used to execute the software of an embodiment of the invention. As
in FIG. 1, computer system 1 includes monitor 3 and keyboard 9, and
mouse 11. Computer system 1 further includes subsystems such as a
central processor 51, system memory 53, fixed storage 55 (e.g.,
hard drive), removable storage 57 (e.g., CD-ROM drive), display
adapter 59, sound card 61, speakers 63, and network interface 65.
Other computer systems suitable for use with the invention may
include additional or fewer subsystems. For example, another
computer system could include more than one processor 51 (i.e., a
multi-processor system) or a cache memory.
[0031] The system bus architecture of computer system 1 is
represented by arrows 67. However, these arrows are illustrative of
any interconnection scheme serving to link the subsystems. For
example, a local bus could be utilized to connect the central
processor to the system memory and display adapter. Computer system
1 shown in FIG. 2 is but an example of a computer system suitable
for use with the invention. Other computer architectures having
different configurations of subsystems may also be utilized.
[0032] The present invention provides methods of aligning scanned
images or image files of hybridized chips including nucleic acid
probes. In a representative embodiment, the scanned image files
include fluorescence data from a biological array, but the files
may also represent other data such as radioactive intensity, light
scattering, refractive index, conductivity, electroluminescence, or
large molecule detection data. Therefore, the present invention is
not limited to analyzing fluorescence measurements of hybridization
but may be readily utilized to analyze other measurements of
hybridization.
[0033] For purposes of illustration, the present invention is
described as being part of a computer system that designs a chip
mask, synthesizes the probes on the chip, labels the nucleic acids,
and scans the hybridized nucleic acid probes. Such a system is
fully described in U.S. Pat. No. 5,571,639 that has been
incorporated by reference for all purposes. However, the present
invention may be used separately from the overall system for
analyzing data generated by such systems.
[0034] FIG. 3 illustrates a computerized system for forming and
analyzing arrays of biological materials such as RNA or DNA. A
computer 100 is used to design arrays of biological polymers such
as RNA and DNA. The computer 100 may be, for example, an
appropriately programmed Sun Workstation or personal computer or
workstation, such as an IBM PC equivalent, including appropriate
memory and a CPU as shown in FIGS. 1 and 2. The computer system 100
obtains inputs from a user regarding characteristics of a gene of
interest, and other inputs regarding the desired features of the
array. Optionally, the computer system may obtain information
regarding a specific genetic sequence of interest from an external
or internal database 102 such as GenBank. The output of the
computer system 100 is a set of chip design computer files 104 in
the form of, for example, a switch matrix, as described in PCT
application WO 92/10092, and other associated computer files.
[0035] The chip design files are provided to a system 106 that
designs the lithographic masks used in the fabrication of arrays of
molecules such as DNA. The system or process 106 may include the
hardware necessary to manufacture masks 110 and also the necessary
computer hardware and software 108 necessary to lay the mask
patterns out on the mask in an efficient manner. As with the other
features in FIG. 3, such equipment may or may not be located at the
same physical site but is shown together for ease of illustration
in FIG. 3. The system 106 generates masks 110 or other synthesis
patterns such as chrome-on-glass masks for use in the fabrication
of polymer arrays.
[0036] The masks 110, as well as selected information relating to
the design of the chips from system 100, are used in a synthesis
system 112. Synthesis system 112 includes the necessary hardware
and software used to fabricate arrays of polymers on a substrate or
chip 114. For example, synthesizer 112 includes a light source 116
and a chemical flow cell 118 on which the substrate or chip 114 is
placed. Mask 110 is placed between the light source and the
substrate/chip, and the two are translated relative to each other
at appropriate times for deprotection of selected regions of the
chip. Selected chemical regents are directed through flow cell 118
for coupling to deprotected regions, as well as for washing and
other operations. All operations are preferably directed by an
appropriately programmed computer 119, which may or may not be the
same computer as the computer(s) used in mask design and mask
making.
[0037] The substrates fabricated by synthesis system 112 are
optionally diced into smaller chips and exposed to marked targets.
The targets may or may not be complementary to one or more of the
molecules on the substrate. The targets are marked with a label
such as a fluorescein label (indicated by an asterisk in FIG. 3)
and placed in scanning system 120. Scanning system 120 again
operates under the direction of an appropriately programmed digital
computer 122, which also may or may not be the same computer as the
computers used in synthesis, mask making, and mask design. The
scanner 120 includes a detection device 124 such as a confocal
microscope or CCD (charge-coupled device) that is used to detect
the location where labeled target (*) has bound to the substrate.
The output of scanner 120 is an image file(s) 124 indicating, in
the case of fluorescein labeled target, the fluorescence intensity
(photon counts or other related measurements, such as voltage) as a
function of position on the substrate. Since higher photon counts
will be observed where the labeled target has bound more strongly
to the array of polymers (e.g., DNA probes on the substrate), and
since the monomer sequence of the polymers on the substrate is
known as a function of position, it becomes possible to determine
the sequence(s) of polymer(s) on the substrate that are
complementary to the target.
[0038] The image file 124 is provided as input to an analysis
system 126 that incorporates the scanned image alignment techniques
of the present invention. Again, the analysis system may be any one
of a wide variety of computer system(s), but in a preferred
embodiment the analysis system is based on a WINDOWS NT workstation
or equivalent. The analysis system may analyze the image file(s) to
generate appropriate output 128, such as the identity of specific
mutations in a target such as DNA or RNA.
[0039] FIG. 4 is a high level flowchart of a process of
synthesizing a chip. At a step 201, the desired chip
characteristics are input to the chip synthesis system. The chip
characteristics may include (such as sequence checking systems) the
genetic sequence(s) or targets that would be of interest. The
sequences of interest may, for example, identify a virus,
microorganism or individual. Additionally, the sequence of interest
may provide information about genetic diseases, cancers or
infectious diseases. Sequence selection may be provided via manual
input of text files or may be from external sources such as
GenBank. In a preferred embodiment that performs de novo sequencing
of target nucleic acids, this steps is not necessary as the chip
includes all the possible n-mer probes (where n represents the
length of the nucleic acid probe).
[0040] For de novo sequencing, a chip may be synthesized to include
cells containing all the possible probes of a specific length. For
example, a chip may be synthesized that includes all the possible
8-mer DNA probes. Such a chip would have 65,536 cells
(4*4*4*4*4*4*4*4), with each cell corresponding to a particular
probe. A chip may also include other probes including all the
probes of other lengths.
[0041] At a step 203 the system determines which probes would be
desirable on the chip, and provides an appropriate "layout" on the
chip for the probes. The layout implements desired characteristics
such as an arrangement on the chip that permits "reading" of
genetic sequence and/or minimization of edge effects, ease of
synthesis, and the like.
[0042] The masks for the chip synthesis are designed at a step 205.
The masks are designed according to the desired chip
characteristics and layout. At a step 207, the system synthesizes
the DNA or other polymer chips. Software controls, among other
things, the relative translation of the substrate and mask, the
flow of the desired reagents through a flow cell, the synthesis
temperature of the flow cell, and other parameters.
[0043] FIG. 5 illustrates the binding of a particular target DNA to
an array of DNA probes 114. As shown in this simple example, the
following probes are formed in the array:
1 3'-AGAACGT AGACCGT AGAGCGT AGATCGT .cndot. .cndot. .cndot.
[0044] As shown, when the fluorescein-labeled (or otherwise marked)
target 5'-TCTTGCA is exposed to the array, it is complementary only
to the probe 3'-AGAACGT, and fluorescein will be primarily found on
the surface of the chip where 3'-AGAACGT is located. The chip
contains cells that include multiple copies of a particular probe.
Thus, the image file will contain fluorescence intensities, one for
each probe (or cell). By analyzing the fluorescence intensities
associated with a specific probe, it becomes possible to extract
sequence information from such arrays using the methods of the
invention disclosed herein.
[0045] For ease of reference, one may call bases by assigning the
bases the following codes:
2 Code Group Meaning A A Adenine C C Cytosine G G Guanine T T(U)
Thymine (Uracil) M A or C aMino R A or G puRine W A or T(U) Weak
interaction (2 H bonds) Y C or T(U) pYrimidine S C or G Strong
interaction (3 H bonds) K G or T(U) Keto V A, C or G not T(U) H A,
C or T(U) not G D A, G or T(U) not C B C, G or T(U) not A N A, C,
G, or T(U) Insufficient intensity to call X A, C, G, or T(U)
Insufficient discrimination to call
[0046] Most of the codes conform to the IUPAC standard. However,
code N has been redefined and code X has been added.
[0047] Scanned Image Alignment
[0048] Before the scanned image alignment of the invention are
discussed, it may be helpful to provide an overview of the overall
process in one embodiment. FIG. 6 illustrates a flowchart of a
process of how a chip is hybridized and analyzed to produce
experimental results. A chip 251 having attached nucleic acid
sequences (or probes) is combined with a sample nucleic acid
sequence (e.g., labeled fragments of the sample) and reagents in a
hybridization step 255. The hybridization step produces a
hybridized chip 257.
[0049] The hybridized chip is scanned at a step 259. For example,
the hybridized chip may be laser scanned to detect where
fluorescein-labeled sample fragments have hybridized to the chip.
Numerous techniques may be utilized to label the sample fragments
and the scanning process will typically be performed according to
the type of label utilized. The scanning step produces a digital
image of the chip.
[0050] In preferred embodiments, the scanned image of the chip
includes varying fluorescent intensities that correspond to the
hybridization intensity or affinity of the sample to the probes in
a cell. In order to achieve more accurate results, it is beneficial
to identify the pixels that belong to each cell on the chip. At an
image alignment step 263, the scanned image is aligned so that the
pixels that correspond to each cell can be identified. Optionally,
the image alignment step includes the alignment of a grid over the
scanned image (see FIG. 7B).
[0051] At a step 267, the analysis system analyzes the scanned
image to calculate the relative hybridization intensities for each
cell of interest on the chip. For example, the hybridization
intensity for a cell, and therefore the relative hybridization
affinity between the probe of the cell and the sample sequence, may
be calculated as the mean of the pixel values within the cell. The
pixel values may correspond to photon counts from the labeled
hybridized sample fragments.
[0052] The cell intensities may be stored as a cell intensity file
269. In preferred embodiments, the cell intensity file includes a
list of cell intensities for the cells. At an analysis step 271,
the analysis system may analyze the cell intensity file and chip
characteristics to generate results 273. The chip characteristics
may be utilized to identify the probes that have been synthesized
at each cell on the chip. By analyzing both the sequence of the
probes and their hybridization intensities from the cell intensity
file, the system is able to extract sequence information such as
the location of mutations, deletions or insertions, or the sequence
of the sample nucleic acid. Accordingly, the results may include
sequence information, graphs of the hybridization intensities of
probe(s), graphs of the differences between sequences, and the
like. See U.S. patent application Ser. No. 08/327,525, which is
hereby incorporated by reference for all purposes.
[0053] In order to align the scanned image, the invention provides
a pattern in the scanned image that will be convolved into a
recognizable pattern. In preferred embodiments, the pattern in the
scanned image is a checkerboard pattern that is generated by
synthesizing alternating cells that include probes that are
complementary to a control nucleic acid sequence. The control
nucleic acid sequence may be a known sequence that is labeled and
hybridized to the chip for the purpose of aligning the scanned
image. Additionally, the brightness of the cells complementary to
the control nucleic acid sequence may be utilized as a baseline or
for comparison to other intensities.
[0054] As an example, FIG. 7A shows a checkerboard pattern in a
hybridized chip. A scanned image 301 of a hybridized chip includes
an active area 303 where the probes were synthesized. At the corner
of the active area is a pattern 305 that is a checkerboard pattern.
Typically, the pattern appears at each corner of the active area of
the scanned image. Although the pattern is shown as being a
checkerboard pattern, in other embodiments the pattern is a circle,
square, plus sign, or any other pattern.
[0055] With regard to FIG. 6, it was stated that a grid may
optionally be placed over the scanned image to show or delineate
the individual cells of the chip. FIG. 7B shows a grid that has
been aligned over the scanned image of FIG. 7A to show the
individual cells of the chip. As shown, a grid 307 has been placed
over active area 303 of hybridized chip 301.
[0056] FIG. 8 illustrates a flowchart of a process of image
alignment. The flowchart shows detail for step 263 of FIG. 6. At a
step 351, the scanned image is convolved with a filter. The filter
is typically a software filter that convolves the scanned image
into a convolved image. When the scanned image is convolved, a
pattern in the scanned image is convolved into a recognizable
pattern. The position of the recognizable pattern in the convolved
image may be utilized to align the scanned image, such as by
placing a grid over the image.
[0057] At a step 353, the convolved image is searched for bright
areas. When the scanned image is convolved, the pattern(s) in the
scanned image will be convolved into a recognizable pattern or
patterns of bright areas. Accordingly, once bright areas are
identified in the convolved image, the system confirms that the
bright areas are in the expected recognizable pattern (e.g., a grid
pattern) at a step 355.
[0058] In order to better understand what is meant by the different
patterns, FIG. 9A shows a checkerboard pattern 401 in a scanned
image 403. FIG. 9B shows a recognizable pattern 451 in convolved
image 453. The convolved image was generated from the scanned image
of FIG. 9A. As shown, recognizable pattern 41 in this embodiment is
a grid pattern that was generated by the checkerboard pattern when
it was convolved with a filter. Additionally, it should be noted
that the filter acted to remove the other pixel intensities so that
the convolved image only includes the recognizable pattern. By
removing pixel intensities pixel intensities that are not part of
the pattern in the scanned image, it is easier to align the scanned
image.
[0059] FIG. 10 illustrates a flowchart of a process of convolving
the scanned image. The flowchart illustrates a process that may be
performed at step 351 of FIG. 8. At a step 501, a pixel is
selected. For simplicity, we will assume that the process selects
pixels of the scanned image from left to right and top to bottom.
Of course, the order that the pixels are analyzed may be
varied.
[0060] Once a pixel selected, neighbor pixels may then be selected
at a step 503. By neighbor pixels, it is meant pixels that the
pixels are near, but not necessarily adjacent to a pixel. For
example, FIG. 11 shows neighbor pixels that may be analyzed to
produce a convolved pixel in a convolved image. As shown in FIG.
11, there are 9 pixels labeled 1-9. In a preferred embodiment,
pixel 1 is the pixel retrieved at step 501 and the neighbor pixels
retrieved at step 503 are pixels 2-9. Of course, any number or
location of different neighbor pixels may be utilized.
[0061] At a step 505, the average of the odd pixels and the average
of the even pixels is determined. Referring again to FIG. 11, the
intensities of pixels 1, 3, 5, 7, and 9 may be averaged to produce
the average of the odd pixels (AVG.sub.O). Similarly, the
intensities of pixels 2, 4, 6, and 8 may be averaged to produce the
average of the odd pixels (AVG.sub.E). Thus, the odd pixels may be
pixels that have an odd number designation and the even pixels may
be pixels that have an even number designation.
[0062] Pixel 1 is convolved into a convolved pixel in a convolved
image by determining if the average of the odd pixels is greater
than the average of the even pixels at a step 507. If the average
of the odd pixels is greater, the convolved pixel is set equal to
the intensity of the minimum of the odd pixels minus the intensity
of the maximum of the even pixels at a step 509. Otherwise, the
convolved pixel is set equal to the intensity of the minimum of the
even pixels minus the intensity of the maximum of the odd pixels at
a step 511.
[0063] Conceptually, the neighbor pixels may be thought of as being
filtered, such as by a software filter in preferred embodiments.
With the filter, the system is searching for a checkerboard pattern
where all the odd pixels are either darker or lighter than the even
pixels. Accordingly, averages of the odd and even pixels are
calculated at step 505. Step 507 acts to determine if the pixels
likely reflect a checkerboard pattern where the odd pixels, and
therefore squares, are light (e.g., high intensity) or dark (e.g.,
low intensity). If the odd pixels likely reflect a checkerboard
pattern where the odd pixels are light, step 509 sets the convolved
pixel to the difference between selected odd and even pixels, where
the selected odd pixel is the minimum of the odd pixels and the
selected even pixel is the maximum of the even pixels. Step 511 is
similar but reversed.
[0064] Therefore, at step 509, if all the odd pixels are much
brighter than all the even pixels, the difference will be a larger
value. Hence, the convolved pixel will be relatively bright (e.g.,
high intensity). The convolved pixel will also be relatively bright
if all the even pixels are much brighter than all the odd pixels at
step 511. However, if the difference at step 509 or 511 is very
small (or negative), the convolved pixel will be set to a
relatively dark intensity. Convolved pixels with negative pixel
values may be set to a zero in preferred embodiments. In short, if
the filter finds a checkerboard pattern, the convolved pixel will
be bright and if the filter finds a relatively random pattern, the
convolved pixel will be dark (thus, filtering out "noise" that is
not the desired pattern).
[0065] The recognizable pattern in FIG. 9B, which is a grid
pattern, was generated by the software filter of FIG. 10. In order
to better see how the recognizable pattern was generated, FIGS.
12A-D show how the filter may be moved over the checkerboard to
produce a grid pattern in the convolved image. As the filter is
convolved over the pattern in the scanned image shown in a square
530 in FIG. 12A, a bright square will be generated in the convolved
image since a checkerboard pattern will be found. Similarly, a
bright square will be generated in the convolved image when the
filter is over the pattern in square 530 of FIG. 12B. Of course,
the checkerboard patterns in square 530 of FIGS. 12A and 12B are
reversed, but both will produce a bright square in the convolved
image as described above in reference to FIG. 10. FIGS. 12C and 12D
will also produce two bright squares. Therefore, a 2.times.2 bright
square grid pattern is generated as shown in FIG. 9B.
[0066] Additionally, as the software filter of FIG. 10 acts to
filter out signals that are not the desired pattern, the
recognizable pattern (e.g., a grid pattern) is easier to identify.
The recognizable patterns in the convolved image are utilized to
align the scanned image. Returning now to FIG. 10, after a selected
pixel is convolved into a convolved pixel by the filter, it is
determined if there is another pixel to process in the scanned
image at a step 513.
[0067] The following shows how well an embodiment of the invention
aligned scanned images of hybridized chips:
3 Previous method With filter convolution Perfect alignment 0% 4% 1
pixel off 8% 96% 2 or more pixels off 20% 0% 1 or more cells off
12% 0% unable to align 60% 0%
[0068] The previous method was to analyze the scanned image
(unfiltered) to locate bright areas or spots in a checkerboard
pattern. As shown, an embodiment of the invention was able to
dramatically increase the accuracy of scanned image alignment.
[0069] Refined Grid Alignment
[0070] In preferred embodiments, refined image alignment may be
performed to further increase the accuracy of the scanned image
alignment. FIG. 13 illustrates a flowchart of a process of refining
grid alignment over a scanned image. Thus, for example, once the
above-described process has been performed to align the scanned
image, the process in FIG. 13 may be utilized to refine the
alignment.
[0071] At a step 551, pixel intensities on grid lines in the grid
are summed. For example, the intensities of the grid in a vertical
direction in the checkerboard pattern in the scanned image may be
summed. FIG. 14 shows the grid lines in the scanned image that may
be analyzed to refine the grid alignment. As shown, the pixel
intensities of vertical lines 601 of a checkerboard pattern 603 may
be summed and stored.
[0072] Then, at a step 553, the system may determine if there are
more positions of the grid to analyze. If there are, the position
of the grid may be adjusted at a step 555. Therefore, the grid may
be moved left and right by one or more pixels before the
intensities are summed along grid lines at step 551. Once all the
positions of the grid have been analyzed, the system selects a grid
position where pixel intensities (e.g., the sum calculated at step
551) are at a minimum. Therefore, if the pixel intensities for grid
lines are lower at another position, the grid is adjusted
accordingly. This refinement will work well if the cells are
typically separated by a darker area or line.
[0073] Although the process in FIG. 13 was described for grid lines
in the vertical direction, preferred embodiments also perform the
same grid alignment for the horizontal direction. The distance that
the grid is able to be moved for refinement may be limited. For
example, the grid may be limited to movement of one-third a cell
size.
[0074] The following shows how well an embodiment of the invention
aligned scanned images of hybridized chips utilizing the refined
grid alignment:
4 Previous method With refined grid alignment Perfect alignment 0%
64% 1 pixel off 8% 36% 2 or more pixels off 20% 0% 1 or more cells
off 12% 0% unable to align 60% 0%
[0075] Once again, the previous method was to analyze the scanned
image (unfiltered) to locate bright areas or spots in a
checkerboard pattern. As shown, an embodiment of the invention was
able to dramatically increase the accuracy of scanned image
alignment. Furthermore, refining grid alignment increased the
percentage of scanned images that were perfectly aligned with the
invention from 4% to 64%. Therefore, performing a refinement of
grid alignment can significantly increase the accuracy of the grid
alignment.
[0076] Conclusion
[0077] While the above is a complete description of preferred
embodiments of the invention, various alternatives, modifications,
and equivalents may be used. It should be evident that the
invention is equally applicable by making appropriate modifications
to the embodiments described above. For example, the invention has
been described in reference to a checkerboard pattern in the
scanned image. However, the invention is not limited to any one
pattern and may be advantageously applied to other patterns
including those described herein. Therefore, the above description
should not be taken as limiting the scope of the invention that is
defined by the metes and bounds of the appended claims along with
their full scope of equivalents.
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