Seminario di presentazione del progetto Eco-AlpsWater Sede … · Seminario di presentazione del...

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Tecniche di Next Generation Sequencing a supporto dei monitoraggi di nuova generazione

Nico SalmasoCoordinatore del progetto Eco-AlpsWater

IASMA Research and Innovation CentreFondazione Mach-Istituto Agrario di S. Michele all'Adige

Unità di ricerca [email protected] – 0461 615323

Innovative Ecological Assessment and Water Management Strategy for the Protection of Ecosystem Services in Alpine Lakes and Rivers

Eco-AlpsWater

Seminario di presentazione del progetto Eco-AlpsWaterSede ISPRA, Sala del Consiglio Federale, Roma, 16 ottobre 2019

Interreg Alpine Space Priority 3: Liveable Alpine Space. SO3.2:Enhance the protection, the conservation and the ecological

connectivity of Alpine Space

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Where were we?

Nico SalmasoSeminario di presentazione del progetto Eco-AlpsWater - Sede ISPRA, Roma, 16 ottobre 2019

• Beyond microscopy: traditional methods to assess planktic biodiversity in water bodies, with case studies

• Limits of traditional methods (microscopy and genetic)

• Classical historical solutions to analyse environmental DNA (eDNA)

• The advent of Next Generation Sequencing approaches

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Beyond microscopy: traditional approaches used to study microscopical biodiversity in lakes and rivers

1. Isolation and cultivation, genetic analyses of strains, and ecological and physiological characterizations (polyphasic approach)

2. Gel based methods

3. Others…

Though with many limits, microscopy allows to obtain immediate information about the diversity of microalgal communities.

More detailed information can be obtained by applying complementary methods that however require more time, based on the genetic analysis of strains or environmental DNA (eDNA)


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Identification of microalgae – Microscopy, culture-dependent

approaches, and genetic (polyphasic approach)

Cultures of cyanobacteria

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3

2

1 2 3 4 50

time (min)

1 2 3 4 50

time (min)

Isolation of single strains (single filaments)SamplingCultivation of algal strains

Light microscopy

Extraction of genomic

DNA

PCR and sequencing (16SrRNA,

housekeeping and cyanotoxins encoding genes)Phylogenetic analyses

Extraction of cyanotoxins and metabolomic profiling (LC-MS)

Complementary methods to microscopy (1)

cyanobacteria


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Toxic phytoplankton in Lake Garda: Problems linked with the correct identification of ATX-a producers in environmental samples

Mean summer concentrations

Microcystinsµg L-1

Anatoxin-aµg L-1

Garda 0.05 0.28

Iseo 0.10 0.24

Como 0.01 0.05

Lugano 0.09 0.000Maggiore 0.05 0.12

Salmaso et al., 2014 – Toxicon 90: 82-96.

Cerasino & Salmaso, 2012 – Ocean. Hydrobiol. Stud. 41: 54-63.

Milan

Switzerland

Iseo

Como

Garda

Maggiore

Lugano

Verona

Italy

50 km Brescia

Austria

Trento

N

Idro

Pusiano

S. Michele all’Adige

?

Case study – Application of the polyphasic approach


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Who was the responsible of the production of anatoxin-a in the large lakes south of the Alps?

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Anatoxin-a

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?

?

Dolichospermum lemmermanniiPlanktothrix rubescens


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Morphological characteristics were consistent with the diacritical features described for Tychonema bourrellyi

Shams et al., 2015 – Water Research 69: 68-79.

Tychonema bourrellyi(microscopic observation: maybe…)

Lake Garda

Nord (e.g. Norway) and Central Europe strains

16S rRNA

The classification based on morphological and morphometrical traits was confirmed by the phylogenetic analyses based on 16S rDNA


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Genetic analyses and metabolomic profiling demonstrated that Tychonema was able to produce ATX

Co

ntr

ol (

+)

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ntr

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-)

1-12: strains of Tychonema bourrellyi; amplicon: anaC residue

100 bp300 bp

Proline adenylation protein

• Positive for the presence of anatoxin-a encoding genes (anaC, and anaF-Norway)

• Negative for the presence of MCs encoding genes (mcyE)

PKS, polyketide synthase

• Almost all the strains were able to produce anatoxin-a


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Phylogenetic analyses (16S rRNA) have confirmed the presence of Tychonema bourrellyi in all the largest lakes south of the Alps

Salmaso et al. (2016)

Tychonema bourrellyiPlanktothrix rubescens

Planktothrix rubescens

Tychonema bourrellyi

N


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up to 1870 µg/L of anatoxin-a

In Lake Garda:< 1µg/L of anatoxin-a


Fastner et al., 2018

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Microscopy, and culture-dependent approaches, have several limits

MICROSCOPY• Morphological simplicity of many organisms and variability of

phenotypic traits within one species• Dependence of certain characters on environmental factors

POLYPHASIC APPROACH• Lengthy and costly method, allowing processing of only individual

isolates• Rare species are missed• Many species are difficult to isolate and/or cultivate (particularly

bacteria!!)

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The limits of approaches based on isolation and cultivation methods is particularly critical for bacterial communities

The comparison of direct microscopic cell count performed after staining of bacteria with 4-6-diamidino-2-phenylindole (DAPI) with the number of the microbial colonies growing on nutrient agar, shows that in natural samples less than one cell in thousand produces a colony

The majority of bacterial organisms is unculturableThe great plate count anomaly

Habitat Cultivability of microorganisms

Sea water 0.001–0.01%

Freshwater 0.25%

Sediment 0.25%

Soil 3% Amann (1995 )

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• Fluorescent In Situ Hybridization (FISH/CARD-FISH) (early 1980s); Fluorescent probes of various colors can be used at the same time for varying bacterial groups.

• Molecular fingerprinting of microbial populations: Denaturing Gradient Gel Electrophoresis (DGGE), polyacrylamide gel with a linear gradient of denaturants (early 1980s)

• Clone and sequencing of 16S rRNA genes from the natural environment to determine phylogenetic diversity of microorganisms (Pace and colleagues, half 1980s). Enzimatically ligate 16S rRNA genes into plasmids, and then transform E. coli cells with plasmids.

Pioneering eDNA analyses: Classical culture-independent approaches

Complementary methods to microscopy (2)

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Esempio di applicazione:Fluorescent In Situ Hybridization (FISH/CARD-FISH)

Hernandez et al., 2018

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DGGE allows the detection of single base-pair changes in a DNA sequence. The technique has been therefore widely applied to study the molecular ecology and diversity of microorganisms, avoiding culture based approaches

Ideally one band on the gel corresponds to one species, and therefore the number of bands gives an estimate of the diversity of the sample

Fig 6: Denaturing gradient gel electrophoresis (DGGE) gel showing cyanobacterial community profiles over the study period. M, marker composed of cyanobacterial clone library from lake Loosdrecht. Labels indicate the corresponding clone label of the marker band. Clone-labels of marker bands corresponding to cyanobacteria are identified (based on 16S rDNA).

Tijdens et al., 2008. Microbial Ecology

Esempio di applicazione:Molecular fingerprinting of microbial populations -Denaturing Gradient Gel Electrophoresis (DGGE)


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In the study of microorganisms, all the traditional techniques used to complement the information obtained by microscopy have several advantages. Nevertheless, they are able to account for only a tiny fraction of the total biodiversity


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Other biological elements: macroinvertebrate, fish

• In the microscopical world (bacteria, cyanobacteria, phytoplankton, protists, fungi…viruses) the identification of organisms is strictly constrained by the simplicity of forms, limitation of discritical characters, and difficulty to isolate species (for genetic characterization), especially bacteria.

• Large forms are more easily to identify, but more difficult to sample (e.g. fish, macroinvertebrates); sampling activities are time-consuming, require personnel and investments.

https://www.flickr.com/photos/usepagov/7984301210https://www.co.ozaukee.wi.us/1232/Electrofishing


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More modern methods push the sequencing approach over the limit obtaining, without isolation and cultivation, without application of classical genetic analyses, and without cloning, hundreds of thousands of sequences from environmental samples.

We are entering the world of metagenomics and High-Throughput Sequencing (HTS).


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eDNA

Modern eDNA analysis methods allows obtaining, without

isolation and cultivation, without application of classical genetic

analyses, and without cloning, hundreds of thousands of

sequences from environmental samples

They provide information on the taxonomic characteristics of

populations, on metagenomes (including protein-coding genes),

and on the genomes of single species


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Next-generation sequencing (NGS), or High-throughput sequencing (HTS) →Metagenomic

1) “full shotgun metagenomics”: functional and sequence-based analysis of the collective microbial genomes contained in an environmental sample

NGS-HTS has led to the establishment of the field of “metagenomics” (environmental genomics, ecogenomics or community genomics), defined as the direct genetic analysis of genomes contained within an environmental sample without the prior need for cultivating clonal cultures

2) “marker gene amplification metagenomics” (“metabarcoding”): studies performing polymerase chain reaction (PCR) amplification of certain genes of interest, e.g. 16S rRNA

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https://www.youtube.com/watch?v=QjuZhPjMnkE

DNA

DNA

DNA

DNA

DNA

DNA

DNA DNA

DNA

TO EXEMPLIFY….


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DNADNA

DNA

DNA

DNADNA

DNA DNA

DNA

Traces left behind……do not think about getting away with it


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DNA

DNA DNA

DNADNA

DNA DNA

DNA

DNA

• Giraffe• Lion• ….• ….• Bear• Zebra


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Marker gene amplification metagenomics (metagenetics or targeted metagenomics), is a high-throughput sequencing (HTS) application focusing on a nucleotide target (e.g. 16S rRNA genes) to describe the taxonomic content of a sample

1) Collect water sample

2) Extract environmental DNA

3.1) Amplify target genes with PCR (e.g. 16S, 18S

rbcL, ITS)

4) Taxonomic inventories and Community analysis 3.2) Sequencing

Environmental DNA

Marker gene amplification metagenomics

FASTQ files

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>LT546463.1 Tychonema bourrellyi partial 16S

ribosomal RNA gene, isolate FEM_GT806GATCCTGGCTCAGGATGAACGCTGGCGGTCTGCTTAACACATGCAAGTCGAACGGAATCCTCGGATTTAG

TGGCGGACGGGTGAGTAACGCGTGAGAATCTACCTTCAGGACGGAGACAACAGTTGGAAACGACTGCTAA

CCCCCGATGTACCGAAAGGGCAAATATTTATAGCCTGAAGAAGAGCTCGCGTCCGATTAGCTAGTTGGAG

AGGTAAAAGCTCACCAAGGCGACGATCGGTAGCTGGTCTGAGAGGACGATCAGCCACACTGGGACTGAGA

CACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTCCGCAATGGGCGAAAGCCTGACGGAGCAA

GACCGCGTGGGGGAAGAAGGCTCTTGGGTTGTAAACTCCTTTTCTCTGGGAAGAACAAAATGACGGTACC

AGAGGAATCAGCATCGGCTAACTCCGTGCCAGCAGCCGCGGTAAGACGGAGGATGCAAGCGTTATCCGGA

ATGATTGGGCGTAAAGCGTCCGCAGGTGGCAGTTCAAGTCTGCTGTCAAAGACCGGGGCTCAACCTCGGA

AAGGCAGTGGAAACTGAACAGCTAGAGTATGGTAGGGGCAAAGGGAATTCCTGGTGTAGCGGTGAAATGC

GTAGAGATCAGGAAGAACATCGGTGGCGAAGGCGCTTTGCTGGACCATAACTGACACTCAGGGACGAAAG

CTAGGGGAGCGAATGGGATTAGATACCCCAGTAGTCCTAGCCGTAAACGATGGATACTAGGTGTTGTCTG

TATCGACCCGGACAGTGCCGTAGCTAACGCGTTAAGTATCCCGCCTGGGGAGTACGCACGCAAGTGTGAA

ACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGATGCAACGCGAAGAA

CCTTACCAGGACTTGACATGTCGCGAATCTTTTTGAAAGAGAAGAGTGCCTTAGGGAGCGCGAACACAGG

TGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGT

GTTTAGTTGCCATCATTAAGTTGGGAACTCTAAACAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGAT

GACGTCAAGTCAGCATGCCCCTTACGTCCTGGGCTACACACGTACTACAATGGTAGGGACAGAGGGCAGC

CAACTCGCGAGAGAGAGCTAATCCCGTAAACCCTGCCTCAGTTCAGATTGCAGGCTGCAACTCGCCTGCA

TGAAGGCGGAATCGCTAGTAATCGCAGGTCAGCATACTGCGGTGAATCCGTTCCCGGGCCTTGTACACAC

CGCCCGTCACACCATGGAAGTTGGCCACGCCCGAAGTCATTACTCTAACCCTTCGGGGGGGAGGATGCCG

AAGGCAGGGCTGATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGTGGCTGGATCACCT

14

70

bp

Traditional culture-dependent approach1st generation, Sanger sequencing

Isolation and Culture

DNA extraction

PCR

Sequencing

Culture-independent - Next Generation Sequencing approach

e.g. Illumina MiSqeq sequencing

DNA extraction

PCR

>DENOVO1 (Tychonema)

TGGGGAATTTTCCGCAATGGGCGAAAGCCTGACGGAGCAAGACCGCGTGGGGGAAGAAGGCTCTTGGGTTGTAAACTCCTT

TTCTCTGGGAAGAACAAAATGACGGTACCAGAGGAATCAGCATCGGCTAACTCCGTGCCAGCAGCCGCGGTAAGACGGAGG

ATGCAAGCGTTATCCGGAATGATTGGGCGTAAAGCGTCCGCAGGTGGCAGTTCAAGTCTGCTGTCAAAGACCGGGGCTCAA

CCTCGGAAAGGCAGTGGAAACTGAACAGCTAGAGTATGGTAGGGGCAAAGGGAATTCCTGGTGTAGCGGTGAAATGCGTAG

AGATCAGGAAGAACATCGGTGGCGAAGGCGCTTTGCTGGACCATAACTGACACTCAGGGACGAAAGCTAGGGGAGCGAATG

>DENOVO2 (Family Anaerolineaceae)

TAGGGAATATTGGTCAATGGGCGAAAGCCTGAACCAGCAACGCCGCGTGCGCGATGAAGGCCTTCGGGTCGTAAAGCGCTT

TTGGGAGGGATGAAATTGACAGTACCTCCCGAATAAGGATCGGCTAACTACGTGCCAGCAGCCGCGGTAAGACGTAGGATC

CAAGCGTTATCCGGAATTACTGGGCGTAAAGGGCGTGTAGGAGGTTGGGCAAGTCGGCCATGAAAGCTCCCGGCTCAACTG

GGAGAGGCTGGTCGATACTGCCTGGCTAGAGGGCAAGAGAGGGAGGTGGAATTCCCGGTGTAGTGGTGAAATGCGTAGATA

TCGGGAGGAACACCAGTGGCGAAGGCGGCCTCCTGGCTTGTACCTGACTCTGAAACGCGAAAGCATGGGGAGCAAACA

>DENOVO3 (Synechococcus)

TGGGGAATTTTCCGCAATGGGCGCAAGCCTGACGGAGCAACGCCGCGTGAGGGATGAAGGCCTCTGGGCTGTAAACCTCTT

TTATCAAGGAAGAAGATCTGACGGTACTTGATGAATAAGCCACGGCTAATTCCGTGCCAGCAGCCGCGGTAATACGGGAGT

GGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGTCCGCAGGCGGTTTTACAAGTCTGTCGTTAAAACGTGGAGCTCAAC

TCCATTTCGGCGATGGAAACTGTAAGACTAGAGTGTGGTAGGGGCAGAGGGAATTCCCGGTGTAGCGGTGAAATGCGTAGA

TATCGGGAAGAACACCAGTGGCGAAGGCGCTCTGCTGGGCCATAACTGACGCTCATGGACGAAAGCCAGGGGAGCGAAAG

>DENOVO4 (Cyanobacteria;Chloroplast)

TAGGGAATTTTCCGCAATGGGCGAAAGCCTGACGGAGCAATACCGCGTGAGGGATGACGGCCTGTGGGTTGTAAACCTCTT

TTCTCAAGGAAGAAGTTCTGACGGTACTTGAGGAATAAGCATCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGA

TGCAAGCGTTATCCGGAATCACTGGGCATAAAGCGTCTGTAGGTGGTTTGGTAAGTCTGCTGTTAAAGACTGGGGCTCAAC

CCCAGAAAAGCAGTGGAAACTGCCAGACTTGAGTGTGGTAGAGGTAGAGGGAATTCCTAGTGTAGCGGTGAAATGCGTAGA

TATTAGGAAGAACACCAATGGCGAAGGCACTCTACTGGACCATAACTGACACTGAGAGACGACAGCTAGGGGAGCAAATG

(….)

(….)

40

0-4

30

bp

Others OTUs/ASVs (up to several thousands)

Sequencing

Bioinformatic pipelinesFASTQ → FASTA

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