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Imagenes: Antonio Fernández-Guerra

(Max Planck Institute for Marine Microbiology)

van den Bogert et al. (2020)

Reconstrucción y análisis de genomas a partir de metagenomas

Miguel Ángel González Arias

Twitter: @miangoar

Correo: gamamiguelangel@gmail.com

miangoar.github.io/year-archive/

SEMINARIOS

WINTER GENOMICS

youtu.be/ckIbT93Qhjc

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Introducción

Ensamble

Evaluación de

calidad

Binning

Taxogenómica

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

Imagen: Nicola Segata Lab - Computational Metagenomics

University of Trento

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Dharamshi et al. (2020)

Extracción

del DNA

Introducción

Amplificación

del gen 16S

Secuenciación

del gen 16S

Agrupación por

similitud de

secuencia

Perfilación taxonómica

de procariontes usando

marcadores genómicos

Operational

Taxonomic

Unit

(OTU)

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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Dharamshi et al. (2020)

Chen et al. (2020)

Extracción

del DNA

Introducción

Secuenciación

Por shotgun

Ensamble

Binning

Metagenómica

Metagenome

Assembled

Genomes

(MAG)

62

MAGs completos

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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Introducción

Primeros pasos (2004)

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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Introducción

Microorganismos

“no cultivables”

Candidatus

Prometheoarchaeum syntrophicum

(12 años para lograr cultivarla)

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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Tecnologías de secuenciación

Reads cortos

Reads largos

Introducción

Sanger

2022

Sequencing

Market

Share

sandiegomics.com

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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Ensamble

Compeau et al. (2011) Baker (2012) Ayling et al. (2019)

Principales paradigmas

de ensamble genómico

(de novo)

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Ensamble

Ensambladores “populares”

Megahit
Meta-SPAdes
IDBA-UD

El mejor ensamblador

NO EXISTE

Depende de:

Origen de las

muestras

Recursos

computacionales

Plataforma de

secuenciación

Número de

muestras

RECOMENDACIÓN:

De ser posible, probar diferentes

Ensambladores y comparar su

rendimiento evaluando:

% de mapeo de reads
Tamaño y distribución de los contigs �así como el número de pares de bases
N50, longitud máxima y mínima
Etc.

Existen ensambladores de ensambladores:

CISA GARM Metassembler

Para comparar

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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Regresión

Lineal

Regresión

Logística

Principal Component

Analysis (PCA)

K-means

Dimensionality reduction

Reinforcement

learning

Supervised learning

Unsupervised learning

RLHF

Self-supervised

learning

Binning

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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Binning

Algoritmos de clustering no supervisado

(Binners)

¿Qué información se usa?

Composición

(GC%, 2D, 4T)

Coverage

Perfil filogenético

O funcional

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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Bowtie2

BBmap

BWA

Mapeo

Nielsen et al.

(2014)

“Coverage”

Also known as “depth of coverage”

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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Mapeo

https://anvio.org/vocabulary/

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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Binning

Se recomienda usar Contigs >2kpb

Alneberg et al.

(2014)

Composición

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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Binning

Existen algoritmos para integrar resultados

De diferentes binners

Sedlar et al. (2017)

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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Ensambladores

Alineadores

Binners

Perfiladores

76 algoritmos distintos

Binning

¿Como elegir la mejor

herramienta?

Pipelines

completos

SqueezeMeta

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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Completness = 5 / 5 🡪 100%

Genes de copia única

Completness = 3 / 5 🡪 60%

Contaminación = 1 x 5 🡪 100%

Calidad

Evaluación de Bins /MAGs

Bacteria

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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Taxogenómica

Hug et al. (2016)

Uncultured

Filogenia de 16

proteínas ribosomales

L2, L3, L4, L5, L6, L14, L16, L18, L22 L24, S3, S8, S10, S17 y S19

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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Taxogenómica

PDB:7OE1

Proteínas

16S rRNA

(V3-V4)

Proteínas ribosomales como

genes marcadores universales

¿Y si en realidad somos arqueas?

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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Taxogenómica

El grado de conservación (a nivel de secuencia) permitido para una familia de proteínas es directamente proporcional a la importancia de su función en la célula

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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Computational

requirements &

Taxogenómica

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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Sant’Anna et al. (2019)

Delsuc et al. (2005)

Diferentes atributos de clasificación

genómica

Intrasp

spp

genero

Taxogenómica

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Taxogenómica

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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Cultivados

No Cultivados

≠

=

Plan A

Plan B

Taxogenómica

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

GENOME TAXONOMY DATABASE

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Polyphasic approach

Genotípico (filo, 16S/MSA)
Quimiotaxonómico (API)
Fenotípico (colonias)
cultivo puro (axénico)

Taxogenómica

Prometheoarchaeum syntrophicum

Candidatus

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Proceso de asignación

taxonómica oficial

Taxogenómica

“ICSP does not currently extend to uncultured microorganisms nor recognise

the ranks of phylum

and superphylum”

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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https://www.microbiologyresearch.org/journal/ijsem/editorial-board

“The current structure of oversight of the Code by the ICSP should be reviewed and re-evaluated. Currently, this group is comprised of a small number of scientists, several of whom are associated with culture collections. A biased representation and potential conflicts of interest may hinder progress and implementing solutions expeditiously. Expansion of this group to increase breadth and diversity of researchers with expertise in genomics, microbial ecology, and uncultivated organisms should be considered”

27 personas

en total

Microbial Taxonomy Run Amok

Trends in Microbiology

Sanford et al. (2021)

Taxogenómica

Premisa: Revisión de 1 sp = 7d

Hay ~85,000 spp en la GTDB

1630 años para revisar todas las spp

(si trabajan 24h/7d)

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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“Hennig proposed […] that the categorical rank of any taxon should denote its geological age, but this suggestion has been neglected, perhaps because of a widespread perception that the nodes in evolutionary trees cannot be dated with reasonable assurance. However, recent studies suggest that multilocus DNA sequence data (complemented by fossil evidence) may help”

“A universal time-based taxonomy would […] facilitate comparative evolutionary studies […]

We propose that the approximate dates of nodes in evolutionary trees should be the universal criterion according to which taxonomic classifications above the level of biological species are erected”

Taxogenómica

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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Taxogenómica

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

taxa with high expected

substitution rates

g__Buchnera

o__Mycoplasmatales

g__Liberibacter

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Selección de “geno-spp”

Taxogenómica

Step 1: Genome (402,709) clustering by Average Nucleotide identity (95% ANI)

Step 2: Gene marker detection and phylogenetic inference

Archaea

53

proteins

Bacteria

120 proteins

Step 3: Calculate the Relative Evolutionary Divergence (RED)

p: RED of its parent

d: Branch length to its parent

u: Average branch length from the parent node to all extant taxa descendant from n

RED = p + (d/u) × (1 – p)

Current taxa = 1 | LCA = 0

Ha demostrado ser robusta a:

Método de inferencia filogenética (IQ-Tree, ExaML, FastTree, etc)
Modelo de sustitución (WAG, LG, JTT, GTR | +G, C20, arC60, PMSF)
SCG set (bac120, rand-bac60, rp16, 16S … ~bac1; ar122, ar253, phylophlan)
Muestreo de taxa
Nodos de enraizado
Expansión de la información genómica (arRefSeq: 2015-2018)
Sitios de rápida evolución

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

GTDB foundations

Briefly, species clusters are formed as follows:�Identify a GTDB representative genome for each validly or effectively published species with one or more genomes passing quality control. In most cases this will be a genome sequenced from the type strain of the species. When this is not possible, the representative genome is selected based on its quality and with consideration to additional metadata (e.g., NCBI reference or representative genome, genome assembled from type strain of subspecies).

Assign genomes to selected GTDB representative genomes using average nucleotide identity (ANI) and alignment fraction (AF) criteria. GTDB uses an ANI circumscription radius of 95%, though permits this to be as high as 97% in order to retain a larger number of existing species names. Species with an ANI >97% are synonyms within the GTDB. Species assignments use an AF of 65%. ANI and AF values are calculated with FastANI v1.1.

Remaining genomes are formed into de novo species clusters using a greedy clustering approach that emphasizes selecting representative genomes of high quality. This clustering consists of 3 steps: i) sort remaining genomes by their estimated genome quality, ii) select the highest-quality genome to form a new species cluster, and iii) assign genomes to this species cluster using the ANI and AF criteria. These steps are repeated until all genomes have been assigned to a species.

We also use the alignment fraction (AF; that is, percentage of

orthologous regions shared between two genomes) as an additional

threshold for circumscribing species18,22,25 to ensure that ANI values

are not based on a small set of conserved genes.

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Taxogenómica

¿Por qué la GTDB es polémica?

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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Taxogenómica

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

Of the 6 phyla, 14 classes, 41 orders, 90 families, and 247 genera examined ,we found that 85% of ranks fell within ±0.1 of the median RED value for taxa at that rank, suggesting they had comparable levels of evolutionary divergence.

Saccharomycotina

¿Qué se ha dicho de la GTDB?

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“Despite claiming that 'Nothing in this Code may be construed to restrict the freedom of taxonomic thought or action', the ICNP is very particular about what counts as type material in naming a species: only living pure cultures meet the requirement an ironic contrast to the […] Code […] for algae, fungi, and plants, which requires that – type material be dead or at least inert! […] organisms grown in pure culture, at best, provide an incomplete view of the natural world and, at worst, represent a laboratory artefact”

“Uncultured organisms should have their own names and that genome sequences should be acceptable as type material […] one day, naming Bacteria and Archaea might be as easy as using Google Translate”

Taxogenómica

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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Taxogenómica

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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@joaocpmorais

�

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≠

Cucaracha (Blattodea)

Termita (Isoptera)

Chino

Himalaya

Taxogenómica

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

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Taxogenómica

Contacto: gamamiguelangel@gmail.com | Twitter: miangoar | GitHub pages: miangoar.github.io

¿what portion of global biodiversity

has been sequenced?

goat.genomehubs.org

Metazoa, Viridiplantae, Fungi