bayNorm: relevant code for producing figures in the paper

code for producing figures in bayNorm

#Purpose of this repository The main purpose of this repository is to provide the analysis procedure used in the paper.

Source code of bayNorm

Source code of bayNorm can be found here

Real datasets used in this paper

This paper involves the following 8 studies:

1. Klein study (https://www.cell.com/cell/abstract/S0092-8674%2815%2900500-0)
2. Grün study (https://www.nature.com/articles/nmeth.2930)
3. Torre study (https://www.cell.com/cell-systems/abstract/S2405-4712(18)30051-6)
4. Bacher study (https://www.nature.com/articles/nmeth.4263)
5. Islam study (https://www.ncbi.nlm.nih.gov/pubmed/21543516)
6. Soumillon study (https://www.biorxiv.org/content/early/2014/03/05/003236)
7. Tung study (https://www.nature.com/articles/srep39921)
8. Patel study (http://science.sciencemag.org/content/344/6190/1396)

Simulated datasets used in this paper:

There are 4 simulated datasets with DE genes. Each one of them consists of 2 two groups of cells, and 100 cells in each group. 2000 out of 10000 genes were simulated to be DE genes in the first group and half of the 2000 genes were upregulated \Simulations\SIM_DE.

1. SIM DE I: mean capture efficiency $<\beta>=10%$ for two groups.
2. SIM DE II: mean capture efficiency $<\beta>=5% \text{ and } 10%$ for two groups respectively.
3. SIM DE III: mean capture efficiency $<\beta>=10% \text{ and } 5%$ for two groups respectively.
4. SIM DE IV: mean capture efficiency $<\beta>=5% \text{ and } 5%$ for two groups respectively.

There are another 2 simulated datasets without DE genes \Simulations\SIM_noDE. Mean capture efficiency $<\beta>=10% \text{ and } 5%$ for two groups respectively. These two simulations were inspired by Bacher study. The purpose is to study the ability of normalization method in terms of correcting different sequencing depths.

1. SIM Bacher I: Parameters were estimated from Klein study.
2. SIM Bacher II: Parameters were estimated from H1_P24 cells from Bacher study.

Datasets and the corresponding figures

Real datasets

1. Klein study: Fig1 (b)-(e), Fig3 (a)-(b); Fig S2, S8a-b.
2. Grün study: Fig2 a,c,e and g; Fig S11a-b, S12-S13
3. Torre study: Fig2 b,d,f and h; Fig S6, S8e-f, S10a, S11c, S14.
4. Bacher study: Fig S7, S9 a-d, S10e, S16, S19a, S23a.
5. Islam study: Fig 3c; Fig S9e-f, S23b.
6. Soumillon study: Fig3d; Fig S21.
7. Tung study: Fig4, FigS3-S5, S8c-d, S10b-d, S25-26
8. Patel study: FigS10f

Simulated datasets with DE genes

1. SIM DE I: FigS15a,e,i, S20c-d, S22a, S24a, S27-29
2. SIM DE II: FigS15b,f,j, S20c-d, S22b, S24b, S27-29
3. SIM DE III: FigS15c,g,k, S20c-d, S22c, S24c, S27-29
4. SIM DE IV: FigS15d,h,l, S20c-d, S22d, S24d, S27-29

Simulated datasets without DE genes

1. SIM Bacher I: S17, S19b, S20a-b
2. SIM Bacher II: S18, S19c

Some notes before running the code

1. You cannot directly run all the code at the same time. The paths in each R file need to be modified accordingly.
2. The normalization and DE detection could take a long time, which depends on the size of raw data. Hence make sure running the code step by step so as to avoid bugs.
3. Useful functions are stored in the file \Functions, some of them need to be loaded in advance.
4. The noramlization method DCA is developed using python. The Jupyter Notebooks for running DCA are stored in the file \DCA. Make sure running DCA normalization and corresponding DE detection, and them feed the DCA normalized data into the other R files.
5. Some R files need several .RData files as input and will also output .RData files used in other cases. Hence make sure the first step is completed so as to produce necessary .RData files to begin with.

The first step

Preparing for the real datasets

1. Klein study: firstly, run \RealData\Klein_study\Klein_bayNorm.R, output Klein_bayNorm.RData.

2.Grün study: run LOAD_Grun_smFISH.R (output smFISH_norm_load.RData), LOAD_Grun_2i.R (output Grun_2014_RAW.RData) and LOAD_Grun_serum.R (output Grun_2014_RAW_serum.RData). Then run Grun_2i_norms.R (output Grun_2i_norms.RData) and Grun_serum_norms.R (output Grun_serum_norms.RData) for normalizing data. Note that the other method DCA needs to be run separately.

3. Torre study: run Load_Torre.R (output Load_Torre.RData). Then run Torre_many_normalizations.R (out put Torre_many_normalizations.RData) for normalizing data.

4. Bacher study: run LOAD_Bacher.R (output RAW_INITIATE.RData) to load H1 and H9 datasets. Then run H1_many_normalizations.R (output "H1_many_normalizations.RData") and H9_many_normalizations.R (output "H9_many_normalizations.RData") respectively.

5. Islam study: run Load_Islam.R (output Load_Islam.RData). Then run Islam_many_normalizations.R (output Islam_many_normalizations.RData).

6. Soumillon study: run LOAD_Soumillon.R (output Soumillon_2014.RData). Then run Soumillon_norms.R (output Soumillon_analysis.RData).

7. Tung study: run Load_Tung.R (output Load_Tung.RData). Then run Tung_many_normalizations.R (output Tung_norms.RData).

8. Patel study: run Load_Patel.R (output Patel2014_bay_out.RData)

Notes before running simulations

Firstly, we need to estimate parameters from the real data. Relevant codes are stored in \bayNorm_papercode\Figure1.

1. For Klein dataset, if you have completed the step 1 as shown above, then Klein_bayNorm.RData stored the parameters you need. Klein_bayNorm.RData is needed in SIM DE I-IV and SIM Bacher I.
2. For Bacher dataset (H1_P24), run a section named REAL DATA 6: Bacher study (H1_P24 cells) in the file Simulations_realdata.R, which output H1p24_bay_sim_allgene.RData used in SIM Bacher II.

Preparing for the simulated datasets (with DE genes)

The codes are stored in: \Simulations\SIM_DE

1. SIM DE I: run DE_sim_01_01.R (output SIM_1.RData and GG_SIM_1.RData).
2. SIM DE II: run SIM_005_01.R (output SIM_005_01.RData and GG_SIM_005_01.RData).
3. SIM DE III: run SIM_01_005.R(output SIM_01_005.RData and GG_SIM_01_005.RData).
4. SIM DE IV: run SIM_005_005.r(output SIM_005_005.RData and GG_SIM_005_005.RData).

Preparing for the simulated datasets (without DE genes)

The codes are stored in: \Simulations\SIM_noDE

1. SIM Bacher I: run SIM_noDE_01_005.R (output SIM_noDE_01_005.RData)
2. SIM Bacher II: run SIM_noDE_01_005_H1.R (output SIM_noDE_01_005_H1.RData)

Next

After the above steps, you can try the other R files which include various code for analysing the data.