Genomics- The Science and Technology Behind the Human Genome Project. Charles R. Cantor, Cassandra L / genomics11-15 / 12

words to reconstruct the sequence. The problem of reconstructing a sequence

from

all

n -

tuple subsegments is highly overdetermined, except for the complications that we will

discuss below. Simulations show that reconstructing DNA sequences

from

oligonu-

cleotide hybridization fingerprints is very robust and very error resistant. Even significant

levels of insertions and deletions can be tolerated without badly degrading the final se-

quence. A key element of SBH that is easily forgotten is that negatives as well as posi-

tives are extremely informative. Knowing that a specific oligonucleotide

like AACTG-

GAC does not exist anywhere in the target provides a constraint that can sometimes

be

quite useful in assembling the data from words that are found to be present.

12.12). If the SBH word length used is

n, these ambiguities arise whenever there is an ex-

act recurrence of any sequence with length

n 1

in the target. What happens, as shown

in Figure 12.12, is that the data produced by the complete pattern of

n -tuple words can be

assembled in two different ways. In general, there is no way to distinguish between the

alternative assemblies. In principle, if one could read the sequence out to the very end of

the SBH target, the particular ambiguity shown

in Figure 12.12

could be resolved.

BRANCH POINT AMBIGUITIES

405

Figure 12.12

A tandem repeating sequence

results in a branch point ambiguity: Two different re-

constructions are possible from the pattern on

n -tuple words detected.

However, there is no guarantee that this will be possible. The moment that there are more

than one recurrences, the ambiguities become almost intractable. For example, the case

shown in Figure 12.13, in which a sequence recurs three times, cannot be resolved even if

one could read all the way to the ends of the SBH target.

The probability of any particular sequence 8 long recurring in a target

of 200 bp

is

very low: A rough estimate gives 192/4

8 3 10

3. However

the probability that the

target will have one or more recurrence once all possible recurrences are considered is ac-

tually quite high. An analogy is asking what is the probability that two people in a room

have the same birthday. If you specify a particular date, or pick a particular person,

the

odds of a match are very low. However, if you allow all possible pairings to be consid-

ered, the odds are quite high that in a room with 30 people, two will share the same birth-

day. So sequence recurrences are a serious problem. They

limit the length of sequence

Figure 12.13

A more serious branch point ambiguity that leads to uncertainty in the arrangement

406

FUTURE DNA SEQUENCING WITHOUT LENGTH FRACTIONATION

There are

4

n possible

words of length

n,

for a

four-letter alphabet. Therefore to se-

quence by hybridization could require examining as many as 65,536 possible 8-mers or

262,144 possible 9-mers. Making complete sets of compounds larger than this and con-

trolling their quality is likely to be challenging with present or currently extrapolated

technology. It turns out that an estimate of the average sequence length that can be read

before a

branch point ambiguity arises is given approximately by the

square

root

of

the

number of words used. When the words are DNA sequences, this is 4

n /2

. For 8-mers, the

average length

of

sequence determined between branch points will

be 256.

This

is

quite

an acceptable size scale. It seems like a losing proposition to increase the word size much

beyond 8, unless technical considerations in the hybridizations demand this. Reducing the

word length below 8 will lead to an unacceptably high frequency of branch points, unless

some specific additional strategy is introduced to resolve these ambiguities.

Branch point ambiguities have one additional implication that must be dealt with in all

attempts to implement a successful SBH strategy. The number of branch points present in

a target

sequence

will grow rapidly as the total length of the

target

increases. Thus one

must subdivide the target into relatively short DNA fragments in order to have a reason-

able chance of sequencing each fragment unambiguously. This is a relatively undesirable

feature of SBH. However, even if branch points could be resolved some other way, short

targets are probably still mandated in order to diminish complications that may arise from

intramolecular secondary or tertiary structure in the target. More will be said about this

later.

SBH USING

OLIGONUCLEOTIDE

CHIPS

times called format I SBH. The ideal array would

be very small to minimize the amount

of sample that was needed. Hence it is conventional

to call the array a chip, by analogy

with a semiconductor chip. A schematic illustration

of an SBH experiment using such a

ever systematic method is used

to produce them. The chip

surface itself could be silicon,

or glass, or plastic. The key aspect is that the

oligonucleotides must be covalently at-

tached

to it, and

the surface must not interfere with

the hybridization. The surface must

not show significant amounts of

nonspecific adsorption of the target, and it must not hin-

der,

sterically

or electrostatically, the approach

of the target to the bound oligonu-

SBH USING OLIGONUCLEOTIDE CHIPS

407

Figure 12.14

An example of the expected hybridization pattern of a labeled target exposed to an

oligonucleotide chip. The actual chip might contain 65,000 or more probes.

array separately, and then develop a parallelized automated device to spot or spray the

compounds onto the right locations on the chip. The disadvantage of this approach is that

the rate of manufacture of each chip could be fairly slow. The major advantage of this ap-

proach is that

the oligonucleotides only have to be made once, and their individual se-

quence and purity can be checked. There is no consensus at the present time what the op-

timal way would be to manufacture chips given samples of all the 65,536 8-mers. A key

variable is how

they will be attached to the chip surface. A long enough spacer must be

used to keep the 8-mers well above the surface. Otherwise, the surface is likely to pose a

steric restriction for the much bulkier target DNA.

The alternate strategies involve synthesizing the array on the chip. One potential gen-

eral way to do this is photolithography, a technique that has been very powerful in the

construction of semiconductor chips. It has been used quite successfully by Steven Fodor

and others at

Affymetrix, Inc. to make dense arrays of peptides, and more recently to

make dense arrays of oligonucleotides. The basic requirement is that nucleotide deriva-

tives are needed that are blocked from extending, say because the 3

OH is esterified. The

block used, however, can be removed by photolysis. A mask is used to allow selective il-

lumination of only those chains that require extension by a particular base in this position

(Fig. 12.15). Thus the light activates just a subset of the oligomers on the chip. The chain

SBH USING OLIGONUCLEOTIDE CHIPS

409

Figure 12.16

Pattern of stepwise DNA synthesis used in Southern’s procedure for in situ synthe-

sis of an oligonucleotide chip. Four successive synthetic steps are indicated. Within each square of

the array, the sequence of the tetranucleotide synthesized is read left to right from the 3

to 5 direc-

tion starting from the upper row and continuing with the lower row.

tages and disadvantages, as we

will illustrate later.

Regardless of the method

of synthesis, the key technical

issue that must be

overcome

is how the oligonucleotides are anchored at their position in the array. Mirzabekov uses

direct chemical coupling to an oxidized ribonucleotide placed at

the 3

-end of the 8-mer

Figure 12.17

Glass plates separated by rubber dams are used to direct the reagents in each step of

initiate the synthesis of the first nucleotide of the 8-mer in standard DNA synthesis proto-

cols. It acts chemically exactly like the 3

OH of a nucleoside in coupling to an activated

phosphate of the next nucleotide.

SEQUENCING BY HYBRIDIZATION TO SAMPLE

CHIPS

The second general SBH approach is to make a large, dense array of samples and probe it

by hybridization with one labeled oligonucleotide at a time. This is sometimes called for-

mat II SBH. In this format, while it takes a long time to complete the sequence, one is ac-

tually sequencing a large number of samples simultaneously. A schematic illustration of

this approach is shown in Figure 12.20. It looks deceptively similar to the use of oligonu-

cleotide chips, but everything is reversed. The array might, for example, correspond to an

entire cDNA library, perhaps 2

10 4

clones in all. Because SBH can only

use relatively

short samples, each cDNA might have to be broken down into fragments. It is not imme-

diately obvious how to do this with large numbers of clones at once. One possibility is to

subclone two different restriction enzyme digests of the cDNA inserts. This scrambles up

connectivity information in the original clones; however, in most cases that information

would be easily restored by the sequencing process itself, or by rehybridization of any

ambiguous fragments back to the original, intact clones. If each 1.5-kb average cDNA

clone yielded six fragments in each of the two digests, one would want to array 3.6

10 5

subclones in order to maintain the redundancy

of coverage

of the original library. This

would constitute the sample chip.

SEQUENCING BY HYBRIDIZATION TO SAMPLE CHIPS

411

Since the individual components are available in any desired quantity, one could, in

principle, make as many copies of the array as could conveniently be handled simultane-

ously. In practice, it does not seem at all unreasonable to suppose that 100 copies of the

array could be processed in parallel. It is envisaged that the sample chips be made by us-

ing the robotic

x y tables common in the semiconductor industry (Fig. 12.21). These are

very accurate and fast. It has been estimated that a sample density of 2

10 4 per 10 to 20

Figure 12.21

How a robotic

x y table can be used in offset printing to construct of dense arrays

412

FUTURE DNA SEQUENCING WITHOUT LENGTH FRACTIONATION

cm

2 is quite practical. Thus the entire array of subclones could be contained in 180 to 360

cm

2, which is about 30% to 60% the size of a typical 8.5

11 inch piece of paper.

If octanucleotides are used as hybridization probes to this large array, only a small

fraction of the samples will show a positive signal. For one 250-bp subclone, the odds of

containing any particular 8-mer are 243/4

8 3.8 10

3; the possibility of a positive hy-

bridization is less than 0.4%. When this is multiplied by the number of subclones in the

array, there should be an average of 1.4

10

3 positive subclones per hybridization. Since

each yields eight bases of DNA sequence data, the rate of sequence acquisition per single

hybridization is 1.1

10 4

bp. If 100 chips can really

be

managed simultaneously, and if

three hybridizations can be done per day, the overall

throughput

is

3.3

6

10 . This is

quite an impressive rate, and many of the variables used to estimate it are probably con-

servative.

A major feature of the use of sample chips in sequencing projects is that the approach

does

not scale down conveniently. Sample chips are only

useful if

entire libraries

are to

be sequenced as a unit. Such a method makes good sense for cDNAs and the genomes of

model organisms. If all 65,536 8-mers must be used, at the rates we estimated above of

300 hybridizations per day, it will take more than half a year to complete the sequencing.

Scaling down would not reduce the time of the effort

at all; it would just

reduce the

amount of sequence data ultimately obtained. In practice, one does not have to use all

65,536 compounds to determine the sequence. Because of the considerable redundancy in

the method, one ought to be able to use just a fraction

of all 8-mers. The exact

fraction

will depend on error rates in the hybridization, how branch points will

be resolved, and

what kind of sequencing accuracy one desires. In some

of the enhanced SBH schemes

that

will be described later, it has been

estimated that one might be

able to operate close

to a redundancy of one rather than eight. However, this remains to be demonstrated in

practice.

EARLY

EXPERIENCES

WITH

SBH

A major difficulty in testing the potential of SBH and evaluating the merits of different

SBH strategies or particular variations on conditions,

sample attachment, and so on, is

that the method does not scale down. A particular problem occurs in the use of oligonu-

cleotide chips. It is difficult to vary parameters using the set of all 65,536 8-mers. Indeed,

no one has yet actually made this set of compounds. Instead, several more limited tests of

SBH have been carried out.

Southern has used the scheme shown in Figure 12.16 to make a chip containing four

copies of all possible octapurine sequences (A and G only). An example of some data ob-

tained with this chip is shown in Figure 12.22. In the actual example used, the labeled tar-

get DNA was a specific sequence of 24 pyrimidines (C and T). This contains 17 different

8-tuples, and so 17 positive hybridization spots would be expected. The actual results in

Figure 12.22 are much more

complex than this. Two problems need

to

be dealt

with,

which illustrate some of the basic issues in trying to

implement SBH on a large scale.

First the amount of oligonucleotide at each position in the array differs. More important,

the strength

of hybridization

to different sequences varies quite

a

bit. Duplexes

rich in

G C will be more stable, under most

ordinary hybridization conditions than duplexes

rich in A

T. There are ways to compensate for this, as we will illustrate later, and one of

these was actually

used

with the samples

in Figure 12.22. But

the compensation

is not

EARLY EXPERIENCES WITH SBH

413

Figure 12.22

Properties of an octapurine chip which contains four replicas of all 256 octapurines.

(a ) Hybridization pattern with an equimolar mixture of all octapyrimidines to show variation in the

amount of attached

purine. (

b ) Pattern of hybridization seen with a labeled 24-base target. From

Southern et al. (1992).

Using

the same approach, with a few immobilized

samples,

Radoje Drmanac

and

Radomir

Crkvenjakov successfully completed two short

pilot sequencing projects

by

SBH.

In the first case, the 100-base sequence was

known

in advance, as was