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 mitochondrial DNA. 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 years

ago (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 higer . Short-tailed 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" web site.

Acknowledgement: 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.