Genomics- The Science and Technology Behind the Human Genome Project. Charles R. Cantor, Cassandra L / genomics1-10 / 8

Figure

8.43

An example of how re-

peated

sequence

hybridization simplifies

the identification of two contiguous cos-

mid clones.

(

a ) Two overlapping

clones.

(b ) Restriction fragments and Southern

blot.

(Adapted

from

Stallings et

al.,

1990.)

MEASUREMENTS OF PROGRESS IN BUILDING ORDERED LIBRARIES

275

MEASUREMENTS OF

PROGRESS

IN

BUILDING ORDERED

LIBRARIES

The process of assembling contigs by fingerprinting clones can be treated in relatively

straightforward mathematical ways. One makes the key assumption that the genome is

being sampled uniformly, and sets, as a parameter, the degree of

overlap between

two

clones necessary to constitute positive evidence that they are contiguous. Lander and

Waterman have modeled this process of clone ordering. The sorts of results they obtained

are shown in Figure 8.44. It is assumed that clones are fingerprinted one at a time, and the

number of clones assembled into contigs of two or more clones is plotted as a function of

the number of clones fingerprinted. At early times in the project, there are almost no con-

tigs because the odds of picking overlapping clones, chosen at

random,

are

small.

Eventually overlaps start to build up, but most contigs contain just two clones. These be-

gin to coalesce into larger contigs as the genome is sampled deeper and deeper. However,

the effectiveness of the contig building begins to saturate long before all clones are as-

sembled into a single contig. This saturation is partly determined by the lack of complete-

ness of the library; if any regions are not represented at all, contigs cannot be built across

them. The saturation is also a function of the effectiveness of overlap detection; due to

chance, some clones that

actually are contiguous may not overlap enough

to be counted

as a positive score. Several ongoing programs in contig building have been evaluated

by

the Lander-Waterman approach. Actual progress on these projects is in remarkably good

agreement with predictions.

Eventually the pure bottom-up approach must be abandoned if a complete ordered li-

brary is desired. The point at which a switch in strategy is profitable is said to be some-

where between 60% and 90% coverage, when almost all progress in typical bottom-up

mapping stops. The early stages of bottom-up mapping are very efficient. DNA prepara-

tions, fingerprinting,

and data analysis have all

been completely automated for

some

of

the schemes we have described. Contig assembly is

also done by computer software.

Once the saturation point is reached, a typical project will still

have hundreds or thou-

sands of separate contigs. The challenge is to close the gaps between them in an efficient

way. Several different approaches are useful at this stage. The contigs can be ordered by

FISH localization of individual clone representatives from each contig. Once one knows

that two contigs

are very

close

to each other, frequently overlap data

that were marginal

SURVEY OF RESTRICTION MAP AND ORDERED LIBRARY CONSTRUCTION

277

SURVEY OF RESTRICTION MAP AND ORDERED

LIBRARY

CONSTRUCTION

Complete macrorestriction maps have been produced for

a

number

of

prokaryotic

genomes, some simpler eukaryotic genomes, and sections of complex genomes. The first

of these

maps,

a

Not I map of

E. coli,

is shown in Figure 8.46. The most complex of all

these maps, that for human chromosome 21q, is shown in Figure 8.47. A number of fea-

tures of this map are of interest. Note that small

Not I fragments and large

Not I frag-

ments tend to cluster. This must eflect wide oscillations

in the density of HTF islands

along the chromosome, since

Not I sites occur almost exclusive in these islands.

The

Not

I

map of human chromosome

21 was actually executed,

not in a single cell

line but in a set of eight cell lines. Polymorphisms among these lines were helpful in es-

tablishing the map as described earlier in the chapter. The

full

pattern

of

polymorphisms

is illustrated in Figure 8.48. While the extent of polymorphism is considerable, almost all

of it is consistent with varying degrees of methylation in the cell lines studied. There is

little or no compelling evidence for major shifts in the lengths of DNA between existing

Not I sites. Most important, there is no evidence

that

any significant amounts of DNA

have been rearranged or lost in these cell lines.

Figure 8.46 Not I restriction map of

E. coli. (Adapted from Smith et al., 1987.)

280

PHYSICAL MAPPING

A number of successful projects have been reported that have produced complete, or

almost complete ordered clone libraries. The first of these was the ordered bacteriophage

lambda library covering the

E. coli

genome. Other model organisms now mapped include

the

yeasts

S. cerevisiae,

and

S. pombe,

and the nematode

C. elegans.

Extensive map data

also exists for Drosophila and for the human genome. A relatively complete YAC map

covering the informative part of the

Y chromosome has been reported, and a complete

YAC maps exist that cover most

human chromosomes. Extensive cosmid ordering

projects on chromosomes 16 and 19 are virtually complete. Gaps not covered in cosmids

are mostly covered in YACs or BACs.

An example of some of the data used to construct the chromosome YAC 21 map is shown

in Figure 8.49. It is apparent that at the present stage some of the overlap evidence would be

strengthened by interpolating results from additional clones, and some YACs used show evi-

dence of rearrangements that are potential sources of error. Indeed, when the YAC contig for

chromosome 21 is compared with the

Not

I restriction map, several YACs appear to be as-

signed to the wrong locations on the chromosome (Fig. 8.50). This is almost certainly partly

the result of YAC chimeras which can seriously confuse

clone

ordering (see Chapter

9).

Other discrepancies appear to result from the use of several probes with confused identi-

ties. Nevertheless, a remarkable amount of information and a goodly number of useful

clones are now available for this chromosome.

A complete YAC map and three complete cosmid maps are available for the yeast

S.

pombe.

The tiling path YACs

from this map are shown in Figure 8.51, alongside the

Not

I

restriction map of this

organism and a sketch of the genetic map. This view,

which presents a very simple looking map, hides

the complex

process that

actually

went

into

the construction of the

map. Figure 8.52 illustrates

the actual YAC clones