Hello,
I would like your comments/suggestions for my strategy.
I have F0 samples with two different phenotype. I have F2 samples with unknown phenotype. I would like to create a library with homozygous F0 genotypes.
Then i would like to genotype my F2 samples using this previously created library.
I already pre-processed BAM files (I have all raw data if required) : bowtie2 and samtools.
STRATEGY: 1) Create genotype library with F0 samples:
- GATK HaplotypeCaller for both F0 phenotype samples : java -Xmx30g -jar GenomeAnalysisTK_3-8.jar -nct 16 -T HaplotypeCaller -R GENOME --emitRefConfidence GVCF -I INPUT.bam -o OUTPUT.g.vcf
- Merge the results: java -Xmx16g -jar GenomeAnalysisTK_3-8.jar -nt 16 -T GenotypeGVCFs -R GENOME --variant F0Variant1.g.vcf --variant F0Variant2.g.vcf -o Results_Merge_F0.vcf
- then i used a homemade script to select only position with homozygous genotype and different genotype between both F0 phenotype samples (like 1/1 for a F0 sample and 0/0 for the other one): Results_Merge_F0_filtered.vcf
2) Genotype F2 sample with the library:
- GATK HaplotypeCaller : java -Xmx30g -jar GenomeAnalysisTK_3-8.jar -nct 16 -T HaplotypeCaller -R GENOME --emitRefConfidence GVCF -I INPUT.bam -o OUTPUT.g.vcf -L Results_Merge_F0_filtered.vcf
- then i used a homemade script to identify genotype related to one (or the other) F0 phenotype.
BUUUUUUT ... At this last step i mostly got homozygous SNP for my F2 samples... I should get around 25% phenotype1 -- 25% phenotype2 -- 50% phenotype 1/2 I miss something but I don't know where.
Dear Nicolas,
So, you suppose to obtain segregation for F2 samples, generated from F0, in each of the tested loci. So, what I would do would be
1 - to identify homozygous SNP loci in first and then in the second F0 genotypes, excluding those common for both.
2 - At this step, you may have a kind of control step, checking SNP loci in F2 which were common from F0 samples. And you would expect such F2 loci also to be the same (it doesnot matter if they are homo or heterozygous).
3 - then to perform the comparison as you've decided to do by loci, checking which genotype is presented in F2 - F2 vs first F0, F2 vs second F0 etc, verifying whether you received close to 1:2:1.
4 - use Chisq to test the reliability of the results.
Hope my comments will help you
Good luck!!!
Thank you for your advices.
I will try theses steps using homemade Perl scripts.
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