U.S. patent application number 10/133888 was filed with the patent office on 2002-12-19 for methods and apparatus for dna sequencing and dna identification.
This patent application is currently assigned to HYSEQ, Inc.. Invention is credited to Drmanac, Radoje.
Application Number | 20020192691 10/133888 |
Document ID | / |
Family ID | 23389627 |
Filed Date | 2002-12-19 |
United States Patent
Application |
20020192691 |
Kind Code |
A1 |
Drmanac, Radoje |
December 19, 2002 |
Methods and apparatus for DNA sequencing and DNA identification
Abstract
Sequencing by Hybridization (SBH) methods and apparatus
employing subdivided filters for discrete multiple probe analysis
of multiple samples may be used for DNA identification and for DNA
sequencing. Partitioned filters are prepared. Samples are affixed
to sections of partitioned filters and each sector is probed with a
single probe or a multiplexed probe for hybridization scoring.
Hybridization data is analyzed for probe complementarity, partial
sequencing by SBH or complete sequencing by SBH.
Inventors: |
Drmanac, Radoje; (Palo Alto,
CA) |
Correspondence
Address: |
PENNIE AND EDMONDS
1155 AVENUE OF THE AMERICAS
NEW YORK
NY
100362711
|
Assignee: |
HYSEQ, Inc.
|
Family ID: |
23389627 |
Appl. No.: |
10/133888 |
Filed: |
April 25, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10133888 |
Apr 25, 2002 |
|
|
|
09503442 |
Feb 14, 2000 |
|
|
|
6403315 |
|
|
|
|
09503442 |
Feb 14, 2000 |
|
|
|
08920295 |
Aug 28, 1997 |
|
|
|
6025136 |
|
|
|
|
Current U.S.
Class: |
435/6.11 ;
435/6.12; 435/91.2 |
Current CPC
Class: |
B01J 2219/00504
20130101; C40B 40/06 20130101; C12Q 1/6827 20130101; B01J
2219/00659 20130101; B01J 2219/00513 20130101; B01J 2219/00387
20130101; B01J 2219/00722 20130101; B01J 19/0046 20130101; B01J
2219/00527 20130101; B01J 2219/00315 20130101; B01J 2219/00378
20130101; Y02P 20/582 20151101; C12Q 1/6874 20130101; B01J
2219/00497 20130101; C40B 60/14 20130101; B01J 2219/00369 20130101;
C12Q 1/6874 20130101; C12Q 2565/518 20130101; C12Q 1/6827 20130101;
C12Q 2565/518 20130101 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Claims
1. A method for analyzing nucleic acids by hybridization,
comprising the steps of: arraying a first plurality of nucleic acid
segments on a first sector of a substrate; disposing a second
plurality of nucleic acid segments on a second sector of said
substrate; exposing, under conditions discriminating between full
complementarity and a one base mismatch, said first plurality of
nucleic acid segments to a first hybridization probe in said first
sector, said first hybridization probe being shorter than one from
among said first plurality of nucleic acid segments, to said
plurality of nucleic acid segments; incubating under conditions
discriminating between full complementarity and a one base
mismatch, a second hybridization probe in said second sector, said
second hybridization probe being shorter than a segment from among
said second plurality of nucleic acid segments and said second
hybridization probe being different in sequence from said first
hybridization probe; detecting hybridization of a hybridization
probe to a nucleic acid segment; and analyzing the result.
2. The method as recited in claim 1, further comprising, prior to
said disposing step, the step of introducing a barrier to movement
of a nucleic acid.
3. The method as recited in claim 1 further comprising, after said
arraying and said disposing step but before said incubating step,
the step of introducing a barrier to movement of a nucleic
acid.
4. The method as recited in claim 3 wherein said introducing step
comprises pressing a physical barrier against said substrate.
5. The method as recited in claim 2 wherein said introducing step
comprises the step of applying a direction-switching electrical
field perpendicular to said support to prevent the mixing of probes
between sectors.
6. The method as recited in claim 3 wherein said introducing step
comprises the step of applying a direction-switching electrical
field perpendicular to said support to prevent the mixing of probes
between sectors.
7. The method as recited in claim 1 wherein said arraying step
comprises the step of spotting nucleic acid samples by means of a
pin array.
8. The method as recited in claim 1 wherein said arraying step
comprises the step of dispensing nucleic acid samples by an array
of tubes.
9. The method as recited in claim 1 wherein said arraying step
comprises the step of jet printing nucleic acid samples.
10. The method as recited in claim 1 wherein said exposing step
comprises the step of applying a plurality of contiguously
hybridizing probes.
11. The method as recited in claim 1 wherein said incubating step
comprises the step of applying a plurality of contiguously
hybridizing probes.
12. The method as recited in claim 10 further comprising the step
of ligating at least two of said plurality of contiguously
hybridizing probes.
13. The method as recited in claim 11 further comprising the step
of ligating at least two of said plurality of contiguously
hybridizing probes.
14. The method as recited in claim 1 wherein said exposing step
comprises the step of applying a plurality of competitively
hybridizing probes having overlapping nucleic acid sequences.
15. The method as recited in claim 1 wherein said incubating step
comprises the step of applying a plurality of competitively
hybridizing probes having overlapping nucleic acid sequences.
16. The method as recited in claim 1 wherein a least two of said
first plurality of nucleic acid segments are arrayed as a
mixture.
17. The method as recited in claim 1 wherein a least two of said
second plurality of nucleic acid segments are disposed as a
mixture.
18. The method as recited in claim 1 further comprising the steps
of preparing samples by digestion with an Hga I type restriction
enzyme and ligating the resulting restriction fragments with an
anchor.
19. The method as recited in claim 1 further comprising the step of
selecting probes from a universal set of probes of a given
length.
20. The method as recited in claim 1 further comprising the step of
selecting probes from an incomplete set of probes of a given
length.
21. The method as recited in claim 1 further comprising the step of
selecting deoxyribonucleotide probes.
22. The method as recited in claim 1 further comprising the step of
selecting ribonucleotide probes.
23. The method as recited in claim 1 further comprising the step of
selecting a nucleic acid analog selected from the group consisting
of protein nucleic acid probes and probes containing base
analogs.
24. The method as recited in claim 1 further comprising the step of
multiplex labelling of probes.
25. The method as recited in claim 1 further comprising the step of
degrading a label on an unhybridized probe.
26. The method as recited in claim 19 wherein said exposing or said
incubating step comprises the step of assembling a set of universal
probes 6, 7, 8, 9 or 10 bases in length.
27. The method as recited in claim 19 wherein said exposing or said
incubating step comprises the step of assembling a set of universal
probes 6, 7, 8, 9 or 10 bases in length.
28. The method as recited in claim 20 wherein said exposing or said
incubating step comprises the step of assembling an incomplete set
of probes 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 bases in length.
29. Apparatus analyzing nucleic acids by hybridization comprising a
substrate having points of attachment for nucleic acid fragments,
said substrate being segmented by hydrophobic regions.
30. The method as recited in claim 20 wherein said disposing step
comprises the step of assembling an incomplete set of probes 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29 or 30 bases in length.
31. The method of claim 1 further comprising the step of confirming
the relative order of at least two bases in a segment by detecting
hybridization of two or more probes having overlapping nucleic acid
sequences including said at least two bases.
32. A method for nucleotide sequence analysis comprising the steps
of: introducing a sample to an array of probes; adjusting the
temperature to be one at which a majority of sample molecules are
unassociated with ligated probes at any given time; adding a
labelled probe to the mixture; incubating the mixture with ligase;
removing free probes; and detecting ligation products.
33. The method as recited in claim 1 further comprising the steps
of defining additional probes for improving a desired result and
repeating said exposing, incubating, detecting and analyzing
steps.
34. The method as recited in claim 1 further comprising the step of
stripping the substrate of probes for reuse of said pluralities of
nucleic acid segments.
Description
FIELD OF THE INVENTION
[0001] This invention relates in general to methods and apparatus
for nucleic acid analysis, and, in particular to, methods and
apparatus for DNA sequencing.
BACKGROUND
[0002] The rate of determining the sequence of the four nucleotides
in DNA samples is a major technical obstacle for further
advancement of molecular biology, medicine, and biotechnology.
Nucleic acid sequencing methods which involve separation of DNA
molecules in a gel have been in use since 1978. The only other
proven method for sequencing nucleic acids is sequencing by
hybridization (SBH).
[0003] The array-based approach of SBH does not require, single
base resolution in separation, degradation, synthesis or imaging of
a DNA molecule. In the most commonly discussed variation of this
method, using mismatch discriminative hybridization of short
oligonucleotides K bases in length, lists of constituent K-mer
oligonucleotides may be determined for target DNA. The sequence may
be assembled through uniquely overlapping scored
oligonucleotides.
[0004] In SBH sequence assembly, K-1 oligonucleotides which occur
repeatedly in analyzed DNA fragments due to chance or biological
reasons may be subject to special consideration. If there is no
additional information, relatively small fragments of DNA may be
fully assembled in as much as every base pair (bp) is read several
times. In assembly of relatively longer fragments, ambiguities may
arise due to repeated occurrence of a K-1 nucleotide. This problem
does not exist if mutated or similar sequences have to be
determined. Knowledge of one sequence may be used as a template to
correctly assemble a similar one.
[0005] There are several approaches for sequencing by
hybridization. In SBH Format 1, DNA samples are arrayed and
labelled probes are hybridized with the samples. Replica membranes
with the same sets of sample DNAs may be used for parallel scoring
of several probes and/or probes may be multiplexed. Arraying and
hybridization of DNA samples on the nylon membranes are well
developed. Each array may be reused many times. Format 1 is
especially efficient for batch processing large numbers of
samples.
[0006] In SBH Format 2, probes are arrayed and a labelled DNA
sample fragment is hybridized to the arrayed probes. In this case,
the complete sequence of one fragment may be determined from
simultaneous hybridization reactions with the arrayed probes. For
sequencing other DNA fragments, the same oligonucleotide array may
be reused. The arrays may be produced by spotting or in situ
variant of Format 2, DNA anchors are arrayed and ligation is used
to determine oligosequences present synthesis. Specific
hybridization has been demonstrated. In a variant of Format 2, DNA
anchors are arrayed and ligation is used to determine
oligosequences present at the end of target DNA.
[0007] In Format 3, two sets of probes are used. One set may be in
the form of arrays and another, labelled set is stored in multiwell
plates. In this case, target DNA need not be labelled. Target DNA
and one labelled probe are added to the arrayed set of probes. If
one attached probe and one labelled probe both hybridize
contiguously on the target DNA, they are covalently ligated,
producing a sequence twice as long to be scored. The process allows
for sequencing long DNA fragments, e.g. a complete bacterial
genome, without DNA subcloning in smaller pieces.
[0008] In the present invention, SBH is applied to the efficient
identification and sequencing one or more DNA samples in a short
period of time. The procedure has many applications in DNA
diagnostics, forensics, and gene mapping. It also may be used to
identify mutations responsible for genetic disorders and other
traits, to assess biodiversity and to produce many other types of
data dependent on DNA sequence.
SUMMARY OF THE INVENTION
[0009] As mentioned above, Format 1 SBH is appropriate for the
simultaneous analysis of a large set of samples. Parallel scoring
of thousands of samples on large arrays may be applied to one or a
few samples are in thousands of independent hybridization reactions
using small pieces of membranes. The identification of DNA may
involve 1-20 probes and the identification of mutations may in some
cases involve more than 1000 probes specifically selected or
designed for each sample. For identification of the nature of the
mutated DNA segments, specific probes may be synthesized or
selected for each mutation detected in the first round of
hybridizations.
[0010] According to the present invention, DNA samples may be
prepared in small arrays which may be separated by appropriate
spacers, and which may be simultaneously tested with probes
selected from a set of oligonucleotides kept in multiwell plates.
Small arrays may consist of one or more samples. DNA samples in
each small array may consist of mutants or individual samples of a
sequence. Consecutive small arrays which form larger arrays may
represent either replication of the same array or samples of a
different DNA fragment. A universal set of probes consists of
sufficient probes to analyze any DNA fragment with prespecified
precision, e.g. with respect to the redundancy of reading each bp.
These sets may include more probes than are necessary for one
specific fragment, but fewer than are necessary for testing
thousands of DNA samples of different sequence.
[0011] DNA or allele identification and a diagnostic sequencing
process may include the steps of:
[0012] 1) Selection of a subset of probes from a dedicated,
representative or universal set to be hybridized with each of a
plurality small arrays;
[0013] 2) Adding a first probe to each subarray on each of the
arrays to be analyzed in parallel;
[0014] 3) Performing hybridization and scoring of the hybridization
results;
[0015] 4) Stripping off previously used probes and repeating
remaining probes that are to be scored;
[0016] 5) Processing the obtained results to obtain a final
analysis or to determine additional probes to be hybridized;
[0017] 6) Performing additional hybridizations for certain
subarrays; and
[0018] 7) Processing complete sets of data and computing obtaining
a final analysis.
[0019] The present invention solves problems in fast identification
and sequencing of a small number of nucleic acid samples of one
type (e.g. DNA, RNA) and in parallel analysis of many sample types
by using a presynthesized set of probes of manageable size and
samples attached to a support in the form of subarrays. Two
approaches have been combined to produce an efficient and versatile
process for the determination of DNA identity, for DNA diagnostics,
and for identification of mutations. For the identification of
known sequences a small set of shorter probes may be used in place
of a longer unique probe. In this case, there may be more probes to
be scored, but a universal set of probes may be synthesized to
cover any type of sequence. For example, a full set of 6-mers or
7-mers are only 4,096 and 16,384 probes, respectively.
[0020] Full sequencing of a DNA fragment may involve two levels.
One level is hybridization of a sufficient set of probes that cover
every base at least once. For this purpose, a specific set of
probes may be synthesized for a standard sample. This hybridization
data reveals whether and where mutations (differences) occur in
non-standard samples. To determine the identity of the changes,
additional specific probes may be hybridized to the sample. In
another embodiment, all probes from a universal set may be
scored.
[0021] A universal set of probes allows scoring of a relatively
small number of probes per sample in a two step-process without
unacceptable expenditure of time. The hybridization process
involves successive probings, in a first step of computing an
optimal subset of probes to be hybridized first and, then, on the
basis of the obtained results, a second step of determining
additional probes to be scored from among those in the existing
universal set.
[0022] The use of an array of sample arrays avoids consecutive
scoring of many oligonucleotides on a single sample or on a small
set of samples. This approach allows the scoring of more probes in
parallel by manipulation of only one physical object. By combining
the use of the subarray formed with the universal set of probes and
the four step hybridization process, a DNA sample 1000 bp in length
may be sequenced in a relatively short period of time. If the
sample is spotted at 50 subarrays in an array and the array is
reprobed 10 times, 500 probes may be scored. This number of probes
is highly sufficient. In screening for the occurrence of a
mutation, approximately 335 probes may be used to cover each base
three times. If a mutation is present, several covering probes will
be affected. These negative probes may map the mutation with a two
base precision. To solve a single base mutation mapped with this
precision, an additional 15 probes may be employed. These probes
cover any base combination for the two questionable positions
(assuming that deletions and insertions are not involved). These
probes may be scored in one cycle on 50 subarrays which contain the
given sample. In the implementation of a multiple label color
scheme (multiplexing), two to six probes labelled with different
fluorescent dyes may be used as a pool, thereby reducing the number
of hybridization cycles and shortening the sequencing process.
[0023] In more complicated cases, there may be two close mutations
or insertions. They may be handled with more probes. For example, a
three base insertion may be solved with 64 probes. The most
complicated cases may be approached by several steps of
hybridization, and the selecting of a new set of probes on the
basis of results of previous hybridizations.
[0024] If subarrays consists of tens or hundreds of samples of one
type, then several of them may be found to contain one or more
changes (mutations, insertions, or deletions). For each segment
where mutation occurs, a specific set of probes may be scored. The
total number of probes to be scored for a type of sample may be
several hundreds. The scoring of replica arrays in parallel allow
scoring of hundreds of probes in a relatively small number of
cycles. In addition, compatible probes may be pooled. Positive
hybridizations may be assigned to the probes selected to check
particular DNA segments because these segments usually differ in
75% of their constituent bases.
[0025] By using a larger set of longer probes, longer targets may
be conveniently analyzed. These targets may represent pools of
shorter fragments such as pools of exon clones.
[0026] The multiple step approach, which minimizes the number of
necessary probes, may employ a specific hybridization scoring
method to define the presence of heterozygotes (sequence variants)
in a genomic segment to be sequenced from a diploid chromosomal
set. There are two possibilities: i) the sequence from one
chromosome represents a basic type and the sequence from the other
represents a new variant; or, ii) both chromosomes contain new, but
different variants. In the first case, the scanning step designed
to map changes gives a maximal signal difference of two-fold at the
heterozygotic position. In the second case, there is no masking;
only a more complicated selection of the probes for the subsequent
rounds of hybridizations may be required.
[0027] Scoring two-fold signal differences required in the first
case may be achieved efficiently by comparing corresponding signals
with controls containing only the basic sequence type and with the
signals from other analyzed samples. This approach allows
determination of a relative reduction in the hybridization signal
for each particular probe in the given sample. This is significant
because hybridization efficiency may vary more than two-fold for a
particular probe hybridized with different DNA fragments having its
full match target. In addition, heterozygotic sites may affect more
than one probe depending on the number of oligonucleotide probes.
Decrease of the signal for two to four consecutive probes produces
a more significant indication of heterozygotic sites. The leads may
be checked by small sets of selected probes among which one or few
probes are suppose to give full match signal which is on average
eight-fold stronger than the signals coming from
mismatch-containing duplexes.
[0028] Partitioned membranes allow a very flexible organization of
experiments to accommodate relatively larger numbers of samples
representing a given sequence type, or many different types of
samples represented with smaller number of samples. A range of
4-256 samples can be handled with particular efficiency. Subarrays
within this range of numbers of dots may be designed to match the
configuration and size of standard multiwell plates used for
storing and labelling oligonucleotides. The size of the subarrays
may be adjusted for different number of samples, or a few standard
subarray sizes may be used. If all samples of one type do not fit
in one subarray, additional subarrays or membranes may be used and
processed with the same probes. In addition, by adjusting the
number of replicas for each subarray, the time for completion of
identification or sequencing process may be varied.
DETAILED DESCRIPTION
EXAMPLE 1
Preparation of a Universal Set of Probes
[0029] Two types of universal sets of probes may be prepared. The
first is a complete set (or at least a noncomplementary subset) of
relatively short probes. For example, all 4096 (or about 2000
non-complementary) 6-mers, or all 16,384 (or about 8,000
non-complementary) 7-mers. Full noncomplementary subsets of 8-mers
and longer probes are less convenient in as much as they include
32,000 or more probes.
[0030] A second type of probe set is selected as a small subset of
probes still sufficient for reading every bp in any sequence with
at least with one probe. For example, 12 of 16 dimers are
sufficient. A small subset for 7-mers, 8-mer and 9-mers for
sequencing double stranded DNA may be about 3000, 10,000 and 30,000
probes, respectively.
[0031] Probes may be prepared using standard chemistry with one to
three non-specified (mixed A, T, C and G) or universal (e.g. M
base, inosine) bases at the ends. If radiolabelling is used, probes
may have an OH group at the 5' end for kinasing by radiolabelled
phosphorous groups. Alternatively, probes labelled with fluorescent
dyes may be employed. Other types of probes like PNA (Protein
Nucleic Acids)or probes containing modified bases which change
duplex stability also may be used.
[0032] Probes may be stored in barcoded multiwell plates. For small
numbers of probes, 96-well plates may be used; for 10,000 or more
probes, storage in 384- or 864-well plates is preferred. Stacks of
5 to 50 plates are enough to store all probes. Approximately 5 pg
of a probe may be sufficient for hybridization with one DNA sample.
Thus, from a small synthesis of about 50 .mu.g per probe, ten
million samples may be analyzed. If each probe is used for every
third sample, and if each sample is 1000 bp in length, then over 30
billion bases (10 human genomes) may be sequenced by a set of 5,000
probes.
EXAMPLE 2
Preparation of DNA Samples
[0033] DNA fragments may be prepared as clones in M13, plasmid or
lambda vectors and/or prepared directly from genomic DNA or cDNA by
PCR or other amplification methods. Samples may be prepared or
dispensed in multiwell plates. About 100-1000 ng of DNA samples may
be prepared in 2-500 .mu.l of final volume.
EXAMPLE 3
Preparation of DNA Arrays
[0034] Arrays may be prepared by spotting DNA samples on a support
such as a nylon membrane. Spotting may be performed by using arrays
of metal pins (the positions of which correspond to an array of
wells in a microtiter plate) to repeated by transfer of about 20 nl
of a DNA solution to a nylon membrane. By offset printing, a
density of dots higher than the density of the wells is achieved.
One to 25 dots may be accommodated in 1 mm.sup.2 depending on the
type of label used. By avoiding spotting in some preselected number
of rows and columns, separate subsets (subarrays) may be formed.
Samples in one subarray may be the same genomic segment of DNA (or
the same gene) from different individuals, or may be different,
overlapped genomic clones. Each of the subarrays may represent
replica spotting of the same samples. In one example, one gene
segment may be amplified from 64 patients. For each patient, the
amplified gene segment may be in one 96-well plate (all 96 wells
containing the same sample). A plate for each of the 64 patients is
prepared. By using a 96-pin device all samples may be spotted on
one 8.times.12 cm membrane. Subarrays may contain 64 samples, one
from each patient. Where the 96 subarrays are identical, the dot
span may be 1 mm.sup.2 and there may be a 1 mm space between
subarrays.
[0035] Another approach is to use membranes or plates (available
from NUNC, Naperville, Ill.) which may be partitioned by physical
spacers e.g. a plastic grid molded over the membrane, the grid
being similar to the sort of membrane applied to the bottom of
multiwell plates, or hydrophobic strips. A fixed physical spacer is
not preferred for imaging by exposure to flat phosphor-storage
screens or x-ray films.
EXAMPLE 4
Selection and Labelling of Probes
[0036] When an array of subarrays is produced, the sets of probes
to be hybridized in each of the hybridization cycles on each of the
subarrays is defined. For the samples in Example 3, a set of 384
probes may be selected from the universal set, and 96 probings may
be performed in each of 4 cycles. Probes selected to be hybridized
in one cycle preferably have similar G+C contents.
[0037] Selected probes for each cycle are transferred to a 96-well
plate and then are labelled by kinasing or by other labelling
procedures if they are not labelled (e.g. with stable fluorescent
dyes) before they are stored.
[0038] On the basis of the first round of hybridizations, a new set
of probes may be defined for each of the subarrays for additional
cycles. Some of the arrays may not be used in some of the cycles.
For example, if only 8 of 64 patient samples exhibit a mutation and
8 probes are scored first for each mutation, then all 64 probes may
be scored in one cycle and 32 subarrays are not used. These
subarrays may then be treated with hybridization buffer to prevent
drying of the filters.
[0039] Probes may be retrieved from the storing plates by any
convenient approach, such as a single channel pipetting device or a
robotic station such as a Beckman Biomek 1000 (Beckman Instruments,
Fullerton, Calif.) or a Mega Two robot (Megamation, Lawrenceville,
N.J.). A robotic station may be integrated with data analysis
programs and probe managing programs. Outputs of these programs may
be inputs for one or more robotic stations.
[0040] Probes may be retrieved one by one and added to subarrays
covered by hybridization buffer. It is preferred that retrieved
probes be placed in a new plate and labelled or mixed with
hybridization buffer. The preferred method of retrieval is by
accessing stored plates one by one and pipetting (or transferring
by metal pins) a sufficient amount of each selected probe from each
plate to specific wells in an intermediary plate. An array of
individually addressable pipettes or pins may be used to speed up
the retrieval process.
EXAMPLE 5
Hybridization and Scoring Process
[0041] Labelled probes may be mixed with hybridization buffer and
pipetted preferentially by multichannel pipettes to the subarrays.
To prevent mixing of the probes between subarrays (if there are no
hydrophilic strips or physical barriers imprinted in the membrane),
a corresponding plastic, metal or ceramic grid may be firmly
pressed to the membrane. Also, the volume of the buffer may be
reduced to about 1 .mu.l or less per mm.sup.2. The concentration of
the probes and hybridization conditions used may be as described
previously except that the washing buffer may be quickly poured
over the array of subarrays to allow fast dilution of probes and
thus prevent significant cross-hybridization. For the same reason,
a minimal concentration of the probes may be used and hybridization
time extended to the maximal practical level. For DNA detection and
sequencing, knowledge of a "normal" sequence allows the use of the
continuous stacking interaction phenomenon to increase the signal.
In addition to the labelled probe, additional unlabelled probes
which hybridize back to back with a labelled one may be added in
the hybridization reaction. The amount of the hybrid may be
increased several times. The probes may be connected by ligation.
This approach may be important for resolving DNA regions forming
"compressions".
[0042] In the case of radiolabelled probes, images of the filters
may be obtained preferentially by phosphorstorage technology.
Fluorescent labels may be scored by CCD cameras, confocal
microscopy or otherwise. Raw signals are normalized based on the
amount of target in each dot to properly scale and integrate data
from different hybridization experiments. Differences in the amount
of target DNA per dot may be corrected for by dividing signals of
each probe by an average signal for all probes scored on one dot.
Also, the normalized signals may be scaled, usually from 1-100, to
compare data from different experiments. Also, in each subarray,
several control DNAs may be used to determine an average background
signal in those samples which do not contain a full match target.
Furthermore, for samples obtained from diploid (polyploid) scores,
homozygotic controls may be used to allow recognition of
heterozygotes in the samples.
EXAMPLE 6
Diagnostics--Scoring Known Mutations or Full Gene Resequencing
[0043] A simple case is to discover whether some known mutations
occur in a DNA segment. Less than 12 probes may suffice for this
purpose, for example, 5 probes positive for one allele, 5 positive
for the other, and 2 negative for both. Because of the small number
of probes to be scored per sample, large numbers of samples may be
analyzed in parallel. For example, with 12 probes in 3
hybridization cycles, 96 different genomic loci or gene segments
from 64 patient may be analyzed on one 6.times.9 in membrane
containing 12.times.24 subarrays each with 64 dots representing the
same DNA segment from 64 patients. In this example, samples may be
prepared in sixty-four 96-well plates. Each plate may represent one
patient, and each well may represent one of the DNA segments to be
analyzed. The samples from 64 plates may be spotted in four
replicas as four quarters of the same membrane.
[0044] A set of 12 probes may be selected by single channel
pipetting or a single pin transferring device (or by an array of
individually controlled pipets or pins) for each of the 96 segments
and rearranged in twelve 96-well plates. Probes may be labelled if
they are not prelabelled before storing, and then probes from four
plates may be mixed with hybridization buffer and added to the
subarrays preferentially by a 96-channel pipeting device. After one
hybridization cycle it is possible to strip off previously used
probes by incubating the membrane at 37.degree. to 55.degree. C. in
the preferably undiluted hybridization or washing buffer.
[0045] The likelihood that probes positive for one allele are
positive and probes positive for the other allele are negative may
be used to determine which of the two allels is present. In this
redundant scoring scheme, some level (about 10%) of errors in
hybridization of each probe may be tolerated.
[0046] An incomplete set of probes may be used for scoring most of
the alleles, especially if the smaller redundancy is sufficient,
e.g. one or two probes which prove the presence or absence in a
sample of one of the two alleles. For example, with a set of four
thousand 8-mers there is a 91% chance of finding at least one
positive probe for one of the two alleles for a randomly selected
locus. The incomplete set of probes may be optimized to reflect G+C
content and other biases in the analyzed samples.
[0047] For full gene sequencing, genes may be amplified in an
appropriate number of segments. For each segment, a set of probes
(about one probe per 2-4 bases) may be selected and hybridized.
These probes may identify whether there is a mutation anywhere in
the analyzed segments. Segments (i.e., subarrays which contain
these segments) where one or more mutated sites are detected may be
hybridized with additional probes to find the exact sequence at the
mutated sites. If a DNA sample is tested by every second 6-mer, and
a mutation is localized at the position that is surrounded by
positively hybridized probes TGCAAA and TATTCC and covered by three
negative probes: CAAAAC, AAACTA and ACTATT, the mutated nucleotides
must be A and/or C occurring in the normal sequence at that
position. They may be changed by a single base mutation, or by a
one or two nucleotide deletion and/or insertion between bases AA,
AC or CT.
[0048] One approach is to select a probe that extends the
positively hybridized probe TGCAAA for one nucleotide to the right,
and which extends the probe TATTCC one nucleotide to the left. With
these 8 probes (GCAAAA, GCAAAT, GCAAAC, GCAAAG and ATATTC, TTATTC,
CTATTC, GTATTC) two questionable nucleotides are determined.
[0049] The most likely hypothesis about the mutation may be
determined. For example, A is found to be mutated to G. There are
two solutions satisfied by these results. Either replacement of A
with G is the only change or there is in addition to that change an
insertion of some number of bases between newly determined G and
the following C. If the result with bridging probes is negative
these options may then be checked first by at least one bridging
probe comprising the mutated position (AAGCTA) and with an
additional 8 probes: CAAAGA, CAAAGT, CAAAGC, CAAAGG and ACTATT,
TCTATT, CCTATT, GCTATT, I There are many other ways to select
mutation-solving probes.
[0050] In the case of diploid, particular comparisons of scores for
the test samples and homozygotic control may be performed to
identify heterozygotes (see above). A few consecutive probes are
expected to have roughly twice smaller signals if the segment
covered by these probes is mutated on one of the two
chromosomes.
EXAMPLE 7
Identification of Genes (Mutations) Responsible for Genetic
Disorders and Other Traits
[0051] The sequencing process disclosed herein has a very low cost
per bp. Also, using larger universal sets of longer probes (8-mers
or 9-mers), DNA fragments as long as 5-20 kb may be sequenced
without subcloning. Furthermore, the speed of resequencing may be
about 10 million bp/day/hybridization instrument. This performance
allows for resequencing a large fraction of human genes or the
human genome repeatedly from scientifically or medically
interesting individuals. To resequence 50% of the human genes,
about 100 million bp is checked. That may be done in a relatively
short period of time at an affordable cost.
[0052] This enormous resequencing capability may be used in several
ways to identify mutations and/or genes that encode for disorders
or any other traits. Basically, mRNAs (which may be converted into
cDNAs) from particular tissues or genomic DNA of patients with
particular disorders may be used as starting materials. From both
sources of DNA, separate genes or genomic fragments of appropriate
length may be prepared either by cloning procedures or by in vitro
amplification procedures (for example by PCR). If cloning is used,
the minimal set of clones to be analyzed may be selected from the
libraries before sequencing. That may be done efficiently by
hybridization of a small number of probes, especially if a small
number of clones longer than 5 kb is to be sorted. Cloning may
increase the amount of hybridization data about two times, but does
not require tens of thousands of PCR primers.
[0053] In one variant of the procedure, gene or genomic fragments
may be prepared by restriction cutting with enzymes like Hga I
which cuts DNA in following way: GACGC(N5')/CTGCG(N10'). Protruding
ends of five bases are different for different fragments. One
enzyme produces appropriate fragments for a certain number of
genes. By cutting cDNA or genomic DNA with several enzymes in
separate reactions, every gene of interest may be excised
appropriately. In one approach, the cut DNA is fractionated by
size. DNA fragments prepared in this way (and optionally treated
with Exonuclease III which individually removes nucleotides from
the 3' end and increases length and specificity of the ends) may be
dispensed in the tubes or in multiwell plates. From a relatively
small set of DNA adapters with a common portion and a variable
protruding end of appropriate length, a pair of adapters may be
selected for every gene fragment that needs to be amplified. These
adapters are ligated and then PCR is performed by universal
primers. From 1000 adapters, a million pairs may be generated, thus
a million different fragments may be specifically amplified in the
identical conditions with a universal pair of primers complementary
to the common end of the adapters.
[0054] If a DNA difference is found to be repeated in several
patients, and that sequence change is nonsense or can change
function of the corresponding protein, then the mutated gene may be
responsible for the disorder. By analyzing a significant number of
individuals with particular traits, functional allelic variations
of particular genes could be associated by specific traits.
[0055] This approach may be used to eliminate the need for very
expensive genetic mapping on extensive pedigrees and has special
value when there is no such genetic data or material.
EXAMPLE 8
Scoring Single Nucleotide Polymorphisms in Genetic Mapping
[0056] Techniques disclosed in this application are appropriate for
an efficient identification of genomic fragments with single
nucleotide polymorphisms (SNUPs). In 10 individuals by applying the
described sequencing process on a large number of genomic fragments
of known sequence that may be amplified by cloning or by in vitro
amplification, a sufficient number of DNA segments with SNUPs may
be identified. The polymorphic fragments are further used as SNUP
markers. These markers are either mapped previously (for example
they represent mapped STSs) or they may be mapped through the
screening procedure described below.
[0057] SNUPs may be scored in every individual from relevant
families or populations by amplifying markers and arraying them in
the form of the array of subarrays. Subarrays contain the same
marker amplified from the analyzed individuals. For each marker, as
in the diagnostics of known mutations, a set of 6 or less probes
positive for one allele and 6 or less probes positive for the other
allele may be selected and scored. From the significant association
of one or a group of the markers with the disorder, chromosomal
position of the responsible gene(s) may be determined. Because of
the high throughput and low cost, thousands of markers may be
scored for thousands of individuals. This amount of data allows
localization of a gene at a resolution level of less than one
million bp as well as localization of genes involved in polygenic
diseases. Localized genes may be identified by sequencing
particular regions from relevant normal and affected individuals to
score a mutation(s).
[0058] PCR is preferred for amplification of markers from genomic
DNA. Each of the markers require a specific pair of primers. The
existing markers may be convertible or new markers may be defined
which may be prepared by cutting genomic DNA by Hga I type
restriction enzymes, and by ligation with a pair of adapters as
described in Example 7.
[0059] SNUP markers can be amplified or spotted as pools to reduce
the number of independent amplification reactions. In this case,
more probes are scored per one sample. When 4 markers are pooled
and spotted on 12 replica membranes, then 48 probes (12 per marker)
may be scored in 4 cycles.
EXAMPLE 9
Detection and Verification of Identity of DNA Fragments
[0060] DNA fragments generated by restriction cutting, cloning or
in vitro amplification (e.g. PCR) frequently may be identified in a
experiment. Identification may be performed by verifying the
presence of a DNA band of specific size on gel electrophoresis.
Alternatively, a specific oligonucleotide may be prepared and used
to verify a DNA sample in question by hybridization. The procedure
developed here allows for more efficient identification of a large
number of samples without preparing a specific oligonucleotide for
each fragment. A set of positive and negative probes may be
selected from the universal set for each fragment on the basis of
the known sequences. Probes that are selected to be positive
usually are able to form one or a few overlapping groups and
negative probes are spread over the whole insert.
[0061] This technology may be used for identification of STSs in
the process of their mapping on the YAC clones. Each of the STSs
may be tested on about 100 YAC clones or pools of YAC clones. DNAs
from these 100 reactions possibly are spotted in one subarray.
Different STSs may represent consecutive subarrays. In several
hybridization cycles, a signature may be generated for each of the
DNA samples, which signature proves or disproves existence of the
particular STS in the given YAC clone with necessary
confidence.
[0062] To reduce the number of independent PCR reactions or the
number of independent samples for spotting, several STSs may be
amplified simultaneously in a reaction or PCR samples may be mixed,
respectively. In this case more probes have to be scored per one
dot. The pooling of STSs is independent of pooling YACs and may be
used on single YACs or pools of YACs. This scheme is especially
attractive when several probes labelled with different colors are
hybridized together.
[0063] In addition to confirmation of the existence of a DNA
fragment in a sample, the amount of DNA may be estimated using
intensities of the hybridization of several separate probes or one
or more pools of probes. By comparing obtained intensities with
intensities for control samples having a known amount of DNA, the
quantity of DNA in all spotted samples is determined
simultaneously. Because only a few probes are necessary for
identification of a DNA fragment, and there are N possible probes
that may be used for DNA N bases long, this application does not
require a large set of probes to be sufficient for identification
of any DNA segment. From one thousand 8-mers, on average about 30
full matching probes may be selected for a 1000 bp fragment.
EXAMPLE 10
Identification of Infectious Disease Organisms and Their
Variants
[0064] DNA-based tests for the detection of viral, bacterial,
fungal and other parasitic organisms in patients are usually more
reliable and less expensive than alternatives. The major advantage
of DNA tests is to be able to identify specific strains and
mutants, and eventually be able to apply more effective treatment.
Two applications are described below.
[0065] The presence of 12 known antibiotic resistance genes in
bacterial infections may be tested by amplifying these genes. The
amplified products from 128 patients may be spotted in two
subarrays and 24 subarrays for 12 genes may then be repeated four
times on a 8.times.12 cm membrane. For each gene, 12 probes may be
selected for positive and negative scoring. Hybridizations may be
performed in 3 cycles. For these tests, as for the tests in Example
9, a much smaller set of probes is most likely to be universal. For
example, from a set of one thousand 8-mers, on average 30 probes
are positive in 1000 bp fragments, and 10 positive probes are
usually sufficient for a highly reliable identification. As
described in Example 9, several genes may be amplified and/or
spotted together and the amount of the given DNA may be determined.
The amount of amplified gene may be used as an indicator of the
level of infection.
[0066] Another example involves possible sequencing of one gene or
the whole genome of an HIV virus. Because of rapid diversification,
the virus poses many difficulties for selection of an optimal
therapy. DNA fragments may be amplified from isolated viruses from
up to 64 patients and resequenced by the described procedure. On
the basis of the obtained sequence the optimal therapy may be
selected. If there is a mixture of two virus types of which one has
the basic sequence (similar to the case of heterozygotes), the
mutant may be identified by quantitative comparisons of its
hybridization scores with scores of other samples, especially
control samples containing the basic virus type only. Scores twice
as small may be obtained for three to four probes that cover the
site mutated in one of the two virus types present in the sample
(see above).
EXAMPLE 11
Forensic and Parental Identification Applications
[0067] Sequence polymorphisms make an individual genomic DNA
unique. This permits analysis of blood or other body fluids or
tissues from a crime scene and comparison with samples from
criminal suspects. A sufficient number of polymorphic sites are
scored to produce a unique signature of a sample. SBH may easily
score single nucleotide polymorphisms to produce such
signatures.
[0068] A set of DNA fragments (10-1000) may be amplified from
samples and suspects. DNAs from samples and suspects representing
one fragment are spotted in one or several subarrays and each
subarray may be replicated 4 times. In three cycles, 12 probes may
determine the presence of allele A or B in each of the samples,
including suspects, for each DNA locus. Matching the patterns of
samples and suspects may lead to discovery of the suspect
responsible for the crime.
[0069] The same procedure may be applicable to prove or disprove
the identity of parents of a child. DNA may be prepared and
polymorphic loci amplified from the child and adults; patterns of A
or B alleles may be determined by hybridization for each.
Comparisons of the obtained patterns, along with positive and
negative controls, aide in the determination of familial
relationships. In this case, only a significant portion of the
alleles need match with one parent for identification. Large
numbers of scored loci allow for the avoidance of statistical
errors in the procedure or of masking effects of de novo
mutations.
EXAMPLE 12
Assessing Genetic Diversity of Populations or Species and
Biological Diversity of Ecological Niches
[0070] Measuring the frequency of allelic variations on a
significant number of loci (for example, several genes or entire
mitochondrial DNA) permits development of different types of
conclusions, such as conclusions regarding the impact of the
environment on the genotypes, history and evolution of a population
or its susceptibility to diseases or extinction, and others. These
assessments may be performed by testing specific known alleles or
by full resequencing of some loci to be able to define de novo
mutations which may reveal fine variations or presence of mutagens
in the environment.
[0071] Additionally, biodiversity in the microbial world may be
surveyed by resequencing evolutionarily conserved DNA sequences,
such as the genes for ribosomal RNAs or genes for highly
conservative proteins. DNA may be prepared from the environment and
particular genes amplified using primers corresponding to
conservative sequences. DNA fragments may be cloned preferentially
in a plasmid vector (or diluted to the level of one molecule per
well in multiwell plates and than amplified in vitro). Clones
prepared this way may be resequenced as described above. Two types
of information are obtained. First of all, a catalogue of different
species may be defined as well as the density of the individuals
for each species. Another segment of information may be used to
measure the influence of ecological factors or pollution on the
ecosystem. It may reveal whether some species are eradicated or
whether the abundance ratios among species is altered due to the
pollution. The method also is applicable for sequencing DNAs from
fossils.
EXAMPLE 13
DNA Sequencing
[0072] An array of subarrays allows for efficient sequencing of a
small set of samples arrayed in the form of replicated subarrays;
For example, 64 samples may be arrayed on a 8.times.8 mm subarray
and 16.times.24 subarrays may be replicated on a 15.times.23 cm
membrane with 1 mm wide spacers between the subarrays. Several
replica membranes may be made. For example, probes from a universal
set of three thousand seventy-two 7-mers may be divided in
thirty-two 96-well plates and labelled by kinasing. Four membranes
may be processed in parallel during one hybridization cycle. On
each membrane, 384 probes may be scored. All probes may be scored
in two hybridization cycles. Hybridization intensities may be
scored and the sequence assembled as described below.
[0073] If a single sample subarray or subarrays contains several
unknowns, especially when similar samples are used, a smaller
number of probes may be sufficient if they are intelligently
selected on the basis of results of previously scored probes. For
example, if probe AAAAAAA is not positive, there is a small chance
that any of 8 overlapping probes are positive. If AAAAAAA is
positive, then two probes are usually positive. The sequencing
process in this case consists of first hybridizing a subset of
minimally overlapped probes to define positive anchors and then to
successively select probes which confirms one of the most likely
hypotheses about the order of anchors and size and type of gaps
between them. In this second phase, pools of 2-10 probes may be
used where each probe is selected to be positive in only one DNA
sample which is different from the samples expected to be positive
with other probes from the pool.
[0074] The subarray approach allows efficient implementation of
probe competition (overlapped probes) or probe cooperation
(continuous stacking of probes) in solving branching problems.
After hybridization of a universal set of probes the sequence
assembly program determines candidate sequence subfragments (SFs).
For the further assembly of SFs, additional information has to be
provided (from overlapped sequences of DNA fragments, similar
sequences, single pass gel sequences, or from other hybridization
or restriction mapping data). Competitive hybridization and
continuous stacking interactions have been proposed for SF
assembly. These approaches are of limited practical value for
sequencing of large numbers of samples by SBH wherein a labelled
probe is applied to a sample affixed to an array if a uniform array
is used. Fortunately, analysis of small numbers of samples using
replica subarrays allows efficient implementation of both
approaches. On each of the replica subarrays, one branching point
may be tested for one or more DNA samples using pools of probes
similarly as in solving mutated sequences in different samples
spotted in the same subarray (see above).
[0075] If in each of 64 samples described in this example, there
are about 100 branching points, and if 8 samples are analyzed in
parallel in each subarray, then at least 800 subarray probings
solve all branches. This means that for the 3072 basic probings an
additional 800 probings (25%) are employed. More preferably, two
probings are used for one branching point. If the subarrays are
smaller, less additional probings are used. For example, if
subarrays consist of 16 samples, 200 additional probings may be
scored (6%). By using 7-mer probes (N.sub.1-2B.sub.7N.sub.1-2) and
competitive or collaborative branching solving approaches or both,
fragments of about 1000 bp fragments may be assembled by about 4000
probings. Furthermore, using 8-mer probes (NB.sub.8N) 4 kb or
longer fragments may be assembled with 12,000 probings. Gapped
probes, for example, NB.sub.4NB.sub.3N or NB.sub.4NB.sub.4N may be
used to reduce the number of branching points.
EXAMPLE 14
DNA Analysis by Transient Attachment to Subarrays of Probes and
Ligation of Labelled Probes.
[0076] Oligonucleotide probes having an informative length of four
to 40 bases are synthesized by standard chemistry and stored in
tubes or in multiwell plates. Specific sets of probes comprising
one to 10,000 probes are arrayed by deposition or in situ synthesis
on separate supports or distinct sections of a larger support. In
the last case, sections or subarrays may be separated by physical
or hydrophobic barriers. The probe arrays may be prepared by in
situ synthesis. A sample DNA of appropriate size is hybridized with
one or more specific arrays. Many samples may be interrogated as
pools at the same subarrays or independently with different
subarrays within one support. Simultaneously with the sample or
subsequently, a single labelled probe or a pool of labelled probes
is added on each of the subarrays. If attached and labelled probes
hybridize back to back on the complementary target in the sample
DNA they are ligated. Occurrence of ligation will be measured by
detecting a label from the probe.
[0077] This procedure is a variant of the described DNA analysis
process in which DNA samples are not permanently attached to the
support. Transient attachment is provided by probes fixed to the
support. In this case there is no need for a target DNA arraying
process. In addition, ligation allows detection of longer
oligonucleotide sequences by combining short labelled probes with
short fixed probes.
[0078] The process has several unique features. Basically, the
transient attachment of the target allows its reuse. After ligation
occur the target may be released and the label will stay covalently
attached to the support. This feature allows cycling the target and
production of detectable signal with a small quantity of the
target. Under optimal conditions, targets do not need to be
amplified, e.g. natural sources of the DNA samples may be directly
used for diagnostics and sequencing purposes. Targets may be
released by cycling the temperature between efficient hybridization
and efficient melting of duplexes. More preferablly, there is no
cycling. The temperature and concentrations of components may be
defined to have an equilibrium between free targets and targets
entered in hybrids at about 50:50% level. In this case there is a
continuous production of ligated products. For different purposes
different equilibrium ratios are optimal.
[0079] An electric field may be used to enhance target use. At the
beginning, a horizontal field pulsing within each subarray may be
employed to provide for faster target sorting. In this phase, the
equilibrium is moved toward hybrid formation, and unlabelled probes
may be used. After a target sorting phase, an appropriate washing
(which may be helped by a vertical electric field for restricting
movement of the samples) may be performed. Several cycles of
discriminative hybrid melting, target harvesting by hybridization
and ligation and removing of unused targets may be introduced to
increase specificity. In the next step, labelled probes are added
and vertical electrical pulses may be applied. By increasing
temperature, an optimal free and hybridized target ratio may be
achieved. The vertical electric field prevents diffusion of the
sorted targets.
[0080] The subarrays of fixed probes and sets of labelled probes
(specially designed or selected from a universal probe set) may be
arranged in various ways to allow an efficient and flexible
sequencing and diagnostics process. For example, if a short
fragment (about 100-500 bp) of a bacterial genome is to be
partially or completely sequenced, small arrays of probes (5-30
bases in length) designed on the bases of known sequence may be
used. If interrogated with a different pool of 10 labelled probes
per subarray, an array of 10 subarrays each having 10 probes,
allows checking of 200 bases, assuming that only two bases
connected by ligation are scored. Under the conditions where
mismatches are discriminated throughout the hybrid, probes may be
displaced by more than one base to cover the longer target with the
same number of probes. By using long probes, the target may be
interrogated directly without amplification or isolation from the
rest of DNA in the sample. Also, several targets may be analyzed
(screened for) in one sample simultaneously. If the obtained
results indicate occurrence of a mutation(or a pathogen),
additional pools of probes may be used to detect type of the
mutation or subtype of pathogen. This is a desirable feature of the
process which may be very cost effective in preventive diagnosis
where only a small fraction of patients is expected to have an
infection or mutation.
[0081] In the processes described in the examples, various
detection methods may be used, for example, radiolabels,
fluorescent labels, enzymes or antibodies (chemiluminescence),
large molecules or particles detectable by light scattering or
interferometric procedures.
EXAMPLE 15
Oligonucleotide Probes and Targets Suitable for SBH
[0082] In order to obtain experimental sequence data defined as a
matrix of (number of fragments-clones).times.(number of probes),
the number of probes may be reduced depending on the number of
fragments used and vice versa. The optimal ratio of the two numbers
is defined by the technological requirements of a particular
sequencing by hybridization process.
[0083] There are two parameters which influence the choice of probe
length. The first is the success in obtaining hybridization results
that show the required degree of discrimination. The second is the
technological feasibility of synthesis of the required number of
probes.
[0084] The requirement of obtaining sufficient hybridization
discrimination with practical and useful amounts of target nucleic
acid limits the probe length. It is difficult to obtain a
sufficient amount of hybrid with short probes, and to discriminate
end mismatches with long probes. Traditionally the use of probes
shorter than 11-mers in the literature, is limited to very stable
probes [Estivill et al., Nucl. Acids Res.15: 1415 (1987)] On the
other hand, probes longer than 15 bases discriminate end mismatches
with difficulty (Wood et al., Proc. Natl. Acad. Sci. USA 82: 1585
(1985)].
[0085] One solution for the problems of unstable probes and end
mismatch discrimination is the use of a group of longer probes
representing a single shorter probe in an informational sense. For
example, groups of sixteen 10-mers may be used instead of single
8-mers. Every member of the group has a common core 8-mer and one
of three possible variations on outer positions with two variations
at each end. The probe may be represented as 5' (A, T, C, G) (A, T,
C, G) B.sub.8 (A, T, C, G) 3'. With this type of probe one does not
need to discriminate the non-informative end bases (two on 5' end,
and one on 3' end) since only the internal 8-mer is read. This
solution employs a higher mass amounts of probes and label in
hybridization reactions.
[0086] These disadvantages are eliminated by the use of a few sets
of discriminative hybridization conditions for oligomer probes as
short as 6-mers.
[0087] The number of hybridization reactions is dependent on the
number of discrete labelled probes. Therefore in the cases of
sequencing shorter nucleic acids using a smaller number of
fragments-clones than the number of oligonucleotides, it is better
to use oligomers as the target and nucleic acid fragment as
probes.
[0088] Target nucleic acids which have undefined sequences may be
produced as a mixture of representative libraries in a phage or
plasmid vector having inserts of genomic fragments of different
sizes or in samples prepared by PCR. Inevitable gaps and
uncertainties in alignment of sequenced fragments arise from
nonrandom or repetitive sequence organization of complex genomes
and difficulties in cloning poisonous sequences in Escherichia
coli. These problems are inherent in sequencing large complex
molecules using any method. Such problems may be minimized by the
choice of libraries and number of subclones used for hybridization.
Alternatively, such difficulties may be overcome through the use of
amplified target sequences, e.g. by PCR amplification, ligation
reactions, ligation-amplified reactions, etc.
[0089] Nucleic acids and methods for isolating, cloning and
sequencing nucleic acids are well known to those of skill in the
art. See e.g., Ausubel et al., Current Protocols in Molecular
Biology, Vol. 1-2, John Wiley & Sons (1989); and Sambrook et
al., Molecular Cloning A Laboratory Manual, 2nd Ed., Vols. 1-3,
Cold Springs Harbor Press (1989), both of which are incorporated by
reference herein.
[0090] SBH is a well developed technology that may be practiced by
a number of methods known to those skilled in the art.
Specifically, techniques related to sequencing by hybridization of
the following documents is incorporated by reference herein:
Drmanac et al., U.S. Pat. No. 5,202,231 (hereby incorporated by
reference herein)--Issued Apr. 13, 1993; Drmanac et al., Genomics,
4, 114-128 (1989); Drmanac et al., Proceedings of the First Int'l.
Conf. Electrophoresis Supercomputing Human Genome Cantor, DR &
Lim HA eds, World Scientific Pub. Co., Singapore, 47-59 (1991);
Drmanac et al., Science, 260, 1649-1652 (1993); Lehrach et al.,
Genome Analysis: Genetic and Physical Mapping, 1, 39-81 (1990),
Cold Spring Harbor Laboratory Press; Drmanac et al., Nucl. Acids
Res., 4691 (1986); Stevanovic et al., Gene, 79, 139 (1989); Panusku
et al., Mol. Biol. Evol., 1, 607 (1990); Nizetic et al., Nucl.
Acids Res., 19, 182 (1991); Drmanac et al., J. Biomol. Struct.
Dyn., 5, 1085 (1991); Hoheisel et al., Mol. Gen., 4, 125-132
(1991); Strezoska et al., Proc. Nat'l. Acad. Sci. (USA), 88, 10089
(1991); Drmanac et al., Nucl. Acids Res., 19, 5839 (1991); and
Drmanac et al., Int. J. Genome Res., 1, 59-79 (1992).
EXAMPLE 16
Determining Sequence from Hybridization Data
[0091] Sequence assembly may be interrupted where ever a given
overlapping (N-1) mer is duplicated two or more times. Then either
of the two N-mers differing in the last nucleotide may be used in
extending the sequence. This branching point limits unambiguous
assembly of sequence.
[0092] Reassembling the sequence of known oligonucleotides that
hybridize to the target nucleic acid to generate the complete
sequence of the target nucleic acid may not be accomplished in some
cases. This is because some information may be lost if the target
nucleic acid is not in fragments of appropriate size in relation to
the size of oligonucleotide that is used for hybridizing. The
quantity of information lost is proportional to the length of a
target being sequenced. However, if sufficiently short targets are
used, their sequence msy be unambiguously determined.
[0093] The probable frequency of duplicated sequences that would
interfere with sequence assembly which is distributed along a
certain length of DNA may be calculated. This derivation requires
the introduction of the definition of a parameter having to do with
sequence organization: the sequence subfragment (SF). A sequence
subfragment results if any part of the sequence of a target nucleic
acid starts and ends with an (N-1)mer that is repeated two or more
times within the target sequence. Thus, subfragments are sequences
generated between two points of branching in the process of
assembly of the sequences in the method of the invention. The sum
of all subfragments is longer than the actual target nucleic acid
because of overlapping short ends. Generally, subfragments may not
be assembled in a linear order without additional information since
they have shared (N-1)mers at their ends and starts. Different
numbers of subfragments are obtained for each nucleic acid target
depending on the number of its repeated (N-1) mers. The number
depends on the value of N-1 and the length of the target.
[0094] Probability calculations can estimate the interrelationship
of the two factors. If the ordering of positive N-mers is
accomplished by using overlapping sequences of length N-1 or at an
average distance of A.sub.o, the N-1 of a fragment Lf bases long is
given by equation one:
N.sub.sf=1+A.sub.oXEKXP(K, L.sub.f)
[0095] Where K greater than or=2, and P (K, L.sub.f) represents the
probability of an N-mer occurring K-times on a fragment L.sub.f
base long. Also, a computer program that is able to form
subfragments from the content of N-mers for any given sequence is
described below in Example 18.
[0096] The number of subfragments increases with the increase of
lengths of fragments for a given length of probe. Obtained
subfragments may not be uniquely ordered among themselves. Although
not complete, this information is very useful for comparative
sequence analysis and the recognition of functional sequence
characteristics. This type of information may be called partial
sequence. Another way of obtaining partial sequence is the use of
only a subset of oligonucleotide probes of a given length.
[0097] There may be relatively good agreement between predicted
sequence according to theory and a computer simulation for a random
DNA sequence. For instance, for N-1=7, [using an 8-mer or groups of
sixteen 10-mers of type 5' (A, T, C, G) B.sub.8 (A, T, C, G) 3'] a
target nucleic acid of 200 bases will have an average of three
subfragments. However, because of the dispersion around the mean, a
library of target nucleic acid should have inserts of 500 bp so
that less than 1 in 2000 targets have more than three subfragments.
Thus, in an ideal case of sequence determination of a long nucleic
acid of random sequence, a representative library with sufficiently
short inserts of target nucleic acid may be used. For such inserts,
it is possible to reconstruct the individual target by the method
of the invention. The entire sequence of a large nucleic acid is
then obtained by overlapping of the defined individual insert
sequences.
[0098] To reduce the need for very short fragments, e.g. 50 bases
for 8-mer probes. The information contained in the overlapped
fragments present in every random DNA fragmentation process like
cloning, or random PCR is used. It is also possible to use pools of
short physical nucleic acid fragments. Using 8-mers or 11-mers like
5' (A, T, C, G) N.sub.8 (A, T, C, G)3' for sequencing 1 megabase,
instead of needing 20,000 50 bp fragments only 2,100 samples are
sufficient. This number consists of 700 random 7 kb clones (basic
library), 1250 pools of 20 clones of 500 bp (subfragments ordering
library) and 150 clones from jumping (or similar) library. The
developed algorithm (see Example 18) regenerates sequence using
hybridization data of th these described samples.
EXAMPLE 17
Hybridization with Oligonucleotides
[0099] Oligonucleotides were either purchased from Genosys Inc.,
Houston, Tex. or made on an Applied Biosystems 381A DNA
synthesizer. Most of the probes used were not purified by HPLC or
gel electrophoresis. For example, probes were designed to have both
a single perfectly complementary target in interferon, a M13 clone
containing a 921 bp Eco RI-Bgl II human B1-interferon fragment
(Ohno and Tangiuchi, Proc. Natl. Acad. Sci. 74: 4370-4374 (1981)],
and at least one target with an end base mismatch in M13 vector
itself.
[0100] End labelling of oligonucleotides was performed as described
[Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Cold Spring Harbor, N.Y. (1982)] in 10
.mu.l containing T4-polynucleotide kinase (5 units Amersham),
.gamma..sup.32p-ATP (3.3 pM, 10 .mu.Ci Amersham 3000 Ci/mM) and
oligonucleotide (4 pM, 10 ng). Specific activities of the probes
were 2.5-5.times.10 9 cpm/nM.
[0101] Single stranded DNA (2 to 4 .mu.l in 0.5 NaOH, 1.5 M NaCl)
was spotted on a Gene Screen membrane wetted with the same
solution, the filters were neutralized in 0.05 M Na.sub.2HPO.sub.4
pH 6.5, baked in an oven at 80.degree. C. for 60 min. and UV
irradiated for 1 min. Then, the filters were incubated in
hybridization solution (0.5 M Na.sub.2HPO.sub.4 pH 7.2, 7% sodium
lauroyl sarcosine for 5 min at room temperature and placed on the
surface of a plastic Petri dish. A drop of hybridization solution
(10 .largecircle.1, 0.5 M Na.sub.2HPO.sub.4 pH 7.2, 7% sodium
lauroyl sarcosine) with a .sup.32P end labelled oligomer probe at 4
nM concentration was placed over 1-6 dots per filter, overlaid with
a square piece of polyethylene (approximately 1.times.1 cm.), and
incubated in a moist chamber at the indicated temperatures for 3
hr. Hybridization was stopped by placing the filter in 6.times. SSC
washing solution for 3.times.5 minute at 0.degree. C. to remove
unhybridized probe. The filter was either dried, or further washed
for the indicated times and temperatures, and autoradiographed. For
discrimination measurements, the dots were excised from the dried
filters after autoradiography [a phosphoimager (Molecular Dynamics,
Sunnyvale, Calif.) may be used] placed in liquid scintillation
cocktail and counted. The uncorrected ratio of cpms for IF and M13
dots is given as D.
[0102] The conditions reported herein allow hybridization with very
short oligonucleotides but ensure discriminations between matched
and mismatched oligonucleotides that are complementary to and
>therefore bind to a target nucleic acid. Factors which
influence the efficient detection of hybridization of specific
short sequences based on the degree of discriminations (D) between
a perfectly complementary target and an imperfectly complementary
target with a single mismatch in the hybrid are defined. In
experimental tests, dot blot hybridization of twenty-eight probes
that were 6 to 8 nucleotides in length to two M13 clones or to
model oligonucleotides bound to membrane filters was accomplished.
The principles guiding the experimental procedures are given
below.
[0103] Oligonucleotide hybridization to filter bound target nucleic
acids only a few nucleotides longer than the probe in conditions of
probe excess is a pseudo-first order reaction with respect to
target concentration. This reaction is defined by:
S.sub.t/S.sub.o=e.sup.-.sup..sup.k.sup.h.sup..sup.[OP]t
[0104] Wherein S.sub.t and S.sub.o are target sequence
concentrations at time t and t.sub.0, respectively. (OP) is probe
concentration and t is temperature. The rate constant for hybrid
formation, k.sub.h increases only slightly in the 0.degree. C. to
30.degree. C. range (Porschke and Eigen, J. Mol. Biol. 62: 361
(1971); Craig et al., J. Mol. Biol. 62: 383 (1971)]. Hybrid melting
is a first order reaction with respect to hybrid concentration
(here replaced by mass due to filter bound state) as shown in:
H.sub.t/H.sub.o=e.sup.-k.sup..sub.m.sup.t
[0105] In this equation, H.sub.t and H.sub.o are hybrid
concentrations at times t and t.sub.o, respectively; k.sub.m is a
rate constant for hybrid melting which is dependent on temperature
and salt concentration [Ikuta et al., Nucl. Acids Res. 15: 797
(1987); Porsclike and Eigen, J. Mol. Biol. 62: 361 (1971); Craig et
al., J. Mol. Biol. 62: 303 (1971)]. During hybridization, which is
a strand association process, the back, melting, or strand
dissociation, reaction takes place as well. Thus, the amount of
hybrid formed in time is result of forward and back reactions. The
equilibrium may be moved towards hybrid formation by increasing
probe concentration and/or decreasing temperature. However, during
washing cycles in large volumes of buffer, the melting reaction is
dominant and the back reaction hybridization is insignificant,
since the probe is absent. This analysis indicates workable Short
Oligonucleotide Hybridization (SOH) conditions call be varied for
probe concentration or temperature.
[0106] D or discrimination is defined in equation four:
D=H.sub.p(t.sub.w)/H.sub.i(t.sub.w)
[0107] H.sub.p (t.sub.w) and H.sub.i (t.sub.w) are the amounts
hybrids remaining after a washing time, t.sub.w, for the identical
amounts of perfectly and imperfectly complementary duplex,
respectively. For a given temperature, the discrimination D changes
with the 10 length of washing time and reaches the maximal value
when H.sub.i=B which is equation five.
[0108] The background, B, represents the lowest hybridization
signal detectable in the system. Since any further decrease of
H.sub.i may not be examined, D increases upon continued washing.
Washing past t.sub.w just decreases H.sub.p relative to B, and is
seen as a decrease in D. The optimal washing time, t.sub.w, for
imperfect hybrids, from equation three and equation five is:
t.sub.w=-ln(B/H.sub.i(t.sub.0))/k.sub.m,i
[0109] Since H.sub.p is being washed for the same t.sub.w,
combining equations, one obtains the optimal discrimination
function:
D=e.sup.ln(B/Hi(.sup..sup.t.sup.0))km,p/km,iXH.sub.p(t.sub.0)/B
[0110] The change of D as a function, of T is important because of
the choice of an optimal washing temperature. It is obtained by
substituting the Arhenius equation which is:
K-=Ae.sup.-.sup..sup.E.sup..alpha./.sup..sup.RT
[0111] into the previous equation to form the final equation:
D=H.sub.p((t.sub.0)/BX(B/H.sub.i(t.sub.0)).sup.(.sup..sup.A.sup.p/.sup..su-
p.A.sup.i).sup..sup.e(E.sup..alpha.i.sup..sup.-E.sup..alpha.p.sup..sup.)/R-
T;
[0112] Wherein B is less than H.sub.i (t.sub.0).
[0113] Since the activation energy for perfect hybrids,
E.sub..alpha.,p, and the activation energy for imperfect hybrids,
E.sub..alpha.,i, can be either equal, or E.sub..alpha.,i less than
E.sub..alpha.,p D is temperature independent, or decreases with
increasing temperature, respectively. This result implies that the
search for stringent temperature conditions for good discrimination
in SOH is unjustified. By washing at lower temperatures, one
obtains equal or better discrimination, but the time of washing
exponentially increases with the decrease of temperature.
Discrimination more strongly decreases with T, if H.sub.i(t.sub.o)
increases relative to H.sub.p (t.sub.0).
[0114] D at lower temperatures depends to a higher degree on the
H.sub.p (t.sub.0)/B ratio than on the H.sub.p (t.sub.0)/H.sub.i
(t.sub.0) ratio. This result indicates that it is better to obtain
a sufficient quantity of H.sub.p in the hybridization regardless of
the discrimination that can be achieved in this step. Better
discrimination can then be obtained by washing, since the higher
amounts of perfect hybrid allow more time for differential melting
to show an effect. Similarly, using larger amounts of target
nucleic acid a necessary discrimination can be obtained even with
small differences between K.sub.m,p and K.sub.m,i.
[0115] Extrapolated to a more complex situation than covered in
this simple model, the result is that washing at lower temperatures
is even more important for obtaining discrimination in the case of
hybridization of a probe having many end-mismatches within a given
nucleic acid target.
[0116] Using the described theoretical principles as a guide for
experiments, reliable hybridizations have been obtained with probes
six to eight nucleotides in length. All experiments were performed
with a floating plastic sheet providing a film of hybridization
solution above the filter. This procedure allows maximal reduction
in the amount of probe, and thus reduced label costs in dot blot
hybridizations. The high concentration of sodium lauroyl sarcosine
instead of sodium lauroyl sulfate in the phosphate hybridization
buffer allows dropping the reaction from room temperature down to
12.degree. C. Similarly, the 4-6.times. SSC, 10% sodium lauroyl
sarcosine buffer allows hybridization at temperatures as low as
2.degree. C. The detergent in these buffers is for obtaining
tolerable background with up to 40 nM concentrations of labelled
probe. Preliminary characterization of the thermal stability of
short oligonucleotide hybrids was determined on a prototype octamer
with 50% G+C content, i.e. probe of sequence TGCTCATG. The
theoretical expectation is that this probe is among the less stable
octamers. Its transition enthalpy is similar to those of more
stable heptamers or, even to probes 6 nucleotides in length
(Bresslauer et al., Proc. Natl. Acad. Sci. U.S.A. 83: 3746 (1986)).
Parameter T.sub.d, the temperature at which 50% of the hybrid is
melted in unit time of a minute is 18.degree. C. The result shows
that T.sub.d is 15.degree. C. lower for the 8 bp hybrid than for an
11 bp duplex [Wallace et al., Nucleic Acids Res. 6: 3543
(1979)].
[0117] In addition to experiments with model oligonucleotides, an
M13 vector was chosen as a system for a practical demonstration of
short oligonucleotide hybridization. The main aim was to show
useful end-mismatch discrimination with a target similar to the
ones which will be used in various applications of the method of
the invention. Oligonucleotide probes for the M13 model were chosen
in such a way that the M13 vector itself contains the end
mismatched base. Vector IF, an M13 recombinant containing a 921 bp
human interferon gene insert, carries single perfectly matched
target. Thus, IF has either the identical or a higher number of
mismatched targets in comparison to the M13 vector itself.
[0118] Using low temperature conditions and dot blots, sufficient
differences in hybridization signals were obtained between tie dot
containing the perfect and the mismatched targets and the dot
containing the mismatched targets only. This was true for the 6-mer
oligonucleotides and was also true for the 7 and 8-mer
oligonucleotides hybridized to the large IF-M13 pair of nucleic
acids.
[0119] The hybridization signal depends on the amount of target
available on the filter for reaction with the probe. A necessary
control is to show that the difference in sign intensity is not a
reflection of varying amounts of nucleic acid in the two dots.
Hybridization with a probe that has the same number and kind of
targets in both IF and M13 shows that there is an equal amount of
DNA in the dots. Since the efficiency of hybrid formation increases
with hybrid length, the signal for a duplex having six nucleotides
was best detected with a high mass of oligonucleotide target bound
to the filter. Due to their lower molecular weight, a larger number
of oligonucleotide target molecules can be bound to a given surface
area when compared to large molecules of nucleic acid that serves
as target.
[0120] To measure the sensitivity of detection with unpurified DNA,
various amounts of phage supernatants were spotted on the filter
and hybridized with a .sup.32P-labelled octamer. As little as 50
million unpurified phage containing no more than 0.5 ng of DNA gave
a detectable signal indicating that sensitivity of the short
oligonucleotide hybridization method is sufficient. Reaction time
is short, adding to the practicality.
[0121] As mentioned in the theoretical section above, the
equilibrium yield of hybrid depends oil probe concentration and/or
temperature of reaction. For instance, the signal level for the
same amount of target with 4 nM octamer at 13.degree. C. is 3 times
lower than with a probe concentration of 40 nM, and is decreased
4.5-times by raising the hybridization temperature to 25.degree.
C.
[0122] The utility of the low temperature wash for achieving
maximal discrimination is demonstrated. To make the phenomenon
visually obvious, 50 times more DNA was put in the M13 dot than in
the IF dot using hybridization with a vector specific probe. In
this way, the signal after the hybridization step with the actual
probe was made stronger in the, mismatched that in the matched
case. The H.sub.p/H.sub.i ratio was 1:4. Inversion of signal
intensities after prolonged washing at 7.degree. C. was achieved
without a massive loss of perfect hybrid, resulting in a ratio of
2:1. In contrast, it is impossible to achieve any discrimination at
25.degree. C., since the matched target signal is already brought
down to the background level with 2 minute washing; at the same
time, the signal from the mismatched hybrid is still detectable.
The loss of discrimination at 13.degree. C. compared to 7.degree.
C. is not so great but is clearly visible. If one considers the 90
minute point at 7.degree. C. and the 15 minute point at 13.degree.
C. when, the mismatched hybrid signal is near the background level,
which represents optimal washing times for the respective
conditions, it is obvious that the amount of several times greater
at 7.degree. C. than at 13.degree. C. To illustrate this further,
the time course of the change discrimination with washing of the
same amount of starting hybrid at the two temperatures shows the
higher maximal D at the lower temperature. These results confirm
the trend in the change of D with temperature and the ratio of
amounts of the two types of hybrid at the start of the washing
step.
[0123] In order to show the general utility of the short
oligonucleotide hybridization conditions, we have looked
hybridization of 4 heptamers, 10 octamers and an additional 14
probes up to 12 nucleotides in length in our simple M13 system.
These include-the nonamer GTTTTTTAA and octamer GGCAGGCG
representing the two extremes of GC content. Although GC content
and sequence are expected to influence the stability of short
hybrids [Bresslauer et al., Proc. Natl. Acad. Sci. U.S.A. 83: 3746
(1986)], the low temperature short oligonucleotide conditions were
applicable to all tested probes in achieving sufficient
discrimination. Since the best discrimination value obtained with
probes 13 nucleotides in length was 20, a several fold drop due to
sequence variation is easily tolerated.
[0124] The M13 system has the advantage of showing the effects of
target DNA complexity on the levels of discrimination. For two
octamers having either none or five mismatched targets and
differing in only one GC pair the observed discriminations were
18.3 and 1.7, respectively.
[0125] In order to show the utility of this method, three probes 8
nucleotides in length were tested on a collection of 51 plasmid DNA
dots made from a library in Bluescript vector. One probe was
present and specific for Bluescript vector but was absent in M13,
while the other two probes had targets that were inserts of known
sequence. This system allowed the use of hybridization negative or
positive control DNAs with each probe. This probe sequence
(CTCCCTTT) also had a complementary target in the interferon
insert. Since the M13 dot is negative while the interferon insert
in either M13 or Bluescript was positive, the hybridization is
sequence specific. Similarly, probes that detect the target
sequence in only one of 51 inserts, or in none of the examined
inserts along with controls that confirm that hybridization would
have occurred if the appropriate targets were present in the
clones.
[0126] Thermal stability curves for very short oligonucleotide
hybrids that are 6-8 nucleotides in length are at least 15.degree.
C. lower than for hybrids 11-12 nucleotides in length [FIG. 1and
Wallace et al., Nucleic Acids Res. 6: 3543-3557 (1979)]. However,
performing the hybridization reaction at a low temperature and with
a very practical 0.4-40 nM concentration of oligonucleotide probe
allows the detection of complementary sequence in a known or
unknown nucleic acid target. To determine an unknown nucleic acid
sequence completely, an entire set containing 65,535 8-mer probes
may be used. Sufficient amounts of nucleic acid for this purpose
are present in convenient biological samples such as a few
microliters of M13 culture, a plasmid prep from 10 ml of bacterial
culture or a single colony of bacteria, or less than 1 .mu.l of a
standard PCR reaction.
[0127] Short oligonucleotides 6-10 nucleotides long give excellent
discrimination. The relative decrease in hybrid stability with a
single end mismatch is greater than for longer probes. Results with
the octamer TGCTCATG support this conclusion. In the experiments,
the target with a G/T end mismatch, hybridization to the target of
this type of mismatch is the most stable of all other types of
oligonucleotide. This discrimination achieved is the same as or
greater than an internal G/T mismatch in a 19 base paired duplex
greater than an internal G/T mismatch in a 19 paired duplex [Ikuta
et al., Nucl. Acids res. 15: 797 (1987)]. Exploiting these
discrimination properties using the described hybridization
conditions for short oligonucleotide hybridization allows a very
precise determination of oligonucleotide targets.
[0128] In contrast to the ease of detecting discrimination between
perfect and imperfect hybrids, a problem that may exist with using
very short oligonucleotides is the preparation of sufficient
amounts of hybrids. In practice, the need to discriminate H.sub.p
and H.sub.i is aided by increasing the amount of DNA in the dot
and/or the probe concentration, or by decreasing the hybridization
temperature. However, higher probe concentrations usually increase
background. Moreover, there are limits to the amounts of target
nucleic acid that are practical to use. This problems was solved by
the higher concentration of the detergent Sarcosyl which gave an
effective background with 4 nM of probe. Further improvements may
be effected either in the use of competitors for unspecific binding
of probe to filter, or by changing the hybridization support
material. Moreover, for probes having E.sub..alpha. less than 45
Kcal/mol (e.g. for many heptamers and a majority of hexamers,
modified oligonucleotides give a more stable hybrid [Asseline, et
al., Proc. Nat'l Acad. Sci. 81: 3297 (1984)] than their unmodified
counterparts. The hybridization conditions described in this
invention for short oligonucleotide hybridization using low
temperatures give better discriminating for all sequences and
duplex hybrid inputs. The only price paid in achieving uniformity
in hybridization conditions for different sequences is an increase
in washing time from minutes to up to 24 hours depending on the
sequence. Moreover, the washing time can be further reduced by
decreasing the salt concentration.
[0129] Although there is excellent discrimination of one matched
hybrid over a mismatched hybrids, in short oligonucleotide
hybridization, signals from mismatched hybrids exist, with the
majority of the mismatch hybrids resulting from end mismatch. This
may limit insert sizes that may be effectively examined by a probe
of a certain length.
[0130] The influence of sequence complexity on discrimination
cannot be ignored. However, the complexity effects are more
significant when defining sequence information by short
oligonucleotide hybridization for specific, nonrandom sequences,
and can be overcome by using an appropriate probe to target length
ratio. The length ratio is chosen to make unlikely, on statistical
grounds, the occurrence of specific sequences which have a number
of end-mismatches which would be able to eliminate or falsely
invert discrimination. Results suggest the use of oligonucleotides
6, 7, and 8 nucleotides in length on target nucleic acid inserts
shorter than 0.6, 2.5, and 10 kb, respectively.
EXAMPLE 18
Sequencing a Target Using Octamers and Nonamers
[0131] In this example, hybridization conditions that were used are
described supra in Example 17. Data resulting from the
hybridization of octamer and nonamer oligonucleotides shows that
sequencing by hybridization provides an extremely high degree of
accuracy. In this experiment, a known sequence was used to predict
a series of contiguous overlapping component octamer and nonamer
oligonucleotides.
[0132] In addition to the perfectly matching oligonucleotides,
mismatch oligonucleotides, mismatch oligonucleotides wherein
internal or end mismatches occur in the duplex formed by the
oligonucleotide and the target were examined. In these analyses,
the lowest practical temperature was used to maximize hybridization
formation. Washes were accomplished at the same or lower
temperatures to ensure maximal discrimination by utilizing the
greater dissociation rate of mismatch versus matched
oligonucleotide/target hybridization. These conditions are shown to
be applicable to all sequences although the absolute hybridization
yield is shown to be sequence dependent.
[0133] The least destabilizing mismatch that can be postulated is a
simple end mismatch, so that the test of sequencing by
hybridization is the ability to discriminate perfectly matched
oligonucleotide/target duplexes from end-mismatched
oligonucleotide/target duplexes.
[0134] The discriminative values for 102 of 105 hybridizing
oligonucleotides in a dot blot format were greater than 2 allowing
a highly accurate generation of the sequence. This system also
allowed an analysis of the effect of sequence on hybridization
formation and hybridization instability.
[0135] One hundred base pairs of a known portion of a human
$-interferon genes prepared by PCR, i.e. a 100 bp target sequence,
was generated with data resulting from the hybridization of 105
oligonucleotides probes of known sequence to the target nucleic
acid. The oligonucleotide probes used included 72 octamer and 21
nonamer oligonucleotides whose sequence was perfectly complementary
to the target. The set of 93 probes provided consecutive
overlapping frames of the target sequence e displaced by one or two
bases.
[0136] To evaluate the effect of mismatches, hybridization was
examined for 12 additional probes that contained at least one end
mismatch when hybridized to the 100 bp test target sequence. Also
tested was the hybridization of twelve probes with target
end-mismatched to four other control nucleic acid sequences chosen
so that the 12 oligonucleotides formed perfectly matched duplex
hybrids with the four control DNAs. Thus, the hybridization of
internal mismatched, end-mismatched and perfectly matched duplex
pairs of oligonucleotide and target were evaluated for each
oligonucleotide used in the experiment. The effect of absolute DNA
target concentration on the hybridization with the test octamer and
nonamer oligonucleotides was determined by defining target DNA
concentration by detecting hybridization of a different
oligonucleotide probe to a single occurrence non-target site within
the co-amplified plasmid DNA.
[0137] The results of this experiment showed that all
oligonucleotides containing perfect matching complementary sequence
to the target or control DNA hybridized more strongly than those
oligonucleotides having mismatches. To come to this conclusion, we
examined H.sub.p and D values for each probe. H.sub.p defines the
amount of hybrid duplex formed between a test target and an
oligonucleotide probe. By assigning values of between 0 and 10 to
the hybridization obtained for the 105 probes, it was ) apparent
that 68.5% of the 105 probes had an H.sub.p greater than 2.
[0138] Discrimination (D) values were obtained where D was defined
as the ratio of signal intensities between 1) the dot containing a
perfect matched duplex formed between test oligonucleotide and
target or control nucleic acid and 2) the dot containing a mismatch
duplex formed between the same oligonucleotide and a different site
within the target or control nucleic acid. Variations in the value
of D result from either 1) perturbations in the hybridization
efficiency which allows visualization of signal over background, or
2) the type of mismatch found between the test oligonucleotide and
the target. The D values obtained in this experiment were between 2
and 40 for 102 of the 105 oligonucleotide probes examined.
Calculations of D for the group of 102 oligonucleotides as a whole
showed the average D was 10.6.
[0139] There were 20 cases where oligonucleotide/target duplexes
exhibited an end-mismatch. In five of these, D was greater than 10.
The large D value in these cases is most likely due to
hybridization destabilization caused by other than the most stable
(G/T and G/A) end mismatches. The other possibility is there was an
error in the sequence of either the oligonucleotides or the
target.
[0140] Error in the target for probes with low H.sub.p was excluded
as a possibility because such an error would have affected the
hybridization of each of the other eight overlapping
oligonucleotides. There was no apparent instability due to sequence
mismatch for the other overlapping oligonucleotides, indicating the
target sequence was correct. Error in the oligonucleotide sequence
was excluded as a possibility after the hybridization of seven
newly synthesized oligonucleotides was re-examined. Only 1 of the
seven oligonucleotides resulted in a better D value. Low hybrid
formation values may result from hybrid instability or from an
inability to form hybrid duplex. An inability to form hybrid
duplexes would result from either 1) self complementarity of the
chosen probe or 2) target/target self hybridization.
Oligonucleotide/oligonucleo- tide duplex formation may be favored
over oligonucleotide/target hybrid duplex formation if the probe
was self-complementary. Similarly, target/target association may be
favored if the target was self-complementary or may form internal
palindromes. In evaluating these possibilities, it was apparent
from probe analysis that the questionable probes did not form
hybrids with themselves. Moreover, in examining the contribution of
target/target hybridization, it was determined that one of the
questionable oligonucleotide probes hybridized inefficiently with
two different DNAs containing the same target. The low probability
that two different DNAs have a self-complementary region for the
same target sequence leads to the conclusion that target/target
hybridization did not contribute to low hybridization formation.
Thus, these results indicate that hybrid instability and not the
inability to form hybrids was the cause of the low hybrid formation
observed for, specific oligonucleotides. The results also indicate
that low hybrid formation is due to the specific sequences of
certain oligonucleotides. Moreover, the results indicate that
reliable results may be obtained to generate sequences if octamer
and nonamer oligonucleotides are used.
[0141] These results show that using the methods described long
sequences of any specific target nucleic acid may be generated by
maximal and unique overlap of constituent oligonucleotides. Such
sequencing methods are dependent on the content of the individual
component oligomers regardless of their frequency and their
position.
[0142] The sequence which is generated using the algorithm
described below is of high fidelity. The algorithm tolerates false
positive signals from the hybridization dots as is indicated from
the fact the sequence generated from the 105 hybridization values,
which included four less reliable values, was correct. This
fidelity in sequencing by hybridization is due to the "all or none"
kinetics of short oligonucleotide hybridization and the difference
in duplex stability that exists between perfectly matched duplexes
and mismatched duplexes. The ratio of duplex stability of matched
and end-mismatched duplexes increases with decreasing duplex
length. Moreover, binding energy decreases with decreasing duplex
length resulting in a lower hybridization efficiency. However, the
results provided show that octamer hybridization allows the
balancing of the factors affecting duplex stability and
discrimination to produce a highly accurate method of sequencing by
hybridization. Results presented in other examples show that
oligonucleotides that are 6, 7, or 8 nucleotides can be effectively
used to generate reliable sequence on targets that are 0.5 kb (for
hexamers) 2 kb (for septamers) and 6 kb (for octamers). The
sequence of long fragments may be overlapped to generate a complete
genome sequence.
[0143] An algorithm to determine sequence by hybridization is
described in Example 18.
EXAMPLE 19
Algorithm
[0144] This example describes an algorithm for generation of a long
sequence written in a four letter alphabet from constituent k-tuple
words in a minimal number of separate, randomly defined fragments
of a starting nucleic acid sequence where K is the length of an
oligonucleotide probe. The algorithm is primarily intended for use
in the sequencing by hybridization (SBH) process. The algorithm is
based on subfragments (SF), informative fragments (IF) and the
possibility of using pools of physical nucleic sequences for
defining informative fragments.
[0145] As described, subfragments may be caused by branch points in
the assembly process resulting from the repetition of a K-1
Oligomer sequence in a target nucleic acid. Subfragments are
sequence fragments found between any two repetitive words of the
length K-1 that occur in a sequence. Multiple occurrences of K-1
words are the cause of interruption of ordering the overlap of
K-words in the process of sequence generation. Interruption leads
to a sequence remaining in the form of subfragments. Thus, the
unambiguous segments between branching points whose order is not
uniquely determined are called sequence subfragments.
[0146] Informative fragments are defined as fragments of a sequence
that are determined by the nearest ends of overlapped physical
sequence fragments.
[0147] A certain number of physical fragments may be pooled without
losing the possibility of defining informative fragments. The total
length of randomly pooled fragments depends on the length of
k-tuples that are used in the sequencing process.
[0148] The algorithm consists of two main units. The first part is
used for generation of subfragments from the set of k-tuples
contained in a sequence. Subfragments may be generated within the
coding region of physical nucleic acid sequence of certain sizes,
or within the informative fragments defined within long nucleic
acid sequences. Both types of fragments are members of the basic
library. This algorithm does not describe the determination of the
content of the k-tuples of the informative fragments of the basic
library, i.e. the step of preparation of informative fragments to
be used in the sequence generation process.
[0149] The second part of the algorithm determines the linear order
of obtained subfragments with the purpose of regenerating the
complete sequence of the nucleic acid fragments of the basic
library. For this purpose a second, ordering library is used, made
of randomly pooled fragments of the starting sequence. The
algorithm does not include the step of combining sequences of basic
fragments to regenerate an entire, megabase plus sequence. This may
be accomplished using the link-up of fragments of the basic library
which is a prerequisite for informative fragment generation.
Alternatively, it may be accomplished after generation of sequences
of fragments of the basic library by this algorithm, using search
for their overlap, based on the presence of common
end-sequences.
[0150] The algorithm requires neither knowledge of the number of
appearances of a given k-tuple in a nucleic acid sequence of the
basic and ordering libraries, nor does it require the information
of which k-tuple words are present on the ends of a fragment. The
algorithm operates with the mixed content of k-tuples of various
length. The concept of the algorithm enables operations with the
k-tuple sets that contain false positive and false negative
k-tuples. Only in specific cases does the content of the false
k-tuples primarily influence the completeness and correctness of
the generated sequence. The algorithm may be used for optimization
of parameters in simulation experiments, as well as for sequence
generation in the actual SBH experiments e.g. generation of the
genomic DNA sequence. In optimization of parameters, the choice of
the oligonucleotide probes (k-tuples) for practical and convenient
fragments and/or the choice of the optimal lengths and the number
of fragments for the defined probes are especially important.
[0151] This part of the algorithm has a central role in the process
of the generation of the sequence from the content of k-tuples. It
is based on the unique ordering of k-tuples by means of maximal
overlap. The main obstacles in sequence generation are specific
repeated sequences and false positive and/or negative k-tuples. The
aim of this part of the algorithm is to obtain the minimal number
of the longest possible subfragments, with correct sequence. This
part of the algorithm consists of one basic, and several control
steps. A two-stage process is necessary since certain information
can be used only after generation of all primary subfragments.
[0152] The main problem of sequence generation is obtaining a
repeated sequence from word contents that by definition do not
carry information on the number of occurrences of the particular
k-tuples. The concept of the entire algorithm depends on the basis
on which this problem is solved. In principle, there are two
opposite approaches: 1) repeated sequences may be obtained at the
beginning, in the process of generation of pSFs, or 2) repeated
sequences can be obtained later, in the process of the final
ordering of the subfragments. In the first case, pSFs contain an
excess of sequences and in the second case, they contain a deficit
of sequences. The first approach requires elimination of the excess
sequences generated,; and the second requires permitting multiple
use of some of the subfragments in the process of the final
assembling of the sequence.
[0153] The difference in the two approaches in the degree of
strictness of the rule of unique overlap of k-tuples. The less
severe rule is: k-tuple X is unambiguously maximally overlapped
with k-tuple Y if and only if, the rightmost k-1 end of k-tuple X
is present only on the leftmost end of k-tuple Y. This rule allows
the generation of repetitive sequences and the formation of surplus
sequences.
[0154] A stricter rule which is used in the second approach has an
addition caveat: k-tuple X is unambiguously maximally overlapped
with k-tuple Y if and only if, the rightmost K-1 end of k-tuple X
is present only on the leftmost end of k-tuple Y and if the
leftmost K-1 end of k-tuple Y is not present on the rightmost end
of any other k-tuple. The algorithm based on the stricter rule is
simpler, and is described herein.
[0155] The process of elongation of a given subfragment is stopped
when the right k-1 end of the last k-tuple included is not present
on the left end of any k-tuple or is present on two or more
k-tuples. If it is present on only one k-tuple the second part of
the rule is tested. If in addition there is a k-tuple which differs
from the previously included one, the assembly of the given
subfragment is terminated only on the first leftmost position. If
this additional k-tuple does not exist, the conditions are met for
unique k-1 overlap and a given subfragment is extended to the right
by one element.
[0156] Beside the basic rule, a supplementary one is used to allow
the usage of k-tuples of different lengths. The maximal overlap is
the length of k-1 of the shorter k-tuple of the overlapping pair.
Generation of the pSFs is performed starting from the first k-tuple
from the file in which k-tuples are displayed randomly and
independently from their order in a nucleic acid sequence. Thus,
the first k-tuple in the file is not necessarily on the beginning
of the sequence, nor on the start of the particular subfragment.
The process of subfragment generation is performed by ordering the
k-tuples by means of unique overlap, which is defined by the
described rule. Each used k-tuple is erased from the file. At the
point when there are no further k-tuples unambiguously overlapping
with the last one included, the building of subfragment is
terminated and the buildup of another pSF is started. Since
generation of a majority of subfragments does not begin from their
actual starts, the formed pSF are added to the k-tuple file and are
considered as a longer k-tuple. Another possibility is to form
subfragments going in both directions from the starting k-tuple.
The process ends when further overlap, i.e. the extension of any of
the subfragments, is not possible.
[0157] The pSFs can be divided in three groups: 1).Subfragments of
the maximal length and correct sequence in cases of exact k-tuple
set; 2) short subfragments, formed due to the used of the maximal
and unambiguous overlap rule on the incomplete set, and/or the set
with some false positive k-tuples; and 3) pSFs of an incorrect
sequence. The incompleteness of the set in 2) is caused by false
negative results of a hybridization experiment, as well as by using
an incorrect set of k-tuples. These are formed due to the false
positive and false negative k-tuples and can be: a) misconnected
subfragments; b) subfragments with the wrong end; and c) false
positive k-tuples which appears as false minimal subfragments.
[0158] Considering false positive k-tuples, there is the
possibility for the presence of a k-tuple containing more than one
wrong base or containing one wrong base somewhere in the middle, as
well as the possibility for a k-tuple with a wrong base on the end.
Generation of short, erroneous or misconnected subfragments is
caused by the latter k-tuples. The k-tuples of the former two kinds
represent wrong pSFs with length equal to k-tuple length.
[0159] In the case of one false negative k-tuple, pSFs are
generated because of the impossibility of maximal overlapping. In
the case of the presence of one false positive k-tuple with the
wrong base on its leftmost or rightmost end, pSFs are generated
because of the impossibility of unambiguous overlapping. When both
false positive and false negative k-tuples with a common k-1
sequence are present in the file, pSFs are generated, and one of
these pSFs contains the wrong k-tuple at the relevant end.
[0160] The process of correcting subfragments with errors in
sequence and the linking of unambiguously connected pSF is
performed after subfragment generation and in the process of
subfragment ordering. The first step which consists of cutting the
misconnected pSFs and obtaining the final subfragments by
unambiguous connection of pSFs is described below.
[0161] There are two approaches for the formation of misconnected
subfragments. In the first a mistake occurs when an erroneous
k-tuple appears on the points of assembly of the repeated sequences
of lengths k-1. In the second, the repeated sequences are shorter
than k-1. These situations can occur in two variants each. In the
first variant, one of the repeated sequences represents the end of
a fragment. In the second variant, the repeated sequence occurs at
any position within the fragment. For the first possibility, the
absence of some k-tuples from the file (false negatives) is
required to generate a misconnection. The second possibility
requires the presence of both false negative and false positive
k-tuples in the file. Considering the repetitions of k-1 sequence,
the lack of only one k-tuple is sufficient when either end is
repeated internally. The lack of two is needed for strictly
internal repetition. The reason is that the end of a sequence can
be considered informatically as an endless linear array of false
negative k-tuples. From the "smaller than k-1 case", only the
repeated sequence of the length of k-2, which requires two or three
specific erroneous k-tuples, will be considered. It is very likely
that these will be the only cases which will be detected in a real
experiment, the others being much less frequent.
[0162] Recognition of the misconnected subfragments is more
strictly defined when a repeated sequence does not appear at the
end of the fragment. In this situation, one can detect further two
subfragments, one of which contains on its leftmost, and the other
on its rightmost end k-2 sequences which are also present in the
misconnected subfragment. When the repeated sequence is on the end
of the fragment, there is only one subfragment which contains k-2
sequence causing the mistake in subfragment formation on its
leftmost or rightmost end.
[0163] The removal of misconnected subframents by their cutting is
performed according to the common rule: If the leftmost or
rightmost sequence of the length of k-2 of any subfragments is
present in any other subfragment, the subfragment is to be cut into
two subfragments, each of them containing k-2 sequence. This rule
does not cover rarer situations of a repeated end when there are
more than one false negative k-tuple on the point of repeated k-1
sequence. Misconnected subfragments of this kind can be recognized
by using the information from the overlapped fragments, or
informative fragments of both the basic and ordering libraries. In
addition, the misconnected subfragment will remain when two or more
false negative k-tuples occur on both positions which contain the
identical k-1 sequence. This is a very rare situation since it
requires at least 4 specific false k-tuples. An additional rule can
be introduced to cut these subfragments on sequences of length k if
the given sequence can be obtained by combination of sequences
shorter than k-2 from the end of one subfragment and the start of
another.
[0164] By strict application of the described rule, some
completeness is lost to ensure the accuracy of the output. Some of
the subfragments will be cut although they are not misconnected
since they fit into the pattern of a misconnected subfragment.
There are several situations of this kind. For example, a fragment,
beside at least two identical k-1 sequences, contains any k-2
sequence from k-1 or a fragment contains k-2 sequence repeated at
least twice and at least one false negative k-tuple containing
given k-2 sequence in the middle, etc.
[0165] The aim of this part of the algorithm is to reduce the
number of pSFs to a minimal number of longer subfragments with
correct sequence. The generation of unique longer subfragments or a
complete sequence is possible in two situations. The first
situation concerns the specific order of repeated k-1 words. There
are cases in which some or all maximally extended pSFs (the first
group of pSFs) can be uniquely ordered. For example, in fragment
S-R1-a-R2-b-R1-c-R2-E where S and E are the start and end of a
fragment, a, b, and c are different sequences specific to
respective subfragments and R1 and R2 are two k-1 sequences that
are tandemly repeated, five subfragments are generated (S-R1,
R1-a-R2, R2-b-R1, R1-c-R2, and R-E). They may be ordered in two
ways; the original sequence above or S-R1-c-R-b-R1-a-R-E. In
contrast, in a fragment with the same number and types of repeated
sequences but ordered differently, i.e. S-R1-a-R1-b-R-c-R-E, there
is no other sequence which includes all subfragments. Examples of
this type can be recognized only after the process of generation of
pSFs. They represent the necessity for two steps in the process of
pSF generation. The second situation of generation of false short
subfragments on positions of nonrepeated k-1 sequences when the
files contain false negative and/or positive k-tuples is more
important.
[0166] The solution for both pSF groups consists of two parts.
First, the false positive k-tuples appearing as the nonexisting
minimal subfragments are eliminated. All k-tuple subfragments of
length k which do not have an overlap on either end, of the length
of longer than k-a on one end and longer than k-b on the other end,
are eliminated to enable formation of the maximal number of
connections. In our experiments, the values for a and b of 2 and 3,
respectively, appeared to be adequate to eliminate a sufficient
number of false positive k-tuples.
[0167] The merging of subfragments that can be uniquely connected
is accomplished in the second step. The rule tot connection is: two
subfragments may be unambiguously connected if, and only if, the
overlapping sequence at the relevant end or start of two
subfragments is not present at the start and/or end of any other
subfragment.
[0168] The exception is if one subfragment from the considered pair
has the identical beginning and end. In that case connection is
permitted, even if there is another subfragment with the same end
present in the file. The main problem here is the precise
definition of overlapping sequence. The connection is not permitted
if the overlapping sequence unique for only one pair of
subfragments is shorter than k-2, of it is k-2 or longer but an
additional subfragment exists with the overlapping sequence of any
length longer than k-4. Also, both the canonical ends of pSFs and
the ends after omitting one (or few) last bases are considered as
the overlapping sequences.
[0169] After this step some false positive k-tuples (as minimal
subfragments) and some subfragments with a wrong end may survive.
In addition, in very rare occasions where a certain number of some
specific false k-tuples are simultaneously present, an erroneous
connection may take place. These cases will be detected and solved
in the subfragment ordering process, and in the additional control
steps along with the handling of uncut "misconnected"
subfragments.
[0170] The short subfragments that are obtained are of two kinds.
In the common case, these subfragments may be unambiguously
connected among themselves because of the distribution of repeated
k-1 sequences. This may be done after the process of generation of
pSFs and is a good example of the necessity for two steps in the
process of pSF generation. In the case of using the file containing
false positive and/or false negative k-tuples, short pSFs are
obtained on the sites of nonrepeated k-1 sequences. Considering
false positive k-tuples, a k-tuple may contain more than one wrong
base (or containing one wrong base somewhere in the middle), as
well as k-tuple on the end. Generation of short and erroneous (or
misconnected) subfragments is caused by the latter k-tuples. The
k-tuples of the former kind represent wrong pSFs with length equal
to k-tuple length.
[0171] The aim of merging pSF part of the algorithm is the
reduction of the number of pSFs to the minimal number of longer
subfragments with the correct sequence. All k-tuple subfragments
that do not have an overlap on either end, of the length of longer
than k-a on one, and longer than k-b on the other end, are
eliminated to enable the maximal number of connections. In this
way, the majority of false positive k-tuples are discarded. The
rule for connection is: two subfragments can be unambiguously
connected if, and only if the overlapping sequence of the relevant
end or start of two subfragments is not present on the start and/or
end of any other subfragment. The exception is a subfragment with
the identical beginning and end. In that case connection is
permitted, provided that there is another subfragment with the same
end present in the file. The main problem here is of precise
definition of overlapping sequence. The presence of at least two
specific false negative k-tuples on the points of repetition of k-1
or k-2 sequences, as well as combining of the false positive and
false negative k-tuples may destroy or "mask" some overlapping
sequences and can produce an unambiguous, but wrong connection of
pSFs. To prevent this, completeness must be sacrificed on account
of exactness: the connection is not permitted on the end-sequences
shorter than k-2, and in the presence of an extra overlapping
sequence longer than k-4. The overlapping sequences are defined
from the end of the pSFs, or omitting one, or few last bases.
[0172] In the very rare situations, with the presence of a certain
number of some specific false positive and false negative k-tuples,
some subfragments with the wrong end can survive, some false
positive k-tuples (as minimal subfragments) can remain, or the
erroneous connection can take place. These cases are detected and
solved in the subfragments ordering process, and in the additional
control steps along with the handling of uncut, misconnected
subfragments.
[0173] The process of ordering of subfragments is similar to the
process of their generation. If one considers subfragments as
longer k-tuples, ordering is performed by their unambiguous
connection via overlapping ends. The informational basis for
unambiguous connection is the division of subfragments generated in
fragments of the basic library into groups representing segments of
those fragments. The method is analogous to the biochemical
solution of this problem based on hybridization with longer
oligonucleotides with relevant connecting sequence. The connecting
sequences are generated as subfragments using the k-tuple sets of
the appropriate segments of basic library fragments. Relevant
segments are defined by the fragments of the ordering library that
overlap with the respective fragments of the basic library. The
shortest segments are informative fragments of the ordering
library. The longer ones are several neighboring informative
fragments or total overlapping portions of fragments corresponding
of the ordering and basic libraries. In order to decrease the
number of separate samples, fragments of the ordering library are
randomly pooled, and the unique k-tuple content is determined.
[0174] By using the large number of fragments in the ordering
library very short segments are generated, thus reducing the chance
of the multiple appearance of the k-1 sequences which are the
reasons for generation of the subfragments. Furthermore, longer
segments, consisting of the various regions of the given fragment
of the basic library, do not contain some of the repeated k-1
sequences. In every segment a connecting sequence (a connecting
subfragment) is generated for a certain pair of the subfragments
from the given fragment. The process of ordering consists of three
steps: (1) generation of the k-tuple contents of each segment; (2)
generation of subfragments in each segment; and (3) connection of
the subfragments of the segments. Primary segments are defined as
significant intersections and differences of k-tuple contents of a
given fragment of the basic library with the k-tuple contents of
the pools of the ordering library. Secondary (shorter) segments are
defined as intersections and differences of the k-tuple contents of
the primary segments.
[0175] There is a problem of accumulating both false positive and
negative k-tuples in both the differences and intersections. The
false negative k-tuples from starting sequences accumulate in the
intersections (overlapping parts), as well as false positive
k-tuples occurring randomly in both sequences, but not in the
relevant overlapping region. On the other hand, the majority of
false positives from either of the starting sequences is not taken
up into intersections. This is an example of the reduction of
experimental errors from individual fragments by using information
from fragments overlapping with them. The false k-tuples accumulate
in the differences for another reason. The set of false negatives
from the original sequences are enlarged for false positives from
intersections and the set of false positives for those k-tuples
which are not included in the intersection by error, i.e. are false
negative in the intersection. If the starting sequences contain 10%
false negative data, the primary and secondary intersections will
contain 19% and 28% false negative k-tuples, respectively. On the
other hand, a mathematical expectation of 77 false positives may be
predicted if the basic fragment and the pools have lengths of 500
bp and 10,000 bp, respectively. However, there is a possibility of
recovering most of the "lost" k-tuples and of eliminating most of
the false positive k-tuples.
[0176] First, one has to determine a basic content of the k-tuples
for a given segment as the intersection of a given pair of the
k-tuple contents. This is followed by including all k-tuples of the
starting k-tuple contents in the intersection, which contain at one
end k-1 and at the other end k-+ sequences which occur at the ends
of two k-tuples of the basic set. This is done before generation of
the differences thus preventing the accumulation of false positives
in that process. Following that, the same type of enlargement of
k-tuple set is applied to differences with the distinction that the
borrowing is from the intersections. All borrowed k-tuples are
eliminated from the intersection files as false positives.
[0177] The intersection, i.e. a set of common k-tuples, is defined
for each pair (a basic fragment).times.(a pool of ordering
library). If the number of k-tuples in the set is significant it is
enlarged with the false negatives according to the described rule.
The primary difference set is obtained by subtracting from a given
basic fragment the obtained intersection set. The false negative
k-tuples are appended to the difference set by borrowing from the
intersection set according to the described rule and, at the same
time, removed from the intersection set as false positive k-tuples.
When the basic fragment is longer than the pooled fragments, this
difference can represent the two separate segments which somewhat
reduces its utility in further steps. The primary segments are all
generated intersections and differences of pairs (a basic
fragment).times.(a pool of ordering library) containing the
significant number of k-tuples. K-tuple sets of secondary segments
are obtained by comparison of k-tuple sets of all possible pairs of
primary segments. The two differences are defined from each pair
which produces the intersection with the significant number of
k-tuples. The majority of available information from overlapped
fragments is recovered in this step so that there is little to be
gained from the third round of forming intersections, and
differences.
[0178] (2) Generation of the subfragments of the segments is
performed identically as described for the fragments of the basic
library.
[0179] (3) The method of connection of subfragments consists of
sequentially determining the correctly linked pairs of subfragments
among the subfragments from a given basic library fragment which
have some overlapped ends. In the case of 4 relevant subfragments,
two of which contain the same beginning and two having the same
end, there are 4 different pairs of subfragments that can be
connected. In general 2 are correct and 2 are wrong. To find
correct ones, the presence of the connecting sequences of each pair
is tested in the subfragments generated from all primary and
secondary segments for a given basic fragment. The length and the
position of the connecting sequence are chosen to avoid
interference with sequences which occur by chance. They are k+2 or
longer, and include at least one element 2 beside overlapping
sequence in both subfragments of a given pair. The connection is
permitted only if the two connecting sequences are found and the
remaining two do not exist. The two linked subfragments replace
former subfragments in the file and the process is cyclically
repeated.
[0180] Repeated sequences are generated in this step. This means
that some subfragments are included in linked subfragments more
than once. They will be recognized by finding the relevant
connecting sequence which engages one subfragment in connection
with two different subfragments.
[0181] The recognition of misconnected subfragments generated in
the processes of building pSFs and merging pSFs into longer
subfragments is based on testing whether the sequences of
subfragments from a given basic fragment exist in the sequences of
subfragments generated in the segments for the fragment. The
sequences from an incorrectly connected position will not be found
indicating the misconnected subfragments.
[0182] Beside the described three steps in ordering of subfragments
some additional control steps or steps applicable to specific
sequences will be necessary for the generation of more complete
sequence without mistakes.
[0183] The determination of which subfragment belongs to which
segment is performed b comparison of contents of k-tuples in
segments and subfragments. Because of the errors in the k-tuple
contents (due to the primary error in pools and statistical errors
due to the frequency of occurrences of k-tuples) the exact
partitioning of subfragments is impossible. Thus, instead of "all
or none" partition, the chance of coming from the given segment
(P(sf,s)) is determined for each subfragment. This possibility is
the function of the lengths of k-tuples, the lengths of
subfragments, the lengths of fragments of ordering library, the
size of the pool, and of the percentage of false k-tuples in the
file:
P(sf,s)=(Ck-F)/Lsf,
[0184] where Lsf is the length of subfragment, Ck is the number of
common k-tuples for a given subfragment/segment pair, and F is the
parameter that includes relations between lengths of k-tuples,
fragments of basic library, the size of the pool, and the error
percentage.
[0185] Subfragments attributed to a particular segment are treated
as redundant short pSFs and are submitted to a process of
unambiguous connection. The definition of unambiguous connection is
slightly different in this case, since it is based on a probability
that subfragments with overlapping end(s) belong to the segment
considered. Besides, the accuracy of unambiguous connection is
controlled by following the connection of these subfragments in
other segments. After the connection in different segments, all of
the obtained subfragments are merged together, shorter subfragments
included within longer ones are eliminated, and the remaining ones
are submitted to the ordinary connecting process. If the sequence
is not regenerated completely, the process of partition and
connection of subfragments is repeated with the same or less severe
criterions of probability of belonging to the particular segment,
followed by unambiguous connection.
[0186] Using severe criteria for defining unambiguous overlap, some
information is not used. Instead of a complete sequence, several
subfragments that define a number of possibilities for a given
fragment are obtained. Using less severe criteria an accurate and
complete sequence is generated. In a certain number of situations,
e.g. an erroneous connection, it is possible to generate a
complete, but an incorrect sequence, or to generate "monster"
subfragments with no connection among them. Thus, for each fragment
of the basic library one obtains: a) several possible solutions
where one is correct and b) the most probable correct solution.
Also, in a very small number of cases, due to the mistake in the
subfragment generation process or due to the specific ratio of the
probabilities of belonging, no unambiguous solution is generated or
one, the most probable solution. These cases remain as incomplete
sequences, or the unambiguous solution is obtained by comparing
these data with other, overlapped fragments of basic library.
[0187] The described algorithm was tested on a randomly generated,
50 kb sequence, containing 40% GC to simulate the GC content of the
human genome. In the middle part of this sequence were inserted
various All, and some other repetitive sequences, of a total length
of about 4 kb. To simulate an in vitro SBH experiment, the
following operations were performed to prepare appropriate
data.
[0188] Positions of sixty 5 kb overlapping "clones" were randomly
defined, to simulate preparation of a basic library:
[0189] Positions of one thousand 500 bp "clones" were randomly
determined to simulate making the ordering library. These fragments
were extracted from the sequence. Random pools of 20 fragments were
made, and k-tuple sets of pools were determined and stored on the
hard disk. These data are used in the subfragment ordering phase:
For the same density of clones 4 million clones in basic library
and 3 million clones in ordering library are used for the entire
human genome. The total number of 7 million clones is several fold
smaller than the number of clones a few kb long for random cloning
of almost all of genomic DNA and sequencing by a gel-based
method.
[0190] From the data on the starts and ends of 5 kb fragments, 117
"informative fragments" were determined to be in the sequence. This
was followed by determination of sets of overlapping k-tuples of
which the single "informative fragment" consist. Only the subset of
k-tuples matching a predetermined list were used. The list
contained 65% 8-mers, 30% 9-mers; and 5% 10-12-mers. Processes of
generation and the ordering of subfragments were performed on these
data.
[0191] The testing of the algorithm was performed on the simulated
data in two experiments. The sequence of 50 informative fragments
was regenerated with the 100% correct data set (over 20,000 bp),
and 26 informative fragments (about 10,000 bp) with 10% false
k-tuples (5% positive and 5% negative ones).
[0192] In the first experiment, all subfragments were correct and
in only one out of 50 informative fragments the sequence was not
completely regenerated but remained in the form of 5 subfragments.
The analysis of positions of overlapped fragments of ordering
library has shown that they lack the information for the unique
ordering of the 5 subfragments. The subfragments may be connected
in two ways based on overlapping ends, 1-2-3-4-5 and 1-4-3-2-5. The
only difference is the exchange of positions of subfragments 2 and
4. Since subfragments 2, 3, and 4 are relatively short (total of
about 100 bp), the relatively greater chance existed, and occurred
in this case, that none of the fragments of ordering library
started or ended in the subfragment 3 region.
[0193] To simulate real sequencing, some false ("hybridization")
data was included as input in a number of experiments. In oligomer
hybridization experiments, under proposed conditions, the only
situation producing unreliable data is the end mismatch versus full
match hybridization. Therefore, in simulation only those k-tuples
differing in a single element on either end from the real one were
considered to be false positives. These "false" sets are made as
follows. On the original set of a k-tuples of the informative
fragment, a subset of 5% false positive k-tuples are added. False
positive k-tuples are made by randomly picking a k-tuple from the
set, copying it and altering a nucleotide on its beginning or end.
This is followed by subtraction of a subset of 5% randomly chosen
k-tuples. In this way the statistically expected number of the most
complicated cases is generated in which the correct k-tuple is
replaced with a k-tuple with the wrong base on the end.
[0194] Production of k-tuple sets as described leads to up to 10%
of false data. This value varies from case to case, due to the
randomness of choice of k-tuples to be copied, altered, and erased.
Nevertheless, this percentage 3-4 times exceeds the amount of
unreliable data in real hybridization experiments. The introduced
error of 10% leads to the two fold increase in the number of
subfragments both in fragments of basic library (basic library
informative fragments) and in segments. About 10% of the final
subfragments have a wrong base at the end as expected for the
k-tuple set which contains false positives (see generation of
primary subfragments). Neither the cases of misconnection of
subfragments nor subfragments with the wrong sequence were
observed. In 4 informative fragments out of 26 examined in the
ordering process the complete sequence was not regenerated. In all
4 cases the sequence was obtained in the form of several longer
subfragments and several shorter subfragments contained in the same
segment. This result shows that the algorithmic principles allow
working with a large percentage of false data.
[0195] The success of the generation of the sequence from its
k-tuple content may be described in terms of completeness and
accuracy. In the process of generation, two particular situations
can be defined: 1) Some part of the information is missing in the
generated sequence, but one knows where the ambiguities are and to
which type they belong, and 2) the regenerated sequence that is
obtained does not match the sequence from which the k-tuple content
is generated, but the mistake can not be detected. Assuming the
algorithm is developed to its theoretical limits, as in the use of
the exact k-tuple sets, only the first situation can take place.
There the incompleteness results in a certain number of
subfragments that may not be ordered unambiguously and the problem
of determination of the exact length of monotonous sequences, i.e.
the number of perfect tandem repeats.
[0196] With false k-tuples, incorrect sequence may be generated.
The reason for mistakes does not lie in the shortcomings of the
algorithm, but in the fact that a given content of k-tuples
unambiguously represents the sequence that differs from the
original one. One may define three classes of error, depending on
the kind of the false k-tuples present in the file. False negative
k-tuples (which are not accompanied with the false positives)
produce "deletions". False positive k-tuples are producing
"elongations (unequal crossing over)". False positives accompanied
with false negatives are the reason for generation of "insertions",
alone or combined with "deletions". The deletions are produced when
all of the k-tuples (or their majority) between two possible starts
of the subfragments are false negatives. Since every position in
the sequence is defined by k. k-tuples, the occurrence of the
deletions in a common case requires k consecutive false negatives.
(With 10% of the false negatives and k=8, this situation takes
place after every 108 elements). This situation is extremely
infrequent even in mammalian genome sequencing using random
libraries containing ten genome equivalents.
[0197] Elongation of the end of the sequence caused by false
positive k-tuples is the special case of "insertions", since the
end of the sequence can be considered as the endless linear array
of false negative k-tuples. One may consider a group of false
positive k-tuples producing subfragments longer than one k-tuple.
Situations of this kind may be detected if subfragments are
generated in overlapped fragments, like random physical fragments
of the ordering library. An insertion, or insertion in place of a
deletion, can arise as a result of specific combinations of false
positive and false negative k-tuples. In the first case, the number
of consecutive false negatives is smaller than k. Both cases
require several overlapping false positive k-tuples. The insertions
and deletions are mostly theoretical possibilities without sizable
practical repercussions since the requirements in the number and
specificity of false k-tuples are simply too high.
[0198] In every other situation of no meeting the theoretical
requirement of the minimal number an the kind of the false positive
and/or negatives, mistakes in the k-tuples content may produce only
the lesser completeness of a generated sequence.
* * * * *