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A. Dereeper, G. Sarah, F. Sabot
Explore SNPpolymorphism data
Bioinformatics trainings, Vietnam Hanoi, November, 2015
Tablet
• Graphical tools to visualize assemblies
• Accept many formatsACE, SAM, BAM
A. Dereeper, G. Sarah, F. Sabot, Y. Hueber Formation Bio-informatique, 9 au 13 février 2015 A. Dereeper, G. Sarah, F. Sabot Bioinformatics trainings, Vietnam Hanoi, November, 2015
GATK (Genome Analysis ToolKit)
• Software package to analyse NGS data.
• Implemented to analyse human resequencing data, for medical purpose(1000 genomes, The Cancer Genome Atlas)
• Included: depth analyses, quality score recalibration, SNP/InDel detection
• Complementary with other packages: SamTools, PicardTools, VCFtools, BEDtools
PREPROCESS:
* Index human genome (Picard), we used HG18 from UCSC. * Convert Illumina reads to Fastq format * Convert Illumina 1.6 read quality scores to standard Sanger scores
FOR EACH SAMPLE:
1. Align samples to genome (BWA), generates SAI files. 2. Convert SAI to SAM (BWA) 3. Convert SAM to BAM binary format (SAM Tools) 4. Sort BAM (SAM Tools) 5. Index BAM (SAM Tools) 6. Identify target regions for realignment (Genome Analysis Toolkit) 7. Realign BAM to get better Indel calling (Genome Analysis Toolkit) 8. Reindex the realigned BAM (SAM Tools) 9. Call Indels (Genome Analysis Toolkit) 10. Call SNPs (Genome Analysis Toolkit) 11. View aligned reads in BAM/BAI (Integrated Genome Viewer)
A. Dereeper, G. Sarah, F. Sabot, Y. Hueber Formation Bio-informatique, 9 au 13 février 2015 A. Dereeper, G. Sarah, F. Sabot Bioinformatics trainings, Vietnam Hanoi, November, 2015
Global BAM with read group
Cutadapt
Mapping BWA
VCF file
Fastq (RC1)
BAM with read group
Mapping BWA
Fastq (RC2)
BAM with read group
Mapping BWA
Fastq (RC3)
BAM with read group
Mapping BWA
Fastq (RC4)
BAM with read group
….
mergeSam
Add or Replace GroupsAdd or Replace Groups Add or Replace Groups Add or Replace Groups
Cutadapt Cutadapt Cutadapt
TASSEL-GBSPlos One, 2014
A. Dereeper, G. Sarah, F. Sabot, Y. Hueber Formation Bio-informatique, 9 au 13 février 2015 A. Dereeper, G. Sarah, F. Sabot Bioinformatics trainings, Vietnam Hanoi, November, 2015
For GBS data
Tassel pipelineVersion 5
TasselSNP Calling and
genotype assignation
Genotyping dataStorage and mining
Genotyping data analyses and visualization
(GWAS, diversity…)
GBSRAD-Seq
RNA-SeqWGRS
Galaxyworkflow
Reads pre-processing and mapping
+
Format VCF (Variant Call Format)Advantages: Variation description for each position + genotype assignationsIndexed flat files. Binary files also exist: BCF format
A. Dereeper, G. Sarah, F. Sabot, Y. Hueber Formation Bio-informatique, 9 au 13 février 2015 A. Dereeper, G. Sarah, F. Sabot Bioinformatics trainings, Vietnam Hanoi, November, 2015
Autres fonctionalités GATK• Module DepthOfCoverage:Allows to get sequencing depth for each gene, each position and each individual
• Module ReadBackedPhasing:Allows to set, if possible, associations between alleles (phase and haplotypes) when we are in an heterozygote situation.
Et non AGGGGA
A. Dereeper, G. Sarah, F. Sabot, Y. Hueber Formation Bio-informatique, 9 au 13 février 2015
Other GATK functionalities
A. Dereeper, G. Sarah, F. Sabot Bioinformatics trainings, Vietnam Hanoi, November, 2015
Format Pileup
- Another format for variant calling (generated by samtools)- Describe alignment row by row (not line by line like in SAM format)- Used by VarScan like softwares (varscan pileup2snp)- Frequently used for rare variants, with a low frequency (e.g. viral pop)
A. Dereeper, G. Sarah, F. Sabot, Y. Hueber Formation Bio-informatique, 9 au 13 février 2015 A. Dereeper, G. Sarah, F. Sabot Bioinformatics trainings, Vietnam Hanoi, November, 2015
A. Dereeper, G. Sarah, F. Sabot, Y. Hueber Formation Bio-informatique, 9 au 13 février 2015
- Based on NoSQL technology
- Handles VCF files (Variant Call Format) and annotations
- Supports multiple variant types: SNPs, InDels, SSRs, SV
- Powerful genotyping queries
- Easily scalable with MongoDB sharding
- Transparent access
- Takes phasing information into account when importing/exporting in VCF format
Projet Gigwa, pour la gestion des données massives de variants (GBS, RADSeq, WGRS)
« With NGS arise serious computational challenges in terms of storage, search, sharing, analysis, and data visualization, that redefine some practices in data management. »
A. Dereeper, G. Sarah, F. Sabot Bioinformatics trainings, Vietnam Hanoi, November, 2015
A. Dereeper, G. Sarah, F. Sabot, Y. Hueber Formation Bio-informatique, 9 au 13 février 2015
http://gigwa.southgreen.fr/gigwa/
A. Dereeper, G. Sarah, F. Sabot Bioinformatics trainings, Vietnam Hanoi, November, 2015
SNiPlay: Web application for polymorphism analyses
http://sniplay.southgreen.fr
A. Dereeper, G. Sarah, F. Sabot, Y. Hueber Formation Bio-informatique, 9 au 13 février 2015 A. Dereeper, G. Sarah, F. Sabot Bioinformatics trainings, Vietnam Hanoi, November, 2015
• IFB project “Galaxy4Sniplay” (WP4 IFB, Plant node)
A. Dereeper, G. Sarah, F. Sabot, Y. Hueber Formation Bio-informatique, 9 au 13 février 2015 A. Dereeper, G. Sarah, F. Sabot Bioinformatics trainings, Vietnam Hanoi, November, 2015
• Available using Galaxy Toolshed Installable on any Galaxy instance
A. Dereeper, G. Sarah, F. Sabot, Y. Hueber Formation Bio-informatique, 9 au 13 février 2015 A. Dereeper, G. Sarah, F. Sabot Bioinformatics trainings, Vietnam Hanoi, November, 2015
Upload a VCF file in SNiPlay
Upload a VCF file (+ reference if not available in genome collection)
Select rice genome
The reference corresponce to mRNA
A. Dereeper, G. Sarah, F. Sabot Bioinformatics trainings, Vietnam Hanoi, November, 2015
http://sniplay.southgreen.fr
• Filters using VCFtools or Gigwa
• Maf• Missing data• Annotation• Position…
A. Dereeper, G. Sarah, F. Sabot, Y. Hueber Formation Bio-informatique, 9 au 13 février 2015 A. Dereeper, G. Sarah, F. Sabot Bioinformatics trainings, Vietnam Hanoi, November, 2015
SNP annotation using SnpEff
A. Dereeper, G. Sarah, F. Sabot, Y. Hueber Formation Bio-informatique, 9 au 13 février 2015 A. Dereeper, G. Sarah, F. Sabot Bioinformatics trainings, Vietnam Hanoi, November, 2015
A. Dereeper, G. Sarah, F. Sabot, Y. Hueber Formation Bio-informatique, 9 au 13 février 2015 A. Dereeper, G. Sarah, F. Sabot Bioinformatics trainings, Vietnam Hanoi, November, 2015
sNMF
• Test different values of K (estimates the probability (likelihood tests) that samples are structured in K populations)
• For the best value of K, the application shows Q estimates for each individual (admixture percent)(probability that the individual belongs to each population)
MDS (Multi-Dimensional Scale) plot
SNP-based Distance tree with FastME
A. Dereeper, G. Sarah, F. Sabot, Y. Hueber Formation Bio-informatique, 9 au 13 février 2015 A. Dereeper, G. Sarah, F. Sabot Bioinformatics trainings, Vietnam Hanoi, November, 2015
Comparison between individuals
Diversity analysis
Pi: Nucleotide diversity: Average number of nucleotide differences per site between any two DNA sequences chosen randomly from the sample populationUsed to measure the degree of polymorphism within a population
A. Dereeper, G. Sarah, F. Sabot, Y. Hueber Formation Bio-informatique, 9 au 13 février 2015 A. Dereeper, G. Sarah, F. Sabot Bioinformatics trainings, Vietnam Hanoi, November, 2015
=> Can allows the detection of introgression
Introgression = Movement of a exogene region (gene flow) from one species into the gene pool of another by the repeated backcrossing of an interspecific hybrid with one of its parent species
Widely used in agronomy obtained but can occurs naturally
High frequency haplotypes
Low frequency haplotype
Group distribution whithin this haplotype
Distance between 2 haplotypes (nb of mutations)
Haplotypes• Haplotype reconstruction using Gevalt
• Network with Haplophyle
• Available only for regions presenting few variants (short regions, genes)
• Exploit phased VCF (in progress…)
A. Dereeper, G. Sarah, F. Sabot, Y. Hueber Formation Bio-informatique, 9 au 13 février 2015 A. Dereeper, G. Sarah, F. Sabot Bioinformatics trainings, Vietnam Hanoi, November, 2015
Cartesian coordinates
Genotypage file
Fichier de soumission pour Illumina
Analyse with BeadStudio software
Design de puces Illumina
A. Dereeper, G. Sarah, F. Sabot, Y. Hueber Formation Bio-informatique, 9 au 13 février 2015
Illumina ship design Submission file for Illumina
A. Dereeper, G. Sarah, F. Sabot Bioinformatics trainings, Vietnam Hanoi, November, 2015
A. Dereeper, G. Sarah, F. Sabot, Y. Hueber Formation Bio-informatique, 9 au 13 février 2015
• Estimate association between a marker and a phenotypic character
• Manhattan plots: displays GWAS statistical tests(-log10 pvalue) along chromosomes
• TASSEL, MLMM sofwares
• False positives because of the studied structuration panel=> correction using structure population et and kinship
GWAS (Genome-Wide Association Studies)
A. Dereeper, G. Sarah, F. Sabot Bioinformatics trainings, Vietnam Hanoi, November, 2015
A. Dereeper, G. Sarah, F. Sabot, Y. Hueber Formation Bio-informatique, 9 au 13 février 2015
TD: Study of root charaters using GWAS in Oryza sativa japonica. Influence of a correction using structure and kinship
A. Dereeper, G. Sarah, F. Sabot Bioinformatics trainings, Vietnam Hanoi, November, 2015
A. Dereeper, G. Sarah, F. Sabot, Y. Hueber Formation Bio-informatique, 9 au 13 février 2015
Analyse de structure de populations
• Test different values of K (estimates of probability that samples are structured in K populations)
• For the best value of K, the application shows Q estimates for each individual(admixture percent)
Population structure analysis
A. Dereeper, G. Sarah, F. Sabot Bioinformatics trainings, Vietnam Hanoi, November, 2015
A. Dereeper, G. Sarah, F. Sabot, Y. Hueber Formation Bio-informatique, 9 au 13 février 2015
Relatedness between individuals (kinship matrix)
• TASSEL and plink softwares
• Estimation of relatedness between individuals using a distance matrix
A. Dereeper, G. Sarah, F. Sabot Bioinformatics trainings, Vietnam Hanoi, November, 2015