Reconstructing paleoenvironments using metagenomics
-
Upload
rutger-vos -
Category
Education
-
view
708 -
download
1
description
Transcript of Reconstructing paleoenvironments using metagenomics
![Page 1: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/1.jpg)
Reconstructing paleoenvironments using metagenomics
Rutger Vos
![Page 2: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/2.jpg)
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
6 February 2013Metagenomics approaches and data analysis
![Page 3: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/3.jpg)
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
6 February 2013Metagenomics approaches and data analysis
![Page 4: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/4.jpg)
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
6 February 2013Metagenomics approaches and data analysis
![Page 5: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/5.jpg)
Barcode lab
6 February 2013Metagenomics approaches and data analysis
![Page 6: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/6.jpg)
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
6 February 2013Metagenomics approaches and data analysis
![Page 7: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/7.jpg)
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.
6 February 2013Metagenomics approaches and data analysis
![Page 8: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/8.jpg)
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
6 February 2013Metagenomics approaches and data analysis
![Page 9: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/9.jpg)
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
6 February 2013Metagenomics approaches and data analysis
![Page 10: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/10.jpg)
Use case: the woolly mammoth's dietary metagenome
6 February 2013Metagenomics approaches and data analysis
![Page 11: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/11.jpg)
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
6 February 2013Metagenomics approaches and data analysis
![Page 12: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/12.jpg)
Lyuba
Cape BlossomYukagir
Permafrost-preserved mammoth remains
![Page 13: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/13.jpg)
"Lyuba"
6 February 2013Metagenomics approaches and data analysis
• Discovered in May 2007
• One-month old mammoth calf
• Age: 41,910 ± 550 YBP
• Very well-nourished, milk-fed
![Page 14: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/14.jpg)
The Yukagir mammoth
6 February 2013Metagenomics approaches and data analysis
• 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
![Page 15: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/15.jpg)
The Cape Blossom mammoth dung
6 February 2013Metagenomics approaches and data analysis
• Produced during the cold season
• Found among a partial skeleton
• Exact site unknown
• Age: 12,300 YBP
![Page 16: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/16.jpg)
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
6 February 2013Metagenomics approaches and data analysis
![Page 17: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/17.jpg)
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*)
6 February 2013Metagenomics approaches and data analysis
![Page 18: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/18.jpg)
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
6 February 2013Metagenomics approaches and data analysis
![Page 19: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/19.jpg)
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
6 February 2013Metagenomics approaches and data analysis
Marcel Eurlings at Naturalis
![Page 20: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/20.jpg)
IonTorrent chip generations
6 February 2013Metagenomics approaches and data analysis
![Page 21: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/21.jpg)
IonTorrent data pre-processing workflow
6 February 2013Metagenomics approaches and data analysis
Filter out short reads
Splice out low phred scores
Split by primer
sequence
Split by adapter
sequence
FASTA for downstream analysis
![Page 22: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/22.jpg)
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
6 February 2013Metagenomics approaches and data analysis
![Page 23: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/23.jpg)
Pipeline steps
6 February 2013Metagenomics approaches and data analysis
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
![Page 24: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/24.jpg)
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
6 February 2013Metagenomics approaches and data analysis
![Page 25: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/25.jpg)
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
6 February 2013Metagenomics approaches and data analysis
![Page 26: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/26.jpg)
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
6 February 2013Metagenomics approaches and data analysis
![Page 27: Reconstructing paleoenvironments using metagenomics](https://reader037.fdocuments.us/reader037/viewer/2022103116/5584f026d8b42a73618b4d40/html5/thumbnails/27.jpg)
Thank you for listening