use of the genomic data o reconstruction of metabolic properties o nature’s microbiome o ngs in...
TRANSCRIPT
Use of the genomic data
o Reconstruction of metabolic properties
o Nature’s Microbiome
o NGS in Population Genetics
Why are the diatoms so successful?
canonical urea metabolism in animals
duplication of CPS in secondary algae!
Diatoms have somehow reversed urea cycle, which server as a hub for the metabolism of the nitrogen and (to some extent) also the carbon!
Nature’s Microbiome
hologenome
Non specific in situ culture dependent approaches vs Metagenomics
Community composition16S rDNA marker95%, 97%, or 99% for
OTUs/phylotypeOperational Taxonomic Unitbinning
set of abundance histograms or set of binary presence/absence
vectors
Nature’s Microbiome
Community function
Decomposition methods: Principal Component AnalysesCanonical Correlation Analyses
Nature’s Microbiome
the number (richness) and distribution (evenness) of taxa expected within a single populationAlpha Diversity
Beta Diversity
‘‘How much do I need to sequence to completely characterize my microbiome?’’
absolute or relative overlap describe how many taxa are shared between samples
Nature’s Microbiome
Community function
Community composition16S rDNA marker95%, 97%, or 99% for
OTUs/phylotypeOperational Taxonomic Unitbinning
set of abundance histograms or set of binary presence/absence
vectorsDecomposition methods:
Principal Component AnalysesCanonical Correlation Analyses
How many microbial strains and species colonize eukaryotic hosts?
Alpha diversity inflated due to PCR and sequencing errorsX
Diversity involving closely related microbes omitted in NGS data sets (OTUs cutt-off)
Nature’s Microbiome: Chalanges
Raw data „denoising“ Detecting hidden strain diversity
AnalysesSequencing tools (e.g.LEA-Seq)
How many microbial strains and species colonize eukaryotic hosts?Low Error Amplicon Sequencing, LEA-Seq
Nature’s Microbiome:Chalanges
Dilute barcoded primer solution
20x coverage
How to detect sequence libraries plagued by contamination?
Nature’s Microbiome: Chalanges
Results from the source-tracking analysis using a previously published set of mammal gut samples, as well as soil samples, as potential sources to determine which of the 92 samples were potentially contaminated with soil.
Representative source data sets
>10% soil assignment<0.01 mammalian gut
92/27(5)
Negative controlsGeneration of source libraries
marine/fresh watercommon lab microbes
Number of biological replicates
Nature’s Microbiome: Outcomes
NGS and Population Genetics
Problem of genotyping uncertainty on NGS data
machine learning to separate true segregating variation from artifacts
• Sequencing errors• Misalignment errors• Low coverage• Random sampling of
homologous base pairs in heterozygotes
initial read mappinglocal realignment around indelsbase quality score recalibration
SNP discovery and genotyping to find all potential variants
NGS and Population Genetics
Problem of genotyping uncertainty on NGS datainitial read mappinglocal realignment around indelsbase quality score recalibration
Gap enabled alignment with Burrows-Wheeler transform: >15% reads spanning homozygous indels misalgned
NGS and Population Genetics
Problem of genotyping uncertainty on NGS data• Sequencing errors• Misalignment errors• Low coverage• Random sampling of
homologous base pairs in heterozygotes
NGS and Population Genetics: Outcomes