Gene Cloning

190 Gene Cloning
Contig 2
Clone 12
Clone 18
Contig 4
Contig 1
Clone 12 Clone 18
Contig 3
Figure 7.8 Assembly of shotgun sequence reads into contiguous
sequences (contigs). The solid black lines indicate the regions of each insert
that have been sequenced, the dotted lines represent sequence that has not been
determined and is therefore not known. Note some clones have been sequenced
from both ends (e.g. 12 and 18); however, there is still some sequence in the
middle that has not been determined for these clones.
times to give six- to 10-fold coverage. At this point you will have sequenced
tens of thousands of clones, but invariably you will not be able to assemble
your sequence reads into one contiguous sequence, i.e. one contig covering
the whole genome, as you will have gaps in your sequence information.
These gaps will obviously mean that you do not have the complete genome
sequence and also you may not know the relative position of each contig or
their positions on the genome. This “draft” sequence now has to be “finished”.
“Finishing” involves determining the sequence across the gaps to
generate a complete genome sequence. The sequence gaps will fall into
two categories. The first type are called sequencing gaps and these arise
because you have often sequenced one clone from both ends and therefore
have 500 bases of sequence from both ends but no sequence information
from the middle, e.g. clone 12 in Figure 7.8, and it is possible that all or part
of this middle sequence has not been determined from any other clones.
The two different sequence reads from the same clone will therefore fall
into two different contigs because there is no overlapping sequence information
to join up the two reads (e.g. the two reads from clone 12 fall into
contigs 2 and 3). However, you know that the two sequence reads come
from the same clone and so you only need to go back to that clone (clone
12 in this example) and sequence the middle section to fill the gap and join
up the two contigs. Sequencing gaps are thus where you know that you