TITLE: SNP Genotyping and ApoE Genotyping 
AUTHORS: Huw  Morris, Manuela MX Tan, Donald G Grosset, Nigel M Williams

[DESCRIPTION]
This protocol details the steps for DNA extraction from a human blood sample, quality control, and SNP and APOE genotyping. 

The protocol has been adapted from the PRoBaND SNP Genotyping and ApoE Genotyping Protocol.

The overall protocol for PRoBaND /Tracking Parkinson’s is published:

Malek, N., Swallow, D. M. A., Grosset, K. A., Lawton, M. A., Marrinan, S. L., Lehn, A. C., Bresner, C., Bajaj, N., Barker, R. A., Ben-Shlomo, Y., Burn, D. J., Foltynie, T., Hardy, J., Morris, H. R., Williams, N. M., Wood, N., & Grosset, D. G. (2015). Tracking Parkinson’s: Study Design and Baseline Patient Data. Journal of Parkinson’s Disease, 5(4), 947–959. https://doi.org/10.3233/JPD-150662

[BEFORE_START]
Check the planned procedure is covered by existing Research Ethics Committee.
Check that you have the correct PPE, supplies and consumables.

[GUIDELINES]
The 1000 genomes reference panel:

1000 Genomes Project Consortium, Auton, A., Brooks, L. D., Durbin, R. M., Garrison, E. P., Kang, H. M., Korbel, J. O., Marchini, J. L., McCarthy, S., McVean, G. A., & Abecasis, G. R. (2015). A global reference for human genetic variation. Nature, 526(7571), 68–74.https://www.nature.com/articles/nature15393




[STEPS]
SECTION: Quality Control and Principle Component Analysis
4. 
Perform standard quality control procedures in PLINK v1.9: https://www.cog-genomics.org/plink/

SECTION: Quality Control and Principle Component Analysis
7. Remove:

SECTION: Sample Collection and Genotyping
1. At study entry (baseline), collect a 4 mL blood sample in an ethylenediaminetetraacetic acid (EDTA) vacutainer for DNA extraction. 
 

SECTION: Sample Collection and Genotyping
2. Perform DNA extraction. 

 

SECTION: Sample Collection and Genotyping
3. Genotype DNA samples using the Illumina HumanCore Exome array with custom content following manufacturers instructions : https://www.illumina.com/products/by-type/microarray-kits/infinium-exome.html

 


 

SECTION: Quality Control and Principle Component Analysis
5. Remove individuals:

SECTION: Quality Control and Principle Component Analysis
5. With low overall genotyping rates (< 98%), related individuals (Identity-By-Descent PIHAT > 0.1) and heterozygosity outliers (> 2 SDs away from the mean) 
SECTION: Quality Control and Principle Component Analysis
5.1. With low overall genotyping rates (< 98%), related individuals (Identity-By-Descent PIHAT > 0.1) and heterozygosity outliers (> 2 SDs away from the mean) 
SECTION: Quality Control and Principle Component Analysis
5.2. Whose clinically reported biological sex did not match the genetically determined sex. 
SECTION: Quality Control and Principle Component Analysis
6. Conduct principle component analysis on a linkage disequilibrium (LD) pruned set of variants (removing SNPs with an r2 > 0.05 in a 50kb sliding window shifting 5 SNPs at a time) after merging with European samples from the HapMap reference panel. 
SECTION: Quality Control and Principle Component Analysis
7.1. Individuals who are > 6 SDs away from the mean of any of the first 10 principal components. 
SECTION: Quality Control and Principle Component Analysis
7.2. Variants if they had a low genotyping rate (< 99%), Hardy-Weinberg Equilibrium p-value < 1 x 10-5, and minor allele frequency < 1%.
SECTION: 1,000 Genomes Project and APOE Genotyping
8. Following quality control, input genotypes separately to the 1,000 Genomes Project reference panel (phase 3 release 5) using the Michigan Imputation Server (https://imputationserver.sph.umich.edu).
SECTION: 1,000 Genomes Project and APOE Genotyping
10. Tracking Parkinson’s data was lifted over to genome build hg38 using liftOver (https://genome.ucsc.edu/cgi-bin/hgLiftOver). 


 

SECTION: 1,000 Genomes Project and APOE Genotyping
9. Retain only variants with imputation quality >0.8, to keep only high quality calls to merge across the cohorts. 
SECTION: 1,000 Genomes Project and APOE Genotyping
11. Recalculate the genetic principal components after removing outliers, as extreme outliers can substantially affect the calculation of genetic principal components. 
SECTION: 1,000 Genomes Project and APOE Genotyping
11.1. These first 5 new principal components are included as covariates in future GWAS to adjust for population substructure
SECTION: 1,000 Genomes Project and APOE Genotyping
11.2. Additional outliers who are > 6 SDs away from the mean of any of the first 5 principal components are excluded.
SECTION: 1,000 Genomes Project and APOE Genotyping
12. Restrict genotyping of APOE to samples and SNPs that survive these QC procedures based on two common SNPs of the APOE gene: 388 T > C (rs429358) and 526C > T (rs7412). 
SECTION: 1,000 Genomes Project and APOE Genotyping
13. The three haplotypes (ɛ2(388 T–526 T), ɛ3(388 T-526C), ɛ4(388C-526C)) and six genotypes (ɛ2/ɛ2, ɛ2/ɛ3, ɛ2/ɛ4, ɛ3/ɛ3, ɛ3/ɛ4, ɛ4/ɛ4) formed by these SNPs are then used to determine ɛ4 status in each sample.