Explore SNP polymorphism data. A. Dereeper, Y. Hueber
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1 Explore SNP polymorphism data A. Dereeper, Y. Hueber Bioinformatics trainings, Supagro, February, 2016
2 Tablet Graphical tool to visualize assemblies Accept many formats ACE, SAM, BAM
3 GATK (Genome Analysis ToolKit) So>ware package to analyse NGS data. Implemented to analyse human resequencing data, for medical purpose (1000 genomes, The Cancer Genome Atlas) Includes depth analyses, quality score recalibralon, SNP/InDel deteclon 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)
4 Fastq (RC1) Fastq (RC2) Fastq (RC3) Fastq (RC4) Cutadapt Mapping BWA Cutadapt Cutadapt Cutadapt Mapping BWA Mapping BWA Mapping BWA. Add or Replace Groups Add or Replace Groups Add or Replace Groups Add or Replace Groups BAM with read group BAM with read group BAM with read group BAM with read group mergesam Global BAM with read group VCF file
5 For GBS data Tassel pipeline Version 5 TASSEL- GBS Plos One, 2014
6 GBS RAD- Seq RNA- Seq WGRS Reads pre- processing and mapping + SNP Calling and genotype assignalon Tassel Galaxy workflow Genotyping data Storage and mining Genotyping data analyses and visualizalon (GWAS, diversity )
7 Format VCF (Variant Call Format) Advantages: VariaLon descriplon for each posilon + genotype assignalons Indexed flat files. Binary files also exist: BCF format
8 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 so>wares such as Varscan (varscan pileup2snp) - Frequently used for rare variants, with a low frequency (e.g. viral pop)
9 Autres Other GATK fonctionalités functionalities GATK Module DepthOfCoverage: Allows to get sequencing depth for each gene, each posilon and each individual Module ReadBackedPhasing: Allows to set, if possible, associalons between alleles (phase and haplotypes) when we are in an heterozygote situalon. Et non AGG GGA
10 Haplotypes and phasing Haplotype: Specific groups of genes or alleles that progeny inherited from one parent Phasing: DeterminaLon of haplotype phase. Process of stalslcal eslmalon of haplotypes from genotype data. Can be infered by stalslcs methods using non- ambigous haplotypes present in the dataset (Gevalt, ShapeIT, Phase) Can be resolved using physical associalon of alleles within the reads (GATK ReadBackedPhasing, GATK HaplotypeCaller)
11 Projet Gigwa, pour la gestion des données massives de variants (GBS, RADSeq, WGRS) «With NGS arise serious computalonal challenges in terms of storage, search, sharing, analysis, and data visualizalon, that redefine some praclces in data management.» - Based on NoSQL technology - Handles VCF files (Variant Call Format) and annotalons - Supports mullple variant types: SNPs, InDels, SSRs, SV - Powerful genotyping queries - Easily scalable with MongoDB sharding - Transparent access - Takes phasing informalon into account when imporlng/exporlng in VCF format
12 hep://gigwa.southgreen.fr/gigwa/
13 SNiPlay: Web application for polymorphism analyses hep://sniplay.southgreen.fr
14 IFB project Galaxy4Sniplay (WP4 IFB, Plant node)
15 Upload a VCF file in SNiPlay hep://sniplay.southgreen.fr Upload a VCF file (+ reference if not available in genome collec@on) Select rice genome The reference corresponce to mrna A. Dereeper, Y. Hueber Bioinformatics trainings, Supagro, February, 2016
16 Filters using VCFtools or Gigwa Maf Missing data AnnotaLon PosiLon
17 SNP annotation using SnpEff It annotates and predicts the effects of variants on genes (amino acid changes ) Uses as input GFF annoalon file and VCF
18
19 structure (snmf, Admixture) Test different values of K (es.mates the probability (likelihood tests) that samples are structured in K popula.ons) For the best value of K, the applicalon shows Q eslmates for each individual (admixture percent) (probability that the individual belongs to each popula.on)
20 MDS (MulL- Dimensional Scale) plot SNP- based Distance tree with FastME
21 Pi: NucleoLde diversity: Average number of nucleolde differences per site between any two DNA sequences chosen randomly from the sample populalon Used to measure the degree of polymorphism within a populalon Diversity analysis Comparison between individuals Fst: FixaLon index: measure of populalon differenlalon due to genelc structure.
22 SNP density by individuals can allow the deteclon of introgression event. 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
23 Haplotypes Haplotype reconstruclon using Gevalt Network with Haplophyle Available only for regions presenlng few variants (short regions, genes) Exploit phased VCF (in progress ) High frequency haplotypes Group distribution whithin this haplotype Low frequency haplotype Distance between 2 haplotypes (nb of mutations)
24 GWAS (Genome-Wide Association Studies) EsLmate associalon between a marker and a phenotypic character Manhaean plots: displays GWAS stalslcal tests (- log10 pvalue) along chromosomes TASSEL, MLMM sofwares False posilves because of the studied structuralon panel => correclon using structure populalon et and kinship
25 GWAS issues Choice of genotypic panel: phenotypic diversity for target traits must be sufficient (core- colleclon, MAGIC lines, NAM ) Popula@on structure induces high rates of false associalons (false posilves) CorrecLon using structure populalon et and kinship. Mixed models: o Q o K (widely used) o Q+K (widely used) Density of markers must be enough to provide a good genome cover. Density can be also highly variable. Linkage disequilibrium (LD) landscape: level of intra- and inter- chromosomal LD (number of loci in LD with loci from other chromosomes). Ideally, LD profile must be flat to avoid distorsion in associalon paeerns.
26 TD: Study of root characters using GWAS in Oryza sativa japonica. Influence of a correction using structure and kinship
27 Relatedness between individuals (kinship matrix) TASSEL and plink so>wares EsLmaLon of relatedness between individuals using a distance matrix
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