Sequence Assembly

**Part I: The Drosophila Genome Project**

Johannes Jaeger

Outline

1. Drosophila and its Genome

2. Sequencing Strategy

3. References and Links

1. Drosophila and its Genome

Fig. 1: The fruit fly, Drosophila melanogaster (from FlyBase)

Facts about Genomes

Chromosomes: Genomes of higher organisms are arranged on chromosomes.
Diploidy: In animals and plants, each organism contains two copies of each chromosome, one from the mother, one from the father.
Linkage Groups: Genes that lie on the same chromosome are usually inherited together and therefore form a linkage group.
Crossover: Often, maternal and paternal chromosomes exchange pieces of their DNA. This is called genetic crossover.
Genetic Mapping: The relative position (or locus) of a gene on a chromosome can be evaluated using by calculating the probability of crossover events.
Physical Mapping: The absolute locus of a gene on a chromosome is determined by for example:

Cytological mapping: DNA probes are attached to the chromosome and localized under the microscope (you can actually see where the gene is on the chromosome!).
Deletion mapping: Visible chromosome rearrangements are aligned along the chromosomes and compared to gene loci.
Sequence Tagged Site (STS) Mapping: The positions of certain known short sequences in the genome are determined to arrange the STS along the chromosomes.
Sequencing: The actual sequence of the whole genome is determined.

Euchromatin: Is the part of the chromosome that contains most of the genes. This is the part of the genome that is actually sequenced.
Heterochromatin: Contains mostly short repetitive sequences. Its function is unknown. Heterochromatin is difficult to clone and probably impossible to sequence.

Why Drosophila?

Drosophila melanogaster is an excellent genetic model system, because of the tremendous amounts of genetic techniques available:

Drosophila is easy and cheap to grow in a lab and has a short generation time.
Complete genetic and physical (cytological and deletion) maps are available (Fig. 2).

Drosophila genetic and physical
maps
Fig. 2: (A) Genetic map (above) and corresponding cytological map (below).
(B) Shows a picture of the chromosome as you see it under the microscope (taken from 1).

Transgenic fruit flies can be made to study function and interaction of genes.
Methods are available to study the exact timing and location of expression of genes to study developmental processes.

The high evolutionary conservation of a lot of genes can be used to study processes in the fruit fly that are very similar to molecular processes in humans.
Drosophila has a small and compact genome. Therefore, it was ideally suited to test the novel technologies involved in the whole-genome shotgun (WGS) sequencing approach used for the Human Genome Project (see below).

The Drosophila Genome

Length: about 180 million base pairs (humans: more than 3000 mio bp), 120 Mbp euchromatin, 60 Mpb heterochromatin
Contains an estimated 13600 genes (this is a very small number for an organism as complex as a fly)
4 chromosomes: X/Y, 2, 3 and a very small 4th chromosome (Fig. 3)

Fig. 3: The Drosophila genome (taken from 2)

2. Sequencing Strategy

Whole-Genome Shotgun (WGS) Sequencing

Traditional sequencing approaches like the one used by the public Berkeley and European Drosophila Genome Projects have used physical maps to determine the position of the clones to be sequenced on the chromosome. In addition to this, the public projects have created an STS based physical map of chromosomes 2 and 3.
WGS sequencing as used by Celera shears the whole genome into random fragments of equal size, before sequencing only their ends and aligning the sequences obtained using sequence assembly software (3). The main advantage of this approach is that the whole genome can be tackled at once using large arrays of sequencers, which makes the whole process much more efficient than the traditional approaches.
Fragments used in the Celera Drosophila Genome Project were of 2000, 10'000 and 130'000 base pairs length. At both ends of these fragments, stretches of about 500 bp were sequenced using the Polymerase Chain Reaction (PCR) with the flanking vectors sequences as primers (2,3).
The coverage for the 2 kbp and the 10 kbp fragments was 10X, coverage for the 130 kbp fragments was 15X (2).

The Data Sets (3)

Two different data sets were used for the sequence assembly process:

The WGS data set includes all the sequences obtained from sequencing the ends of the 2, 10 and 130 kbp fragments as well as known distances (the clone lengths) between these sequences.
The joint data set includes the WGS data set and the additional sequence data that was obtained from the public genome projects in their traditional approach.

The STS data from the public genome project was not used in the assembly process, but later on to crosscheck the sequence draft and improve the quality of the data.

Scaffolding (3)

The sequences in the data sets were checked for unique sequence overlaps. Contiguous sequences were assembled into contigs.
Contigs can be arranged into scaffolds when distances between contigs can be calculated from the data set (based on the length of the clones).
Within the scaffolds, sequence gaps remain (i.e. the unsequenced interior parts of the inserts). These gaps can often be closed by sequencing whole inserts.
Between scaffolds, physical gaps remain, mostly stretching highly repetitive sequences. The lengths of some of these gaps can be evaluated using the STS data.
The biggest difficulties for sequencing are encountered, when DNA sequences are repeated in the genome. Short stretches of repetitive DNA, such as retrotransposons are easily bridged by the longer (10 and 130 kpb) fragments. However, long stretches of repetitive DNA sequences such as repeated rRNA genes or heterochromatin repeats leave persistent gaps in the sequence and might never be sequenced to completion at all.

3. References

Rubin, G. M and Lewis E. B. (2000): A Brief History of Drosophila's Contributions to Genome Research. Science 287, pp. 2216-2218.
Adams et al. (2000): The Genome Sequence of Drosophila melanogaster. Science 287, pp. 2185-2195
Myers et al. (2000): A Whole-Genome Assembly of Drosophila. Science 287, pp. 2196-2198.

Some additional Drosophila links:

The Berkeley Drosophila Genome Project
The European Drosophila Genome Project
FlyBase
The Interactive Fly

The Drosophila genome sequences were deposited in GenBank, accession numbers AE002566-AE003403.