Last updated: 2019-09-05

workflowr checks: (Click a bullet for more information)
  • R Markdown file: up-to-date

    Great! Since the R Markdown file has been committed to the Git repository, you know the exact version of the code that produced these results.

  • Repository version: e39d12e

    Great! You are using Git for version control. Tracking code development and connecting the code version to the results is critical for reproducibility. The version displayed above was the version of the Git repository at the time these results were generated.

    Note that you need to be careful to ensure that all relevant files for the analysis have been committed to Git prior to generating the results (you can use wflow_publish or wflow_git_commit). workflowr only checks the R Markdown file, but you know if there are other scripts or data files that it depends on. Below is the status of the Git repository when the results were generated:
    
    Ignored files:
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        Ignored:    slurm-50626685.out
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        Ignored:    slurm-50627911.out
        Ignored:    slurm-50627915.out
        Ignored:    slurm-50627916.out
        Ignored:    slurm-50627918.out
        Ignored:    slurm-50627922.out
        Ignored:    slurm-50627928.out
        Ignored:    slurm-50627951.out
        Ignored:    slurm-50627976.out
        Ignored:    slurm-50627995.out
        Ignored:    slurm-50628022.out
        Ignored:    slurm-50628045.out
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        Ignored:    slurm-50628081.out
        Ignored:    slurm-50628089.out
        Ignored:    slurm-50628099.out
        Ignored:    slurm-50628106.out
        Ignored:    slurm-50628112.out
        Ignored:    slurm-50628121.out
        Ignored:    slurm-50628129.out
        Ignored:    slurm-50628135.out
        Ignored:    slurm-50628144.out
        Ignored:    slurm-50628152.out
        Ignored:    slurm-50628159.out
        Ignored:    slurm-50628168.out
        Ignored:    slurm-50628176.out
        Ignored:    slurm-50628184.out
        Ignored:    slurm-50628189.out
        Ignored:    slurm-50628191.out
        Ignored:    slurm-50628196.out
        Ignored:    slurm-50628199.out
        Ignored:    slurm-50628201.out
        Ignored:    slurm-50628204.out
        Ignored:    slurm-50628207.out
        Ignored:    slurm-50628212.out
        Ignored:    slurm-50628221.out
        Ignored:    slurm-50628226.out
        Ignored:    slurm-50628227.out
        Ignored:    slurm-50628232.out
        Ignored:    slurm-50628240.out
        Ignored:    slurm-50628249.out
        Ignored:    slurm-50628257.out
        Ignored:    slurm-50628264.out
        Ignored:    slurm-50628273.out
        Ignored:    slurm-50628280.out
        Ignored:    slurm-50628289.out
        Ignored:    slurm-50628297.out
        Ignored:    slurm-50628305.out
        Ignored:    slurm-50628311.out
        Ignored:    slurm-50628320.out
        Ignored:    slurm-50628328.out
        Ignored:    slurm-50628336.out
        Ignored:    slurm-50628344.out
        Ignored:    slurm-50628348.out
        Ignored:    slurm-50628353.out
        Ignored:    slurm-50628357.out
        Ignored:    slurm-50628358.out
        Ignored:    slurm-50628359.out
        Ignored:    slurm-50628361.out
        Ignored:    slurm-50628364.out
        Ignored:    slurm-50628366.out
        Ignored:    slurm-50628369.out
        Ignored:    slurm-50628374.out
        Ignored:    slurm-50628377.out
        Ignored:    slurm-50628382.out
        Ignored:    slurm-50628387.out
        Ignored:    slurm-50628396.out
        Ignored:    slurm-50628400.out
        Ignored:    slurm-50628408.out
        Ignored:    slurm-50628416.out
        Ignored:    slurm-50628425.out
        Ignored:    slurm-50628434.out
        Ignored:    slurm-50628443.out
        Ignored:    slurm-50628451.out
        Ignored:    slurm-50628460.out
        Ignored:    slurm-50628469.out
        Ignored:    slurm-50628477.out
        Ignored:    slurm-50628485.out
        Ignored:    slurm-50628493.out
        Ignored:    slurm-50628501.out
        Ignored:    slurm-50628508.out
        Ignored:    slurm-50628514.out
        Ignored:    slurm-50628516.out
        Ignored:    slurm-50628519.out
        Ignored:    slurm-50628522.out
        Ignored:    slurm-50628523.out
        Ignored:    slurm-50628527.out
        Ignored:    slurm-50628528.out
        Ignored:    slurm-50628534.out
        Ignored:    slurm-50628536.out
        Ignored:    slurm-50628537.out
        Ignored:    slurm-50628539.out
        Ignored:    slurm-50628541.out
        Ignored:    slurm-50628543.out
        Ignored:    slurm-50628545.out
        Ignored:    slurm-50628547.out
        Ignored:    slurm-50628548.out
        Ignored:    slurm-50628550.out
        Ignored:    slurm-50628552.out
        Ignored:    slurm-50628554.out
        Ignored:    slurm-50628556.out
        Ignored:    slurm-50628557.out
        Ignored:    slurm-50628559.out
        Ignored:    slurm-50628561.out
        Ignored:    slurm-50628563.out
        Ignored:    slurm-50628565.out
        Ignored:    slurm-50628567.out
        Ignored:    slurm-50628569.out
        Ignored:    slurm-50628571.out
        Ignored:    slurm-50628573.out
        Ignored:    slurm-50628576.out
        Ignored:    slurm-50628580.out
        Ignored:    slurm-50628586.out
        Ignored:    slurm-50628593.out
        Ignored:    slurm-50628600.out
        Ignored:    slurm-50628607.out
        Ignored:    slurm-50628616.out
        Ignored:    slurm-50628621.out
        Ignored:    slurm-50628623.out
        Ignored:    slurm-50628631.out
        Ignored:    slurm-50628637.out
        Ignored:    slurm-50628653.out
        Ignored:    slurm-50628677.out
        Ignored:    slurm-50628701.out
        Ignored:    slurm-50628704.out
        Ignored:    slurm-50628712.out
        Ignored:    slurm-50628723.out
        Ignored:    slurm-50628737.out
        Ignored:    slurm-50628751.out
        Ignored:    slurm-50628761.out
        Ignored:    slurm-50628771.out
        Ignored:    slurm-50628782.out
        Ignored:    slurm-50628794.out
        Ignored:    slurm-50628805.out
        Ignored:    slurm-50628817.out
        Ignored:    slurm-50628827.out
        Ignored:    slurm-50628829.out
        Ignored:    slurm-50628841.out
        Ignored:    slurm-50628855.out
        Ignored:    slurm-50628867.out
        Ignored:    slurm-50628872.out
        Ignored:    slurm-50628874.out
        Ignored:    slurm-50628878.out
        Ignored:    slurm-50628882.out
        Ignored:    slurm-50628885.out
        Ignored:    slurm-50628895.out
        Ignored:    slurm-50628908.out
        Ignored:    slurm-50628917.out
        Ignored:    slurm-50628925.out
        Ignored:    slurm-50628936.out
        Ignored:    slurm-50628950.out
        Ignored:    slurm-50628959.out
        Ignored:    slurm-50628961.out
        Ignored:    slurm-50628970.out
        Ignored:    slurm-50628981.out
        Ignored:    slurm-50628989.out
        Ignored:    slurm-50629001.out
        Ignored:    slurm-50629009.out
        Ignored:    slurm-50629020.out
        Ignored:    slurm-50629030.out
        Ignored:    slurm-50629043.out
        Ignored:    slurm-50629055.out
        Ignored:    slurm-50629064.out
        Ignored:    slurm-50629076.out
        Ignored:    slurm-50629086.out
        Ignored:    slurm-50629088.out
        Ignored:    slurm-50629089.out
        Ignored:    slurm-50629091.out
        Ignored:    slurm-50629093.out
        Ignored:    slurm-50629095.out
        Ignored:    slurm-50629096.out
        Ignored:    slurm-50629098.out
        Ignored:    slurm-50629099.out
        Ignored:    slurm-50629103.out
        Ignored:    slurm-50629104.out
        Ignored:    slurm-50629106.out
        Ignored:    slurm-50629108.out
        Ignored:    slurm-50629110.out
        Ignored:    slurm-50629112.out
        Ignored:    slurm-50629115.out
        Ignored:    slurm-50629117.out
        Ignored:    slurm-50629118.out
        Ignored:    slurm-50629120.out
        Ignored:    slurm-50629122.out
        Ignored:    slurm-50629124.out
        Ignored:    slurm-50629126.out
        Ignored:    slurm-50629128.out
        Ignored:    slurm-50629129.out
        Ignored:    slurm-50629131.out
        Ignored:    slurm-50629133.out
        Ignored:    slurm-50629135.out
        Ignored:    slurm-50629137.out
        Ignored:    slurm-50629138.out
        Ignored:    slurm-50629140.out
        Ignored:    slurm-50629142.out
        Ignored:    slurm-50629144.out
        Ignored:    slurm-50629146.out
        Ignored:    slurm-50629148.out
        Ignored:    slurm-50629150.out
        Ignored:    slurm-50629152.out
        Ignored:    slurm-50629154.out
        Ignored:    slurm-50629156.out
        Ignored:    slurm-50629157.out
        Ignored:    slurm-50629158.out
        Ignored:    slurm-50629160.out
        Ignored:    slurm-50629162.out
        Ignored:    slurm-50629164.out
        Ignored:    slurm-50629166.out
        Ignored:    slurm-50629171.out
        Ignored:    slurm-50629176.out
        Ignored:    slurm-50629186.out
        Ignored:    slurm-50629196.out
        Ignored:    slurm-50629206.out
        Ignored:    slurm-50629209.out
        Ignored:    slurm-50629215.out
        Ignored:    slurm-50629220.out
        Ignored:    slurm-50629224.out
        Ignored:    slurm-50629227.out
        Ignored:    slurm-50629229.out
        Ignored:    slurm-50629233.out
        Ignored:    slurm-50629237.out
        Ignored:    slurm-50629240.out
        Ignored:    slurm-50629243.out
        Ignored:    slurm-50629246.out
        Ignored:    slurm-50629249.out
        Ignored:    slurm-50629252.out
        Ignored:    slurm-50629253.out
        Ignored:    slurm-50629256.out
        Ignored:    slurm-50629259.out
        Ignored:    slurm-50629262.out
        Ignored:    slurm-50629265.out
        Ignored:    slurm-50629268.out
        Ignored:    slurm-50629270.out
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        Ignored:    slurm-50629274.out
        Ignored:    slurm-50629277.out
        Ignored:    slurm-50629280.out
        Ignored:    slurm-50629283.out
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        Ignored:    slurm-50629286.out
        Ignored:    slurm-50629289.out
        Ignored:    slurm-50629292.out
        Ignored:    slurm-50629295.out
        Ignored:    slurm-50629298.out
        Ignored:    slurm-50629300.out
        Ignored:    slurm-50629303.out
        Ignored:    slurm-50629306.out
        Ignored:    slurm-50629309.out
        Ignored:    slurm-50629312.out
        Ignored:    slurm-50629314.out
        Ignored:    slurm-50629316.out
        Ignored:    slurm-50629319.out
        Ignored:    slurm-50629322.out
        Ignored:    slurm-50629326.out
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        Ignored:    slurm-50629331.out
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        Ignored:    slurm-50629457.out
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We used this site to collaborate and share our results. Please feel free to explore. The results that made it into the final paper are in the section Finalizing below. Here are some useful links:

Analysis

Supervised method learning: finalizing on data holiding out validation samples

Supervised method learning: building the analysis approach

Approaches to fitting cyclical trend in gene expression data

Cell cycle signal in gene expression data

  1. We investigated cell cycle signals in the sequencing data alone.
  2. We then assign categorical labels of cell cycle and explored the expresson profiles of these categories.
  3. We inferred an angle for each cell on a unit circle using FUCCI intensities alone.
  4. I used nonparametric methods to identify genes that may be cyclical along cell cycle phases.
    • Fit smash and kernel regression on circular variables on a subset of genes with detection rate > .8.
    • Fit trendfilter on a subset of genes (5) that are observed (visually) to have cyclical pattern. trendfilter is robust to small proportion of undetected cells, approx 2 or 3%. In cases of simulation when increasing proportion of undetected cells to 20%, we observed a flat line in gene expression for genes previously identified to tend to a cyclical pattern.
    • Next, we fit trendfilter on all genes after transforming the data to follow standard normal distribution, permutation-based p-values for PVE are used to select 101 significant cyclical genes.
  5. Additional analysis done to identify top cyclical genes in each individual. The top 5 are not shared across the six individuals. Results

RNA-seq data preprcessing

  1. The first step in preprocessing RNA-seq data consists of QC and filtering.
  2. We then analyzed and corrected for batch effect due to C1 plate in the sequencing data

Microscopy image analysis

We evaluated and pre-processed the results of image analysis as follows:

  1. We visually inspect images deteced to have none or more than one nucleus. For cases that are inconsistent with visual inspection, we correct the number of nuclei detected.
  2. We applied background correction to the intensity measurements of GFP, RFP and DAPI based on the following analyses.
  3. We analyzed intensity variation across individuals and batches and considers approaches for removing batch effects in the data.
  4. We investigated the cell time estimates based on FUCCI intensities.



This reproducible R Markdown analysis was created with workflowr 1.0.1