Second-Generation Sequencing of Mutants: the $1000 Mutant Genome (BioSysBio 2009)

J A Pachebat et al.
University of Cambridge

HGP finished 2004, and took $300 million. Same method in 2007 for $10 million. However, there is a new generation of techniques that are much cheaper and faster. Very nice hierarchy, or family tree, of sequencing technologies. However, there are 3rd-generation machines on the horizon (2010-ish?). He works with the Solexa (Illumina) sequencer. In terms of cost, this sequencing method is much cheaper (0.000214 pence/base rather than 0.006 for 454 and 1.56 for sanger sequencing).

What is the use of your 2G sequencing? genome re-sequencing, metagenomics, transcriptional regulation, multi-locus amplicon… In genome resequencing you align your reads against a reference genome. This allows you to look for SNPs or indels. Its affordable, but so far has been used mostly on bacteria. Resequencing of small-medium size bacterial genomes are nicely possible. He uses as an example Dictyostelium discoideum, or an amoeba with interesting properties under different circumstances: amoebae -> aggregation -> mound -> slug -> tipped mound -> spores -> back to amoebae. Originally published in 2005 in Nature and original took 5 years. They resequenced it, and looked at a number of lab strains, and sequenced: the AX4 strain specifically together with the DdB parental strain, and a couple of others.

They managed to sequence at leasst 96.9% of the genome in each strain (the sequencing was hard as it is AT rich). They found a number of errors (at least 4000) in the original sanger-sequenced AX4 genome. They did this by id'ing SNPs common to all 3 strains, and then compared things. You can also identify gene duplications. Showed that there was a bias to G/C-rich reads. Coverage improves with the depth of sequencing – the median depth of coverage was 13. What percentage of the genomes are "solexa-resistant"? around 280,504 bp (0.83 %) between AX2, DdB and AX4. However, when look at all 6 strains, this goes down to 0.41%.

WT amoebae eat bacteria – the AX strians derived from DdB, which was grown on a layer of bacteria, and when preparing for genomic dna, not all bacteria was washed away. Because of this, they got a "serendipitous genome" of non-pathogenic Klebsiella.

Each strain has about 4000 SNPs specific to that strain. Depending on mutation rate, do you still have the same strain you started with after a few months?

Is it $1000? Almost. In practice, you get 4-7X coverage for $1100, but the tech is improving fast.

Personal Comment: I agree with Dan Swan and his tweets: I think Dictyostelium discoideum is a great organism, and glad to see it in this talk. A fun talk in general.

Wednesday Session 3

Please note that this post is merely my notes on the presentation. They are not guaranteed to be correct, and unless explicitly stated are not my opinions. They do not reflect the opinions of my employers. Any errors you can happily assume to be mine and no-one else's. I'm happy to correct any errors you may spot – just let me know!

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