Lecture on metagenomics research at the Naturalis Biodiversity Center, based on the use case of sequencing the metagenome of mammoth stomach contents.

1. Reconstructing paleoenvironments
using metagenomics
Rutger Vos

2. Outline
• About Naturalis Biodiversity
Center
• NBC's facilities and expertises
 Ancient DNA lab
 Barcode lab
 Informatics focus group
• Metagenomics and
paleoecology
• Use case: The mammoth's last
meal
• NGS@Naturalis
• Pipeline development
Metagenomics approaches and data analysis 6 February 2013

3. Naturalis Biodiversity Center
• With 37 million specimens,
NBC holds one of the largest
natural history collections in the
world
• More than just a museum, NBC is
an expert center specializing in:
 Species identification
 Trait harvesting
 Impact modeling
 Ecological intensivation
Metagenomics approaches and data analysis 6 February 2013

4. Ancient DNA lab
• The ancient-DNA facility is equipped
for recovering DNA from plant and
animal material from museum
collections and fossils.
• It permits research that would
otherwise not be possible, such as
the study of ancient populations
and museum material.
• The ancient-DNA lab provides an
environment where the risk of
contamination with contemporary
DNA is minimal.
• The facility, a collaboration of IBL,
the faculty of archeology and
NBC, is unique in the Netherlands
Metagenomics approaches and data analysis 6 February 2013

5. Barcode lab
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6. Informatics focus group
• Exploitation of HPC resources
• Dissemination of best practices
• In-house development of research-
supporting tools:
 NGS data processing
 Clustering, BLASTing
 Custom pipelines
 Visualization
 Image analysis
 Niche modeling
Metagenomics approaches and data analysis 6 February 2013

7. HPC infrastructure
• Dell T7500 and T7600 workstations
• Intel® Xeon® Processor
(QuadCore, 2.40GHz) x 2
• 128Gb RAM
• TESLA/NVIDIA GPU
• RedHat/Ubuntu Linux
• Always looking for extra
numbercrunching power, e.g. from
NBIC Galaxy, CIPRES, BioPortal,
etc.
Metagenomics approaches and data analysis 6 February 2013

8. Paleoenvironments
• Reconstructing the
paleoenvironment is useful for:
 Understanding the dynamics
of ecosystem change
 Reconstructing pre-
industrialization ecosystems
• Many public policy decision-
makers have pointed to the
importance of using
palaeoecological studies as a
basis for choices made in
conservation ecology
Metagenomics approaches and data analysis 6 February 2013

9. Metagenomics
• Taxonomic identification is
one of the main challenges
surrounding metagenomics,
and one of NBC’s core
strengths
• Conversely, a better
understanding of the
metagenome feeds back into
our other research interests
and expertises
• Consequently, a lot of research
activity and ongoing capacity
building
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10. Use case: the woolly mammoth's dietary
metagenome
Metagenomics approaches and data analysis 6 February 2013

11. The research programme
• To test hypotheses about the structure of the ancient environment
of the woolly mammoth, i.e. productive, continuous grassland
steppe or sparsely covered herb tundra
• Finding frozen mammoths with forensically identiable food,
parasites, and microorganisms in their gastrointestinal tracts or
feces has the potential of adding data to the extinction debate
• To integrate the findings from ancient DNA with those obtained
from macro- and micro-fossils
Metagenomics approaches and data analysis 6 February 2013

12. Lyuba
Cape Blossom
Yukagir
Permafrost-preserved mammoth remains

13. "Lyuba"
• Discovered in May 2007
• One-month old mammoth calf
• Age: 41,910 ± 550 YBP
• Very well-nourished, milk-fed
Metagenomics approaches and data analysis 6 February 2013

14. The Yukagir mammoth
• Male woolly mammal
• Discovered in 2002
• Very well preserved in the
permafrost
• Age: 18,560 ± 50 YBP
• Head, front legs, parts of
stomach and intestinal tract
• Last meal still preserved
Metagenomics approaches and data analysis 6 February 2013

15. The Cape Blossom mammoth dung
• Produced during the cold season
• Found among a partial skeleton
• Exact site unknown
• Age: 12,300 YBP
Metagenomics approaches and data analysis 6 February 2013

16. DNA extraction and sequencing
• In all studies, macro-fossils
(stems, leaves, seeds), micro-
fossils (pollen) and ancient
DNA were compared
• DNA was extracted in the
ancient DNA facility using
multiple extraction protocols
• Several commonly-used
markers were amplified (trnL,
rbcL, nrITS1)
• Sanger sequencing was done
on an ABI 3730xl
Metagenomics approaches and data analysis 6 February 2013

17. Data analysis
• Sequences were assembled using
Sequencher
• Taxa were assigned using a
combination of GenBank BLAST
searches and phylogenetic
inference
• BLAST hits were only accepted if
they covered the full query
sequence and differed by at most
1 nucleotide
• Phylogenetic placement was
determined on the basis of
bootstrap support (1000 replicates
using paup*)
Metagenomics approaches and data analysis 6 February 2013

18. Findings
• Ancient DNA could assign 7 ("Lyuba"), 12 ("dung") and
8 ("Yukagir") plant families, with several determinations
down to genus level
• Molecules complemented and confirmed fossils
• Identified vegetation composition is generally
supportive of a productive "mammoth steppe"
• Micro-fossils of specific dung fungi showed that
mammoths appear, unlike elephants, to be habitually
coprophagous
Metagenomics approaches and data analysis 6 February 2013

19. Next generation applications
• The results of the mammoth research
so far have been obtained using
Sanger sequencing
• Similar, as yet unpublished, research is
being undertaken with the newly
acquired IonTorrent "sequencing by
synthesis" platform
Marcel Eurlings at Naturalis
Metagenomics approaches and data analysis 6 February 2013

20. IonTorrent chip generations
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21. IonTorrent data pre-processing workflow
Filter out
short reads
Splice out low
phred scores
Split by Split by
primer adapter
sequence sequence
FASTA for downstream analysis
Metagenomics approaches and data analysis 6 February 2013

22. Taxonomic identification pipeline
• Taxonomic identification of the
contents of samples is a generic
problem for which we have
developed a re-usable pipeline
• It replicates some of the
functionality of QIIME but integrates
more conveniently in our HPC
configuration
• Requirements:
 Python 2.7 or 3.2
 Biopython 1.58
 NCBI-Blast-2.2.25+
 Clustering programs, e.g. TGICL,
Usearch, Octupus, cd-hit
Metagenomics approaches and data analysis 6 February 2013

23. Pipeline steps
Optional: tag FASTA for provenance retracing across files
Cluster sequences into OCTUs of at least 10 reads
Pick exemplar sequence (random, consensus or hybrid)
BLAST exemplar sequences (local or remote)
Optional: retrace provenance
Report
Metagenomics approaches and data analysis 6 February 2013

24. Pipeline extensions
• NBC frequently deals with samples
that may contain materials from
endangered species, for example:
 Putative FSC wood
 Traditional Chinese medicine
 Incense
• We are therefore extending the
taxonomic identification pipeline to
check automatically whether any taxa
from the sample are listed in CITES
appendices
• This, however, poses additional
challenges of taxonomic name
reconciliation
Metagenomics approaches and data analysis 6 February 2013

25. Other metagenomics work
• Phylogenies from
metagenomic sequence data
can grow to immense sizes
• For example, the GreenGenes
16S rRNA tree has ~400k tips
• We are developing novel
algorithms for pruning these
trees using (Google’s)
MapReduce programming
model
Metagenomics approaches and data analysis 6 February 2013

26. Acknowledgements
• I am grateful to:
• Dr. Barbara Gravendeel for her input in
developing this talk
• Youri Lammers for his great working in developing
a well-documented taxonomic identification
pipeline
• And to NBIC for giving me the opportunity to
present this story
Metagenomics approaches and data analysis 6 February 2013

27. Thank you for listening