The genome team state: "Our method for generating the sequence data
was very similar to how we sequenced the woolly mammoth. One critical
difference was that with the mammoth we started with a large ball of hair,
whereas with the rarer thylacine specimens we had only tiny amounts of
hair to work with. Thus, we were not able to enrich the thylacine
material for long pieces, but instead tried to sequence even very short
fragments. As a result, while our woolly mammoth sequences averaged
120 nucleotides in length, for the Smithsonian thylacine skin the average
was 87.5, and for the ethanol-stored specimen it was only 67. In
addition to the problems that stemmed from working with shorter sequences,
analysis of the thylacine
A computer model of
Note the ring-shaped structure
of the chromosome.
|data was complicated
by the lack of a genome sequence from a close relative to use as a reference
point. With mammoth, we could use the sequence from the African savanna
elephant, which is the same as the woolly mammoth sequence in over 99%
of the genomic positions. With the thylacine, the closest sequenced
genome was from a South American opossum, Monodelphis domestica, whose
genome sequence is quite different from the thylacine's. In this
regard, sequencing the thylacine was more representative of sequencing
an arbitrarily chosen animal, so we were glad to address the challenge.
By solving the problem of assembling the thylacine mitochondrial sequence
without having a closely related 'template sequence', we have moved closer
to the day when sequencing museum specimens will be commonplace".
With regard to the long-term possibility of sequencing the entire nuclear
genome, the team state: "To assess the feasibility of determining all
of the thylacine's DNA sequence (that is, not only the mitochondrial sequence,
but also the much larger nuclear genome), we estimated how much of our
data was thylacine nuclear DNA. What made this tricky was that we
did not know the full genome sequence of any immediate relative.
In contrast, when sequencing the woolly mammoth we could compare our data
with the available sequences from the African savanna elephant. But
for the thylacine, the closest sequenced genome is from a South American
marsupial called the short-tailed opossum (Monodelphis domestica).
The two lineages have been separated at least since the land bridge between
South America and Australia was broken, which happened perhaps 60 million
(mammoths and elephants have been separated only about 6 million years).
The 60 million years of separation, together with the small sizes of many
of the sequence fragments that we generated, made it difficult to be certain
that a particular DNA fragment was actually from the thylacine, as opposed
to being, say, human contamination. To improve the odds, we used
the fact that for nuclear-genome sequences that encode a protein, the similarity
between the thylacine and Monodelphis sequences will almost always be much
opossum (Monodelphis domestica).
|than for an arbitrary
region of the genome. In summary, our strategy was to see how much
of the thylacine sequence aligned to Monodelphis protein-coding intervals,
and then extrapolate to the full genome. This resulted in an estimate
that roughly 30% of our sequence data was from the thylacine nuclear genome.
Given the plummeting costs of genome sequencing, this indicates that it
should be possible to determine the thylacine's nuclear genome sequence
even without other improvements to our approach".
Continued research on the thylacine genome not only offers the possibility
of determining the entire nuclear genome, but may identify the unknown
causative organism responsible for the mange-like
illness responsible for the deaths of many thylacines
the latter part of the 19th and early part of the 20th centuries.
The thylacine mitochondrial genomes have "GenBank" accession numbers FJ515780
and FJ515781, and can be accessed at the "GenBank"
This section of the Thylacine Museum has been prepared [in part] from edited
material supplied by the Pennsylvania State University, Centre
for Comparative Genomics and Bioinformatics.