SNP Calling. Tuesday 4/21/15

Transcription

1 SNP Calling Tuesday 4/21/15

2 Why Call SNPs? map mutations, ex: EMS, natural variation, introgressions associate with changes in expression develop markers for whole genome QTL analysis/ GWAS access diversity within/between species

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4 SNP Callers Samtools SOAPsnp FreeBayes Atlas-SNP2 GATK HaplotypeCaller Varscan GATK UnifiedGenotyper Dindel (indels)

5 only 19% ~28% for non-dbsnps agree!

6 Conclusion Moreover, among the four calling programs, GATK and Atlas-SNP2 show a relatively higher positive calling rate and sensitivity when compared to the others, and GATK tends to call more SNVs than Atlas-SNP2. Therefore, if users intend to use only one calling program, we recommend GATK. However, in order to increase the overall accuracy, we advocate for employing more than one SNP calling algorithms.

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8 org_broadinstitute_gatk_tools_walkers_haplotypecaller_haplotypecaller.php

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10 GATK Pipeline

11 Many tools including: Picard duplicate read tagging/removal Adding read group info

12 Exercise 1: run_snpcalling.sh 1. cp run_snpcalling.sh to ~/Desktop/ch4_demo_dataset 2. edit line 25: -targetintervals snps/realign.intervals 3. run, you may need to make it executable with chmod 755./run_snpcalling.sh

13 Exercise 1: run_snpcalling.sh 1. Merge all accepted_hits.bam into one file and then sort, use samtools 2. Mark duplicate reads from the sorted bam file using Picard MarkDuplicates 3. Add read groups using Picard AddOrReplaceReadGroups 4. Create a sequence dictionary using Picard CreateSequenceDictionary 5. Index the bam file from part 3 with samtools 6. Create targets for local realignment using GATK RealignerTargetCreator 7. Do realignment with GATK IndelRealigner 8. Call raw variants using GATK HaplotypeCaller

14 Exercise 1 Solutions 1. samtools merge all_hits.bam breaker/srr404334/srr404334_ch4_thout/accepted_hits.bam breaker/srr404336/ SRR404336_ch4_thout/accepted_hits.bam immature_fruit/srr404331/srr404331_ch4_thout/accepted_hits.bam immature_fruit/srr404333/srr404333_ch4_thout/accepted_hits.bam 2. samtools sort all_hits.bam all_hits_sort 3. java -jar /home/bioinfo/software/picard-tools-1.87/markduplicates.jar INPUT=all_hits.bam OUTPUT=all_hits_md.bam REMOVE_DUPLICATES=FALSE VALIDATION_STRINGENCY=SILENT ASSUME_SORTED=TRUE METRICS_FILE=markdups.metrics 4. java -jar /home/bioinfo/software/picard-tools-1.87/addorreplacereadgroups.jar INPUT=all_hits_md.bam OUTPUT=all_hits_md_rg.bam SORT_ORDER=coordinate RGID=1 RGLB=1 RGPL=illumina RGPU=run RGSM=pimpi RGCN=sra RGDS=pimpi_fruit RGDT=0 5. java -jar /home/bioinfo/software/picard-tools-1.87/createsequencedictionary.jar REFERENCE=bwt2_index/SL2.40ch04.fa OUTPUT=bwt2_index/SL2.40ch04.dict 6. samtools index all_hits_md_rg.bam 7. java -jar /home/bioinfo/software/genomeanalysistk.jar -T RealignerTargetCreator -R /home/bioinfo/desktop/ ch4_demo_dataset/bwt2_index/sl2.40ch04.fa -I all_hits_md_rg.bam -o realign.intervals 8. java -jar /home/bioinfo/software/genomeanalysistk.jar -T IndelRealigner -R bwt2_index/sl2.40ch04.fa -I all_hits_md_rg.bam -targetintervals realign.intervals -o all_hits_md_rg_realn.bam 9. java -jar /home/bioinfo/software/genomeanalysistk.jar -T HaplotypeCaller -R bwt2_index/sl2.40ch04.fa -I all_hits_md_rg_realn.bam -o all_hits_hapcall.vcf

15 SNP calling Exercise 2 1. Call SNPs from bam file and convert to vcf format $ samtools mpileup -C 50 -uf reference.fa alignment.bam bcftools view -bvcg - > raw_var.bcf mpileup computes the likelihood of data given each possible genotype and stores the likelihoods in the BCF format. It does not call variants. $ bcftools view raw_var.bcf vcfutils.pl varfilter -D 100 > filtered_var.vcf bcftools does the actual SNP calling, and converts the BCF to VCF run_snpcalling.sh already ran this

16 VCF Format

17 Exercise 3 1. Compare the GATK vcf file and the samtools vcf file using CombineVariants (run_snpcalling.sh tried to run this, but see if you can find the error) org_broadinstitute_gatk_tools_walkers_variantutil s_combinevariants.php 2. How many SNPs intersect?

18 Exercise 3 Solution 1. #Compare GATK and Samtools SNPs with combine variants 1. java -jar /home/bioinfo/software/ GenomeAnalysisTK.jar -T CombineVariants -R bwt2_index/sl2.40ch04.fa -o snps/ hapcall_vs_samtools_snps.vcf --variant:hapcall snps/ all_hits_hapcall.vcf --variant:samtools snps/snps/ samtools_snp_filt.vcfg 2. grep Intersect hapcall_all_hits_hapcall.vcvf 1. 6,647 Intersect

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20 SNP calling: effect prediction Exercise 4 SnpEff Read the manual! SnpEff is a variant annotation and effect prediction tool. It annotates and predicts the effects of genetic variants (such as amino acid changes). 1. Build a snpeff database for the reference genome 2. Use snpeff to determine if SNPs occur in genes

21 Exercise 4 1. Build snpeff database 1. run make_snpeff_db.sh 2. emacs /home/bioinfo/software/snpeff/snpeff.config change data.dir to ~/Software/snpEff/data and add: #Tomato ch04 SL2.40ch04.genome : SL2.40ch04 3.java -jar ~/Software/snpEff/snpEff.jar build -gtf22 -c snpeff.config -v SL2.40ch04

22 SNP calling: effect prediction 2. Use snpeff to determine if SNPs occur in genes. $ java -jar snpeff.jar eff SL2.40 snps.vcf -c snpeff.config -v > snpeff.out Exercise 5.out file has the snpeff stats snpeff_genes.txt : SNPs in genes (remember the genes.gtf file? ) snpeff_summary.html Look at the output and Count the number of genes with SNPs How many synonymous SNPs? How many are non-synonymous?

23 Exercise 5 Solution 1. Run snpeff 1. cd ~/Desktop/ch4_demo_dataset/ 2. java -jar /home/bioinfo/software/snpeff/snpeff.jar eff SL2.40ch04 snps/ hapcall_vs_samtools_snps.vcf -c /home/bioinfo/software/snpeff/ snpeff.config -v > snps/hapcall_vs_samtools_snps.snpeff.out 2. no. of genes with SNPs 1. awk '$7!= 0 {print $0}' snpeff_genes.txt wc 3. no. nonsynonymous: 1. grep "missense" hapcall_vs_samtools_snps.snpeff.out wc 4. non. synonymous: 1. grep "synonymous" hapcall_vs_samtools_snps.snpeff.out wc

24 Other Useful Tools Bedtools - useful for coverage assessment, ex: how many reads map to a genomic location. Used to detect copy number variation or structural variation like large deletions Breakdancer SVDetect Hydra Plink - QTL detection and analysis R - programming language useful for statistics and graphing of results