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ANOMALY: AmplicoN wOrkflow for Microbial community AnaLYsis

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HAL Id: hal-02340484

https://hal.archives-ouvertes.fr/hal-02340484

Submitted on 5 Jun 2020

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ANOMALY: AmplicoN wOrkflow for Microbial community AnaLYsis

Sébastien Theil, Etienne Rifa

To cite this version:

Sébastien Theil, Etienne Rifa. ANOMALY: AmplicoN wOrkflow for Microbial community AnaLYsis.

5. Colloque de Génomique Environnementale, Oct 2019, La Rochelle, France. �hal-02340484�

(2)

ANOMALY :

AMPLICON WORKFLOW FOR MICROBIAL COMMUNITY ANALYSIS

UMRF

20, côte de Reyne 15000 AURILLAC

https://www6.ara.inra.fr/umrf

04.71.45.64.10

● Amplicon sequence variant allows more resolution in taxonomic unit detection

● Double taxonomic assignment with environment dedicated and general database increases accuracy

● Contamination control reduces interpretation errors

● Integrated statistical analysis allowing reproducibility, all global analysis in one tool.

● Constant development with regular updates.

https://forgemia.inra.fr/umrf/anomaly

Sebastien Theil 1 , Etienne Rifa 1

1 Université Clermont Auvergne, INRA, VetAgro Sup, UMRF Unité Mixte de Recherche sur le Fromage, 20 côte de Reyne, 15000 Aurillac, France

Robjects

ITS 16S

DESeq2

metacoder metagenomeSeq 7

3

4 2

3 1 10

Phyloseq Object ASV counts

Metadata Taxonomy Sequences

Tree

Context Features

Conclusions ANOMALY

Available on repository at :

one-way, two-ways ANOVA,

PERMANOVA, Non parametric

tests

Bioinformatic tools for amplicon sequencing data analysis are continuously and rapidly evolving, thus integrating most recent techniques and tools is challenging.

We present ANOMALY, an R workflow for 16S and ITS amplicons sequencing. This workflow is based on several Rscripts using mainly DADA2 and phyloseq packages amongst others. This workflow allows to perform an analysis from raw fastq sequence files to final statistical analysis. The objective was to automate bioinformatic analyses to ensure reproducibility between projects and trying to be versatile and simple enougth to integrate new bioinformatic tools or statistical techniques.

● All integrated analyse 16S & ITS in R

● DADA2 Amplicon Sequence Variant

● Decontamination from control samples (decontam)

● Filtering : frequency, prevalence

● Taxonomic assignment from 2 references with validation

● Integrated statistics and visualization

● Multiple differential analyse

● Versatility (easy to integrate new features)

● Use well established methods

FASTQ FASTQ

FASTQ

Quality filtering

&

Inferring ASVs

Taxonomic Assignment

Merge assignation from two databases

ITS

16S

UTOPIA

Amplification

&

Illumina Sequencing

FASTA TSV

Fasta ASV sequences

&

Raw ASV table IDTAXA

Phylogenetic tree

Phangorn package

Neighbour joining

DECONTAM

● Control versus sample

● Control prevalence

● DNA concentration

● Prevalence

● Frequency

Visualization Bar plot Rarefaction curve Alpha diversity Beta diversity

DESeq2 Metacoder Boxplot

STAMP

MetagenomeSeq Aggregate analysis

Zero-inflated log-normal model with Cumulative Sum Scaling Normalization Negative binomial generalized

linear models with Variance Statbilizing Transformation

Interactive statistical analysis and visualization, with total sum

scaling normalization

Non parametric methods for differential analysis, with different normalization methods

Richness, Shannon, Simpson indexes. BrayCurtis, Unifrac, weightedUnifrac distances plotted with PCoA andd NMDS Sampling effort checking

Community distribution overview

Regrouping in tabulated file differential abundant features detected by the different methods, with taxonomy and

sequences

Differential analysis

ANOMALY uses latest approved methods like :

Références

Télécharger maintenant ( PDF - 2 Page - 1.26 MB )

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