SNP Genotyping and ApoE Genotyping

Huw Morris, Manuela MX Tan, Donald G Grosset, Nigel M Williams

Published: 2022-08-05 DOI: 10.17504/protocols.io.by9ypz7w

Single Nucleotide Polymorphisms (SNPs)

Abstract

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.

Steps

Sample Collection and Genotyping

At study entry (baseline), collect a 4mL blood sample in an ethylenediaminetetraacetic acid (EDTA) vacutainer for DNA extraction.

Safety information

The process of venepuncture has the potential to expose research and clinical personnel and other health care professionals and participants to blood from other people, thereby putting them at risk from blood borne pathogens. In order to minimise risk personnel performing a venepuncture must have received appropriate training and deemed competent. This procedure, the use of PPE and the use and disposal of sharps etc., must be in accordance institution policies, approved risk assessments and procedures.

Perform DNA extraction.

Note

DNA extraction was carried out at DNA extraction was carried out at K biosciences, UK, UK

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

Note

This covered approximately 250,000 common variants, 250,000 rare variants, and over 27,000 custom variants that have been implicated in neurological and psychiatric disorders.Purcell, S., Neale, B., Todd-Brown, K., Thomas, L., Ferreira, M. A. R., Bender, D., Maller, J., Sklar, P., de Bakker, P. I. W., Daly, M. J., & Sham, P. C. (2007). PLINK: a tool set for whole-genome association and population-based linkage analyses. American Journal of Human Genetics, 81(3), 559–575. Purcell, S., Neale, B., Todd-Brown, K., Thomas, L., Ferreira, M. A. R., Bender, D., Maller, J., Sklar, P., de Bakker, P. I. W., Daly, M. J., & Sham, P. C. (2007). PLINK: a tool set for whole-genome association and population-based linkage analyses. American Journal of Human Genetics, 81(3), 559–575. https://doi.org/10.1086/519795

Note

Genotyping was performed Cardiff University: Genotyping was performed Cardiff University: https://www.cardiff.ac.uk/mrc-centre-neuropsychiatric-genetics-genomics

Quality Control and Principle Component Analysis

Perform standard quality control procedures in PLINK v1.9:https://www.cog-genomics.org/plink//

Remove individuals:

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)

5.2.

Whose clinically reported biological sex did not match the genetically determined sex.

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.

Remove:

7.1.

Individuals who are > 6 SDs away from the mean of any of the first 10 principal components.

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%.

1,000 Genomes Project and APOE Genotyping

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).

Retain only variants with imputation quality >0.8, to keep only high quality calls to merge across the cohorts.

10.

Tracking Parkinson’s data was lifted over to genome build hg38 using liftOver (https://genome.ucsc.edu/cgi-bin/hgLiftOver).

Note

Twenty genetic principal components are generated from a linkage-pruned SNP set (removing SNPs with an r2 > 0.02 in a 1000kb sliding window shifting 10 SNPs at a time).Extreme outliers are removed according to the first 5 principal components (> 6 SDs away from the mean).

11.

Recalculate the genetic principal components after removing outliers, as extreme outliers can substantially affect the calculation of genetic principal components.

11.1.

These first 5 new principal components are included as covariates in future GWAS to adjust for population substructure

11.2.

Additional outliers who are > 6 SDs away from the mean of any of the first 5 principal components are excluded.

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).

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.

SNP Genotyping and ApoE Genotyping

Abstract

Before start

Steps

Sample Collection and Genotyping

Quality Control and Principle Component Analysis

1,000 Genomes Project and APOE Genotyping

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