1 / 17

Comprehensive SNP Analysis Pipeline for Drosophila melanogaster Using VCF Data

This project details the development of a SNP analysis pipeline specifically designed for Drosophila melanogaster. The pipeline processes VCF files, calculates genomic summary statistics across user-defined sliding windows, and implements modules to assess nucleotide frequencies and Tajima’s D across populations. Using simulated data for chromosome 2R, the pipeline outputs key metrics like allele frequencies, Fst values, and GO annotations for identified SNPs. Furthermore, it visualizes the results using Integrated Genomics Viewer (IGV) to enhance the understanding of genetic differentiation and selection.

ramya
Télécharger la présentation

Comprehensive SNP Analysis Pipeline for Drosophila melanogaster Using VCF Data

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. ParSNP Hash Pipeline to parse SNP data and output summary statistics across sliding windows

  2. Objective • Parse VCF files • Calculate summary statistics across sliding windows throughout the genome • Implement NTFreq module to calculate nucleotide frequencies for each population and combined populations • Implement TajimasD module to calculate Tajima’s D • Implement GO module to annotate identified SNPs

  3. Data set • Simulated data set for chromosome 2R in Drosophila melanogaster • 1.4 Mbp • 2 populations • Pooled individuals per population • 75bp reads, error rate 1% • 10,000 simulated SNPs • 100x coverage per variant • At least 100bp apart • Allelic Frequencies ranging from .1 to .9 per population

  4. FastQ -> sai -> SAM -> BAM - > .bcf -> VCF Data to Variant Call Format Index Reference Genome Only chromosome 2R of D. melanogaster -Genome build Dmel 3 from Flybase Use BWA to Align FastQ to Reference Genome Gap open penalty = 1 Disallowing deletion within 12bp of 3’UTR Gap extension max = 12 Maximum level of gap extensions = 12 Seeding length of 35 bp Use SAMTools to Remove Ambiguously mapped Regions (MAPQ >= 20) Use BCFToolsmpileup to Generate a Binary Code Format (BCF) BCF -> VCF

  5. Formatting data: Parse VCF For each window: • Fetch the VCF rows from each BCF file • Convert the VCF rows into hashes of arrays • Compute the Theta, Pi, Tajima’s D for eachpopulation • Compute Fst for each window between each population

  6. Sliding windows • Sliding window size is specified by user • Called modules are calculated across specified window size

  7. Module 1: Calculate allele frequencies • Input is taken from parsed VCF file • Hashes are created for each population with the following structure • {SNP_location} {nucleotide} -> frequency; • Hashes created for full dataset • {SNP_location}{Population} -> {nucleotide} -> frequency

  8. Output site frequency spectra • Site frequency spectrum (SFS) output as the following hash: • $SFS{frequency}->count; • Allows us to calculate a histogram for the non-reference allele frequencies • Send output to R to generate SFS graphs

  9. Module 2: Calculate Summary Statistics and Tajima’s D • theta_pi (index of diversity) • theta_watterson (index of diversity)

  10. Module 2: Calculate Summary Statistics and Tajima’s D • Tajima’s D (index of selection/population expansion)

  11. Module 3: FST for DNA sequence • Calculate FST (index of differentiation) according to Hudson et al. 1992 1 – Hw/Hb Hw: average number of differences within each population Hb: average number of differences between the 2 populations

  12. Module 4: GO annotations • Module takes SNP list as input • Outputs the following: • List of genes that have overlap with SNP positions • Gene Ontology (GO) IDs and terms associated with each SNP matched gene • List of genes for a selected window • Visualization using GOSlim

  13. Data visualization • Integrated Genomics Viewer (IGV) • Broad Institute • http://www.broadinstitute.org/igv/

  14. SFS for population 1 and 2

  15. Sliding window for summary statistics Phist greater than 0.1 in window 1080001 - 1100000 Go Accession ID Ontology Specific GO:0000124 Cellular Component Spt-Ada-Gcn5-acetyltransferase complex GO:0005703 Cellular Component (Thought to be a site of active transcription) GO:0005634 Cellular Component (Nucleus) GO:0006911 Biological Process Phagosome biosynthesis/formation GO:0045747 Biological Process Up regulation of Notch signaling pathway GO:0006355 Biological Process Regulation of cellular transcription, DNA-dependent GO:0000910 Biological Process (Cytoplasm division) GO:0016773 Molecular Function (Intermolecular transfer of phosphorus group to an alcohol group) GO:0005700 Cellular Component (Polytene associated) GO:0005488 Molecular Function (Ligand, non-covalent partner) GO:0005737 Cellular Component (Ambiguous) GO:0035222 Biological Process (Patterning in wing imaginal disc) GO:0005875 Cellular Component (Microtubule associated) GO:0004672 Molecular Function Protaminekinase activity GO:0000123 Cellular Component Histoneacetylase complex

  16. Identify differentiated genomic regions • For each window with a Fst > 0.1, print the name of the SNP and associated GO term Phist (Fst) greater than 0.1 in window 1080001 - 1100000 Go Accession ID Ontology Specific GO:0000124 Cellular Component Spt-Ada-Gcn5-acetyltransferase complex GO:0005703 Cellular Component (Thought to be a site of active transcription) GO:0005634 Cellular Component (Nucleus) GO:0006911 Biological Process Phagosome biosynthesis/formation GO:0045747 Biological Process Regulation of cellular transcription, DNA-dependent GO:0000910 Biological Process (Cytoplasm division) GO:0016773 Molecular Function (Intermolecular transfer of phosphorus group to an alcohol group)GO:0005700 Cellular Component (Polytene associated) GO:0005488 Molecular Function (Ligand, non-covalent partner) GO:0005737 Cellular Component (Ambiguous) GO:0035222 Biological Process (Patterning in wing imaginal disc) GO:0005875 Cellular Component (Microtubule associated) GO:0004672 Molecular Function Protaminekinase activity GO:0000123 Cellular Component Histoneacetylase complex

  17. Thank You Use PERLordie,print“ (X_x)”; ##Hashes to Hashes## Print“ % 2 %”; __END__

More Related