Bridges Lab Protocols - Recent changes [en]

Mendelian Randomization

Davebrid1 — Tue, 14 Apr 2026 01:21:00 GMT

Added link to STROBE-MR

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 This SOP outlines the standardized pipeline for conducting two-sample Mendelian Randomization (MR) analyses, from instrument selection through to causal inference adjudication.
 == 1. Genetic Instrument Selection ==
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 | '''Unresolved / Inconclusive'''. State that genetic evidence is too pleiotropic to reliably disentangle.
 |}

Mendelian Randomization

Davebrid1 — Tue, 14 Apr 2026 00:24:03 GMT

Generated intitial SOP for MR analyses

New page

= Mendelian Randomization Standard Operating Procedure (SOP) =

This SOP outlines the standardized pipeline for conducting two-sample Mendelian Randomization (MR) analyses, from instrument selection through to causal inference adjudication.

== 1. Genetic Instrument Selection ==
Instruments must be robustly associated with the exposure and independent of one another.

=== 1.1 Stringent Criteria (Primary Analysis) ===
For exposures with well-powered GWAS, apply the following strict inclusion criteria:
* '''P-value Threshold:''' <math>p < 5 \times 10^{-8}</math> (Genome-wide significance)
* '''Linkage Disequilibrium (LD) Clumping:''' <math>r^2 < 0.001</math>
* '''Clumping Distance:''' > 10,000 kb
* '''Reference Panel:''' 1000 Genomes European (or population-matched to the GWAS)

=== 1.2 Loose Criteria (Relaxed Analysis) ===
If the stringent criteria yield fewer than 5 Single Nucleotide Polymorphisms (SNPs), the analysis lacks the degrees of freedom required for standard sensitivity tests. In this case, relax the threshold:
* '''P-value Threshold:''' <math>p < 5 \times 10^{-6}</math>
* '''Requirement:''' If this relaxed threshold is used, '''MR-RAPS''' becomes a mandatory primary reporting model due to the guaranteed introduction of weak instruments and measurement error.

== 2. Estimating Instrument Strength ==
Before conducting MR, validate the strength of the genetic instruments to rule out weak instrument bias.

=== 2.1 Variance Explained (R-squared) ===
Calculate the proportion of variance in the exposure explained by each SNP using the Minor Allele Frequency (MAF) and the effect size (<math>\beta</math>):

<math>R^2 = 2 \times MAF \times (1 - MAF) \times \beta^2</math>

''Note: Review the average MAF. If the average MAF is very low (< 0.05), the instruments rely on rare variants, which may have less stable effect estimates.''

=== 2.2 F-Statistics ===
Calculate the individual F-statistic for each SNP to measure instrument strength, where <math>N</math> is the sample size of the exposure GWAS:

<math>F = \frac{R^2 \times (N - 2)}{1 - R^2}</math>

* '''Individual F-statistic:''' Any single SNP with <math>F < 10</math> should be excluded from the standard IVW analysis.
* '''Total/Mean F-statistic:''' Calculate the average F-statistic across all retained SNPs. If the Mean <math>F < 10</math>, the overall instrument is weak, and MR-RAPS must be prioritized.

== 3. Analytical Models to Test ==
Run the following suite of models to assess causality, heterogeneity, and horizontal pleiotropy.

=== 3.1 The Baseline Model ===
* '''Inverse Variance Weighted (IVW):''' The primary meta-analysis of all SNPs.
** Check Cochran's Q statistic. If <math>p > 0.05</math>, report the '''IVW Fixed Effects (FE)''' model.
** If Cochran's Q <math>p < 0.05</math>, switch to the '''IVW Multiplicative Random Effects (RE)''' model to account for heterogeneity.

=== 3.2 Standard Sensitivity Models (The Big Three) ===
* '''MR-Egger:''' Used to detect directional pleiotropy via the intercept.
* '''Weighted Median:''' Provides a valid estimate if up to 50% of the instrument weight comes from invalid (pleiotropic) SNPs.
* '''Weighted Mode:''' Provides a valid estimate if the largest single cluster of SNPs is valid (ZEro InSIDE assumption).

=== 3.3 Advanced Robustness Models ===
* '''MR-PRESSO:''' Run if Cochran's Q is significant. It detects and removes specific outlier SNPs driving horizontal pleiotropy and provides an outlier-corrected estimate.
* '''MR-RAPS:''' Run if Mean F < 10 or if loose inclusion criteria were used. It handles measurement error from weak instruments.
* '''CAUSE:''' Run as the ultimate robustness check to differentiate true causality from correlated pleiotropy (shared genetic architecture).

=== 3.4 Calculating I-squared GX for MR-Egger ===
To determine if the MR-Egger slope is reliable, you must test the No Measurement Error (NOME) assumption by calculating <math>I^2_{GX}</math>. If <math>I^2_{GX} < 0.90</math>, the MR-Egger slope suffers from dilution bias and should not be trusted.

<syntaxhighlight lang="R">
# R Script for calculating I^2_GX using a harmonised TwoSampleMR dataframe ('dat')

calculate_i2gx <- function(dat) {
beta_x <- dat$beta.exposure
se_x <- dat$se.exposure

# Variance of the exposure estimates
var_beta_x <- var(beta_x)

# Mean of the squared standard errors
mean_se_x2 <- mean(se_x^2)

# Calculate I^2_GX
I2gx <- 1 - (mean_se_x2 / var_beta_x)

# Constrain between 0 and 1
I2gx <- max(0, I2gx)

return(I2gx)
}

i2gx_value <- calculate_i2gx(dat)
print(paste("I^2_GX =", round(i2gx_value, 3)))
</syntaxhighlight>

== 4. Decision Matrix: Which Results to Report ==
Use the following adjudication criteria to synthesize the results from the models above.

{| class="wikitable"
|-
! Scenario !! Data Presentation !! Adjudicated Result to Report
|-
| '''1. Clean Signal'''
| F > 10. Cochran's Q p > 0.05. Egger Intercept p > 0.05. CAUSE Causal Model p < 0.05.
| '''IVW (Fixed Effects)'''. All assumptions are met.
|-
| '''2. Balanced Pleiotropy'''
| F > 10. Cochran's Q p < 0.05. Egger Intercept p > 0.05.
| '''MR-PRESSO (Outlier Corrected)''' or '''IVW (Random Effects)'''. Support with Weighted Median.
|-
| '''3. Weak Instruments'''
| Mean F < 10 OR Relaxed criteria (p < 5e-6) used.
| '''MR-RAPS'''. Baseline IVW is vulnerable to weak instrument bias.
|-
| '''4. Directional Pleiotropy'''
| Egger Intercept p < 0.05.
| '''Weighted Mode''' (if I2GX < 0.90) OR '''MR-Egger Slope''' (if I2GX >= 0.90). IVW is discarded.
|-
| '''5. Correlated Pleiotropy'''
| CAUSE model comparison p > 0.05 (Sharing Model wins).
| '''Null Result (Discard Causality)'''. The traits are correlated due to shared genetics, not a direct causal pathway. Any significant IVW results are false positives.
|-
| '''6. Inconclusive'''
| MR-PRESSO removes >30% of SNPs causing power loss, OR Median and Mode significantly contradict each other.
| '''Unresolved / Inconclusive'''. State that genetic evidence is too pleiotropic to reliably disentangle.
|}

LDLR Genotyping

Davebrid1 — Wed, 01 Apr 2026 12:40:56 GMT

Removed table of contents

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	Modified from https://www.jax.org/Protocol?stockNumber=002207&protocolID=27075		Modified from https://www.jax.org/Protocol?stockNumber=002207&protocolID=27075

			__NOTOC__

	* [[SOP - Ethidium Bromide]]		* [[SOP - Ethidium Bromide]]

Preparing an Agarose Gel

Davebrid1 — Fri, 27 Mar 2026 12:45:48 GMT

Added SOP

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			[[ SOP - Ethidium Bromide ]]

	==Materials==		==Materials==
	*Agarose		*Agarose
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	*Thermo Apparatus (Mold, Holder, Comb and Gel Rig)		*Thermo Apparatus (Mold, Holder, Comb and Gel Rig)

	==Prepare Gel ~~(BioRad Gel Rig)~~==		==Prepare Gel==
	#Place plastic mold securely in holder		#Place plastic mold securely in holder
	#Put well comb in mold		#Put well comb in mold
	#Weight out 1.0g agarose and add 100 mL of TAE (1% solution) ~~then return TAE to shelf~~		#Weight out 2.0g agarose and add 100 mL of TAE (2% solution).
	#Microwave 90s until dissolved		#Microwave 90s until dissolved
	#Add 2 uL Gel Red. Swirl to mix and eliminate sediments		#Add 2 uL Ethidium Bromide or Gel Red. Swirl to mix and eliminate sediments
	#Pour into mold		#Pour into mold, add the comb, and wait for it to solidify
	#Place gel in cold room if in a hurry		#Place gel in cold room if in a hurry
	#After loading, run gel at 100V		#After loading, run gel at 100V

QPCR

Davebrid1 — Mon, 23 Mar 2026 20:08:13 GMT

Formatting for GCal link

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	===Plate Preparation===		===Plate Preparation===
	#Book 2h on qPCR machine by signing up ~~on the sheet in room 7013 and~~ on the [https://calendar.google.com/calendar/~~embed~~?~~src~~=~~umich.edu_jkmcg58uidmngdh0sutt1d36og%40group.calendar.google.com&ctz=America%2FDetroit google calendar~~]		#Book 2h on qPCR machine by signing up on the [https://calendar.google.com/calendar/u/0?cid=Y19jN2RhYmJlMjJiNmQ1YTY0YjAzYzU1NzVmOGUxNmEyZmZjNjAwOTJjYWIyZmYwM2VjY2M1NjkyNzljYmQwY2QzQGdyb3VwLmNhbGVuZGFyLmdvb2dsZS5jb20 Google Calendar]
	#Prepare cDNA and dilute in water in a 96 well plate. Typically a 20x dilution of cDNA leaves enough to be detected.		#Prepare cDNA and dilute in water in a 96 well plate. Typically a 20x dilution of cDNA leaves enough to be detected.
	#Get optically clear 384 well plate and keep on paper towel. Do not touch bottom of plate.		#Get optically clear 384 well plate and keep on paper towel. Do not touch bottom of plate.

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