Professor Alan Cooper presents the sixth instalment of the Science Seminar Series. The accurate and rapid assessment of biodiversity is a critical aspect for modern science, and ranges from the measurement of environmental and climate change, to microbes in water systems or at the point of care in medical centres. Similarly, the need for the rapid and responsible economic development of primary resources, and the monitoring of invasive species and biosecurity, also place a premium on the ability to quickly assess and quantify biodiversity across a range of diverse habitats. We have combined methods developed to detect trace amounts of ancient DNA with 2nd Generation Sequencing technology to design a vertical barcoding system capable of rapidly screening the genetic and taxonomic diversity of modern, complex biological samples. In parallel with studies of taxonomically identified museum and herbaria material, this approach promises to provide the first genetic audits of diverse Australian environments.

1. The Environment Institute
Where ideas grow
Professor Alan Cooper
Environmental genomics

2. Environmental genomics:
Rapid biodiversity assessment of any sample
Australian Centre for Ancient DNA,
The University of Adelaide

3. Environmental genomics:
Current Funding
ARC LINKAGE project (2009). $0.5M,
plus industry contributions (ca. 0.6M) =
$1.1M
Future Fellowship (2009). $1M

4. What is ACAD?
• Internationally leading centre for study of preserved
genetic information
• Expertise with multiplex analysis (many genes) of
biologically complex, trace-level samples, and 2nd
Generation Sequencing
• Able to use existing museum and herbaria collections to
generate reference database of taxonomically ID’d taxa
• AQIS approved facility
• Suitable for forensics (standard and environmental),
sedimentary, and water analysis

6. Individual (still-air) working rooms • 3 Laboratories – incl. high-
tech ancient facility, museum
grade specimens, modern lab
• Trambarn - AQIS approved
• Positive air-pressure
• UV sterilisation
• Controlled personnel flow
• 16 researchers
-20’C • International visitors
workrooms • Evolution, archaeology,
biodiversity/climate change,
Air
shower forensics - collaboration with
AFP, DEH, NGS,
Air flow
UV
Entry

7. ancient DNA lab
= ultra-low DNA environment

8. Environmental Genomics
• Takes advantage of massive increase in sequencing power of 2nd and
now 3rd Generation sequencing to perform vertical genetic bar-coding
• Capable of working with diverse taxonomic groups rapidly and
simultaneously – using deep (multi-marker) characterisation of genetic
diversity within any sample.
• Do not need to know what is present a priori, and can map species
across a broad scale (eg landscape, soil, water, complex samples),
and without need for prior taxonomic knowledge
• De novo assessment of biodiversity, eg mining, developing world
• Rapid, powerful, high resolution - using flexible, standard platform
• Remove constraints (esp. time) of traditional morphological
approaches, ideal for mining, primary industries
• Needs active involvement of existing taxonomic and ecological
expertise to identify accuracy, and potential uses

9. What is 2nd Generation Sequencing?
• It is already out of date.
• Parallel analysis of millions of different DNA sequences
using chip-based arrays, or microwells
• Very cheap per base. 1st human genome = $Bn, currently
<$50k
• Produces massive amounts of data, only few % of current
products analysed
• 3rd Generation released next year. Active moves to locate
machine at Adelaide for metagenomics. Promises of
human genome in 3 minutes, for $5k.
• Key constraint in this revolution in biological science – who
is going to analyse the data??

10. 3rd Generation Sequencing - Oxford Nanopore Technologies
a-hemolysin nanopore (ribbon diagram) with covalently attached cyclodextrin (teal)
transiently binds a DNA base (red) traversing the pore. A, G, T, C and CM are
separated according to charge and mass. No need for dyes, CCD cameras etc

11. Current partners
• PIRSA: Paul Heithersay – sediment-based survey of biodiversity
across South Australia, focusing on plant and animal taxa
• Australian Federal Police: Paul Kirkbride – forensic analysis of soils for
geographic predictions, evidence analysis
• SA Water: Chris Saint – microbial diversity of water systems, including
re-use, de-salination plants, and reservoirs. Unknown pathogens
• SA Pathology: Hamish Scott, Tuckweng Kok (James Paton) –
microbial diversity within hospital/medical systems, unknown
pathogens
• DEH: Hugh Cross, Andy Lowe – analysis of herbarium specimens,
grasses
• José Facelli – ecological interpretation of EG data vs field sites
• Biomatters Ltd (Geneious): Shane Sturrock – design and
implementation of software interface between raw genomics output,
and end-users. Training postdocs and PhDs in bioinformatics.
• Daniel Huson (MEGAN) – leading metagenomics analytical software

12. Sample extraction
Ancient DNA methods developed to extract trace signals from
complex biological samples – eg sediments, old bones (mostly
microbes), faeces
Bulk processing (eg sediments), and selective hybridisation or
primer-based capture techniques to pull out useful sequences from
high background levels. Connect to High throughput sequencing
Water = bulk processing and filtering, perhaps with semi-permanent
sensors (IPAS)
R+D on DNA extraction and isolation procedures

13. Informative loci
The environmental genomics approach harnesses
to power of genomics technology, by focusing the
amplified loci to contain only taxonomically
informative genes.
Candidate loci, and suitable databases, already
exist although generally short and with limited
coverage.
Examples include COI/12S/cytb (vertebrates,
invertebrates), 16S (bacterial), rbcL/trnL, matK
(plants).
We will use extra domains within these loci, and
additional loci (eg nuclear introns, vWF, RAG-1,
others) as necessary. The system has large
capacity.

14. Future directions
• Genographic project
• newly collected material (!), needs rates and demographic analysis

15. Metagenomic studies of ancient
samples confirm that most of
the DNA is exogenous/microbial

16. Voucher specimens
The environmental signals must be identified
through comparison with voucher specimens – eg
taxonomically identified material. The same genetic
loci must be sequenced to allow identification
Suitable material includes museums, herbaria,
microbial cultures and collections – but new
samples will need to be gathered and processed
throughout the project

17. Current constraints
Need locally sited 2nd or 3rd Generation machine
Desperate need for bioinformaticians (students and
trained maths/engineers/physicists
PhD and postdocs

18. Acknowledgements
Kyle Armstrong
Paul Brotherton
Wolfgang Haak
Jeremy Austin

19. The Environment Institute
Where ideas grow
Next Seminar: 16 October
Assoc. Prof. Bronwyn Gillanders
Giant Australian cuttlefish: a globally unique species
under threat?