ParSNP HashParSNP Hash
Pipeline to parse SNP data and outputsummary statistics across slidingwindowsPipeline to parse SNP data and outputsummary statistics across slidingwindows
ObjectiveObjective
•Parse VCF files•Parse VCF files
•Calculate summary statistics across sliding windowsthroughout the genome•Calculate summary statistics across sliding windowsthroughout the genome
•Implement NTFreq module to calculate nucleotidefrequencies for each population and combinedpopulation•Implement NTFreq module to calculate nucleotidefrequencies for each population and combinedpopulation
•Implement TajimasD module to calculate Tajima’s D•Implement TajimasD module to calculate Tajima’s D
•Implement GO module to annotate identified SNPs•Implement GO module to annotate identified SNPs
Data setData set
•Simulated data set for chromosome 2R inDrosophila melanogaster•Simulated data set for chromosome 2R inDrosophila melanogaster
•1.4 Mbp•1.4 Mbp
–2 populations–2 populations
•Pooled individuals per population•Pooled individuals per population
–75bp reads, error rate 1%–75bp reads, error rate 1%
–10,000 simulated SNPs–10,000 simulated SNPs
•100x coverage per variant•100x coverage per variant
•At least 100bp apart•At least 100bp apart
•Allelic Frequencies ranging from .1 to .9 per population•Allelic Frequencies ranging from .1 to .9 per population
Data to Variant Call FormatData to Variant Call Format
Index Reference GenomeIndex Reference Genome
Only chromosome 2R of D. melanogasterOnly chromosome 2R of D. melanogaster
-Genome build Dmel 3 from Flybase-Genome build Dmel 3 from Flybase
Use BWA to Align FastQ to Reference GenomeUse BWA to Align FastQ to Reference Genome
Gap open penalty = 1Disallowing deletion within 12 bp of 3’UTRGap open penalty = 1Disallowing deletion within 12 bp of 3’UTR
Gap extension max = 12Maximum level of gap extensions = 12 Gap extension max = 12Maximum level of gap extensions = 12
Use SAMTools to Remove Ambiguously mapped RegionsUse SAMTools to Remove Ambiguously mapped Regions
(MAPQ >= 20) (MAPQ >= 20)
Use BCFTools mpileup to Generate a Binary Code Format (BCF)Use BCFTools mpileup to Generate a Binary Code Format (BCF)
BCF -> VCFBCF -> VCF
FastQ -> sai -> SAM -> BAM - > .bcf -> VCF
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Formatting data: Parse VCFFormatting data: Parse VCF
For each window:For each window:
•Fetch the VCF rows from each BCF file•Fetch the VCF rows from each BCF file
•Convert the VCF rows into hashes of arrays•Convert the VCF rows into hashes of arrays
•Compute the Theta, Pi, Tajima’s D for eachpopulation•Compute the Theta, Pi, Tajima’s D for eachpopulation
•Compute Fst for each window between eachpopulation•Compute Fst for each window between eachpopulation
Sliding windowsSliding windows
•Sliding window size is specified, and calledmodules are calculated across specifiedwindow size•Sliding window size is specified, and calledmodules are calculated across specifiedwindow size
Module 1: Calculate allele frequenciesModule 1: Calculate allele frequencies
•Input is taken from parsed VCF file•Input is taken from parsed VCF file
•Hashes are created for each population withthe following structure•Hashes are created for each population withthe following structure
–{SNP_location} {nucleotide} -> frequency;–{SNP_location} {nucleotide} -> frequency;
•Hashes created for full dataset•Hashes created for full dataset
–{SNP_location}{Population} -> {nucleotide} ->frequency–{SNP_location}{Population} -> {nucleotide} ->frequency
Output site frequency spectraOutput site frequency spectra
•Site frequency spectrum (SFS) output as thefollowing hash:•Site frequency spectrum (SFS) output as thefollowing hash:
–{nonref_allele}{frequency}->count;–{nonref_allele}{frequency}->count;
•Allows us to calculate a histogram for the non-reference allele frequencies•Allows us to calculate a histogram for the non-reference allele frequencies
•Send output to R to generate SFS graphs•Send output to R to generate SFS graphs
Module 2: Calculate SummaryStatistics and Tajima’s DModule 2: Calculate SummaryStatistics and Tajima’s D
•theta_pi (index of diversity)•theta_pi (index of diversity)
•theta_watterson (index of diversity)•theta_watterson (index of diversity)
Module 2: Calculate SummaryStatistics and Tajima’s DModule 2: Calculate SummaryStatistics and Tajima’s D
•Tajima’s D (index of selection/populationexpansion)•Tajima’s D (index of selection/populationexpansion)
Module 3: FST for DNA sequenceModule 3: FST for DNA sequence
•Calculate FST (index of differentiation) accordingto Hudson et al. 1992•Calculate FST (index of differentiation) accordingto Hudson et al. 1992
1 – Hw/Hb1 – Hw/Hb
Hw: average number of differences withineach populationHw: average number of differences withineach population
Hb: average number of differences betweenthe 2 populationsHb: average number of differences betweenthe 2 populations
Module 4: GO annotationsModule 4: GO annotations
•Module takes SNP list as input•Module takes SNP list as input
•Outputs the following:•Outputs the following:
–List of genes that have overlap with SNP positions–List of genes that have overlap with SNP positions
–Gene Ontology (GO) IDs and terms associatedwith each SNP matched gene–Gene Ontology (GO) IDs and terms associatedwith each SNP matched gene
–List of genes for a selected window–List of genes for a selected window
•Visualization using GOSlim•Visualization using GOSlim
Data visualizationData visualization
•Integrated Genomics Viewer (IGV)•Integrated Genomics Viewer (IGV)
•Broad Institute•Broad Institute
•http://www.broadinstitute.org/igv/•http://www.broadinstitute.org/igv/
SFS for population 1 and 2SFS for population 1 and 2
Sliding window for summary statisticsSliding window for summary statistics
Phist greater than 0.1 in window 1080001 - 1100000
Go Accession IDOntologySpecific
GO:0000124Cellular ComponentSpt-Ada-Gcn5-acetyltransferase complex
GO:0005703Cellular Component(Thought to be a site of active transcription)
GO:0005634Cellular Component(Nucleus)
GO:0006911Biological ProcessPhagosome biosynthesis/formation
GO:0045747Biological ProcessUp regulation of Notch signaling pathway
GO:0006355Biological ProcessRegulation of cellular transcription, DNA-dependent
GO:0000910Biological Process(Cytoplasm division)
GO:0016773Molecular Function(Intermolecular transfer of phosphorus group to an alcohol group)
GO:0005700Cellular Component(Polytene associated)
GO:0005488Molecular Function(Ligand, non-covalent partner)
GO:0005737Cellular Component(Ambiguous)
GO:0035222Biological Process(Patterning in wing imaginal disc)
GO:0005875Cellular Component(Microtubule associated)
GO:0004672Molecular FunctionProtamine kinase activity
GO:0000123Cellular ComponentHistone acetylase complex
Identify differentiated genomic regionsIdentify differentiated genomic regions
•For each window with a Fst > 0.1, print thename of the SNP and associated GO term•For each window with a Fst > 0.1, print thename of the SNP and associated GO term
Phist (Fst) greater than 0.1 in window 1080001 - 1100000
Go Accession IDOntologySpecific
GO:0000124Cellular ComponentSpt-Ada-Gcn5-acetyltransferase complex
GO:0005703Cellular Component(Thought to be a site of active transcription)
GO:0005634Cellular Component(Nucleus)
GO:0006911Biological ProcessPhagosome biosynthesis/formation
GO:0045747Biological ProcessRegulation of cellular transcription, DNA-dependent
GO:0000910Biological Process(Cytoplasm division)
GO:0016773Molecular Function(Intermolecular transfer of phosphorus group to an alcoholgroup)GO:0005700Cellular Component(Polytene associated)
GO:0005488Molecular Function(Ligand, non-covalent partner)
GO:0005737Cellular Component(Ambiguous)
GO:0035222Biological Process(Patterning in wing imaginal disc)
GO:0005875Cellular Component(Microtubule associated)
GO:0004672Molecular FunctionProtamine kinase activity
GO:0000123Cellular ComponentHistone acetylase complex
Thank YouThank You
Use PERL or die , print “ (X_x)”;Use PERL or die , print “ (X_x)”;
##Hashes to Hashes####Hashes to Hashes##
Print “ % 2 %”;Print “ % 2 %”;
