Genome sequencing 8/1/03. By Giles Newton How the full genetic code of an organism is read.

Whether bacterium or human, the genome of any organism to too large to be deciphered in one go. The genome is therefore broken into smaller pieces of DNA, each piece is sequenced and computers fit all the sequences back together.

Back	Next
1 of 8 The human chromosome to be sequenced.

Back	Next
2 of 8 The chromosome is first chopped randomly into conveniently sized chunks.

Back	Next
3 of 8 These large fragments are inserted into bacterial artificial chromosomes (BACs) and cloned in bacteria.

Back	Next
4 of 8 These fragments are then mapped so it is known which region of the chromosome they came from.

Back	Next
5 of 8 Each BAC is shotgunned - broken randomly into many small pieces. This process is repeated several times to give different sets of fragments. (The whole-genome shotgun method goes directly to this stage.)

Back	Next
6 of 8 The fragments are cloned in small vectors and then sequenced. About 500 bases of sequence information is produced from each fragment.

Back	Next
7 of 8 The sequences are fed into a computer, which looks for overlaps at the end of the sequence to find neighbouring fragments.

Back	Next
8 of 8 When many fragments have been sequenced the sequence of the original BAC insert can be assembled. The process is carried out for all the BACs to give a complete chromosomal sequence.

For example, the human genome is about 3 billion base pairs, arrayed in 24 chromosomes. The chromosomes themselves are 50–250 million bases (megabases) long. These tracts of DNA are much too large for even the latest automated machines, which sequence fragments of DNA between 400 and 700 bases long.

The genome is first broken into conveniently sized chunks, fragments of about 150 kilobases. Each fragment is inserted into a bacterial artificial chromosome (BAC), a cloning vector used to propagate DNA in bacteria grown in culture.

The BACs are then mapped, so that it is known exactly where the inserts have come from. This process makes re-assembling the sequenced fragments to reflect their original position in the genome easier and more accurate, and any one piece of human DNA sequence can automatically be placed to an accuracy of 1 part in 30 000.

Each of the large clones is then 'shotgunned' - broken into pieces of perhaps 1500 base pairs, either by enzymes or by physical shearing - and the fragments are sequenced separately. Shotgunning the original large clone randomly several times ensures that some of the fragments will overlap; computers then analyse the sequences of these small fragments, looking for end sequences that overlap - indicating neighbouring fragments - and assembling the original sequence of the clone.

An alternative approach, 'whole genome shotgun sequencing', was first used in 1982 by the inventor of shotgun sequencing, Fred Sanger, while working on phages (viruses of bacteria). As its name suggests, in this technique the whole genome is broken into small fragments that can be sequenced and reassembled. This method is very useful for organisms with smaller genomes, or when a related genome is already known.