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MONOD2

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MONOD2 is a toolkit for methylation haplotype analysis of bisulfite sequencing data

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# MONOD2
MONOD2 is a toolkit for methylation haplotype analysis of bisulfite sequencing data. The analysis can be divided into three parts. First sequence alignment files (bam files) are analyzed to generate methylation haplotypes, which include haplotype strings and haplotype counts. Next, a collection of methylation haplotype files are analyzed for identifying methylation haplotype blocks or for calculating differential methylation haplotype load. Methylation haplotype blocks are regions with high linkage disequilibrium between pairs of CpGs. For the analysis of differentially methylation haplotype load, we performed tumor load estimation and plasma tissue of origin prediction on the methylation haplotype load profile of plasma DNA.

MONOD2 is comprised of executable `bash`, `perl`, and `R` programs. No installation is necessary except for the required dependencies listed below.

## Contact
email: hdinhdp@gmail.com

## Download

You can use git to download the entire codebase and datasets. Only the processed datasets are provided here, please contact us if you would like to obtain the BAM or methylation haplotype files.

```
git clone https://github.com/dinhdiep/MONOD2

```

## Required R packages

```
require(MASS)
require(pheatmap)
require(RColorBrewer)
require(randomForest)
require(impute)
require(abind)
require(ggplot2)
require(skmeans)
require(ModelMetrics)
require(caret)
require(ROCR)
require(reshape)
require(compiler)
require(LDheatmap)
require(RANN)
require(pROC)

```

## Required software

The following two software must be installed.

1. [samtools version 1.2 or above](http://samtools.sourceforge.net/)
2. [bedtools version 2.26 or above](http://bedtools.readthedocs.io/en/latest/)

## Important Notes

MONOD2 is based on the concepts and methods described in [Guo et al. 2017 Nature Genetics](http://www.nature.com/ng/journal/v49/n4/full/ng.3805.html). However, we have made additional modifications on the original methods and codes that was hosted on the [Supplementary Website](http://genome-tech.ucsd.edu/public/MONOD_NG_TR44413). A copy of the processed data, codes, and supplementary tables are available here in the ng.3805 directory. Some notable modifications are listed below:

1. The original code for Figure 2 was not producing the correct values from the Figure. Note that there are two errors in Figure 2 as follows: the epipolymorphism for panel 4 should be 0.9375 (the published figure has 0.375 due to an editing error), the MHL value for panel 5 should be 0.1167 (the published figure have a rounded up value of 0.1200).

2. The original comparison of AMF versus MHL at tissue specific regions were slightly biased because the comparison was based on the differentially methylated regions identified by MHL. In MONOD2 we used the MHBs overlapping with published tissue specific DMRs for a truly unbiased comparison.

3. The original method for tumor load estimation performed features selection without a clear separation of the plasma samples to be estimated. In MONOD2 we identified the features using a subset of 50 normal plasma, and then computed tumor load on the remaining normal and cancer patient plasma samples.

4. The original method for classification of plasma DNA did not have proper separation of training and test data during features selection, although in model building the training and test data were separated. In MONOD2 we separated training and test data for both features selection and model building.

## Usage

### Extracting methylation haplotypes from sequence alignment files

Sequence alignment files in BAM format should have the expected sam flags for Watson (forward) and Crick (reverse) strands. The commands below will generate CpG haplotype files without clonal removal (any clonal removal must be performed upstream).

```
bam2cghap.sh [cpg position file] [bam file] [output file prefix name]

```

Run example

```
./scripts/bam2cghap.sh allcpg/cpg.small.txt.gz BAMfiles/Colon_primary_tumor_sept9_promoter.bam test

```
Version 1 code used in ng.3805 is also provided but user must specify RRBS or WGBS mode and can only use BAM that have been mapped to hg19. RRBS and WGBS data are treated differently in that reads which are considered clonal in WGBS would be removed.

Run RRBS example

```
./scripts/bam2cghap_v1.sh RRBS Colon_primary_tumor_sept9_promoter.RD1_80up.genomecov.bed allcpg/cpg.small.txt.gz BAMfiles/Colon_primary_tumor_sept9_promoter.bam test

```
Run WGBS example

```
./scripts/bam2cghap_v1.sh WGBS Colon_primary_tumor_sept9_promoter.RD1_80up.genomecov.bed allcpg/cpg.small.txt.gz BAMfiles/Colon_primary_tumor_sept9_promoter.bam test

```

### Plotting the LD scores for a region

Inputs are a haploinfo file and the region to be plotted. Output will be a PDF file named as follows: "chromosome,start,end.pdf"

Run example

```
./bin/hapinfo2LDplot_signed.pl results/HaploInfo/chr22.sub.hapInfo.txt chr22:16053662-16058136

```

### Making mappable bin file

Empirically determine which regions in the genome have high mappability using whole genome bisulfite sequencing datasets.

```
make-mappable-bins.sh [bam file] [minimum depth cutoff]

```

Run example

```
./scripts/make-mappable-bins.sh BAMfiles/Colon_primary_tumor_sept9_promoter.bam 5

```

### Identifying the methylation haplotype blocks

Methylation haplotypes were split into continuous mappable bins and then an algorithm greedily selects the largest possible continuous region with a minimum linkage disequilibrium score to be considered methylation haplotype blocks. Blocks must have at least 3 CpGs sites. The concept for methylation haplotype blocks were described in details by [Shoemaker et al. 2010 Genome Research](http://genome.cshlp.org/content/20/7/883.full.pdf).

```
cghap2mhbs.sh [haplotype file] [target bed] [minimum LD R2 cutoff] [output name prefix]

```
Run example

```
./scripts/cghap2mhbs.sh results/HaploInfo/chr22.sub.hapInfo.txt example/N37_10_tissue_pooled.autosomes.RD10_80up.genomecov.bed 0.3 chr22

```

### Generating the data matrices

First, a list of paths to haplotype files should be generated with the `ls` tool in unix. The script allows for `AMF`, `MHL`, or `IMF` to be the output metric for the data matrix and a regions definition (BED) file should be provided to make the matrix.

```
cghap2matrix.sh [list of haplotype files] <AMF|MHL|IMF> [output name prefix] [target bed file]

```
Run example

```
./scripts/cghap2matrix.sh example/hapinfo_list MHL chr22 data/ng.3805/MHBS.txt

```
Output file would have the extension `mhl.txt`, `amf.txt`, or `imf.txt` depending on the metric specified.

### Compare AMF versus MHL

We compared the signal to noise levels between a matrix calculated using the average methylation frequency (AMF) and a matrix calculated using the methylation haplotype load (MHL) to demonstrate the advantage of using MHL on heterogeneous samples. The following files are required.

1. WGBS MHL matrix. `data/ng.3805/WGBS.adult_tissues.mhl.txt.gz`
2. WGBS AMF matrix. `data/ng.3805/WGBS.adult_tissues.amf.txt.gz`
3. List of MHBs overlapping with published DMRs: `ng.3805/RRBS_MHBs.sorted.DMR.withID.bed`

The only requirements for (1) and (2) are that the region indices and the sample IDs are the same between the two matrices. The region file (3) is a list of regions (subset of the region indices used to make the matrices).

Run example

```
Rscript src/mhl_vs_amf.R data/ng.3805/WGBS.adult_tissues.mhl.txt.gz data/ng.3805/WGBS.adult_tissues.amf.txt.gz ng.3805/RRBS_MHBs.sorted.DMR.withID.bed

```

The output is a pdf file that includes two heatmaps similar to Figure 3 from Guo et al. 2017, while subsetting at regions with tissue specific DMRs and also includes a scatterplot showing the relative signal to noise levels for AMF versus MHL.

### Preprocess the data matrices

Analysis cannot be performed across RRBS and WGBS datasets due to technical differences that may bias the results. Therefore we generated two separate matrices, one for each dataset. We then performed data pruning where samples with too few region coverage and regions with too few sample covered are removed. After pruning, imputation using k-nearest neighbor to fill all the missing values was performed. To run the provided preprocessing script, the following files are required.

1. The metadata for the WGBS tissue samples: `data/ng.3805/WGBS.getHaplo.sampleInfo.txt`
2. The metadata for the RRBS tissue/plasma samples: `data/ng.3805/RRBS.getHaplo.sampleInfo.txt`
3. WGBS MHL matrix: `data/ng.3805/WGBS_180118.gethaplo.mhl.mhbs1.0.useSampleID.txt.gz`
4. RRBS MHL matrix: `data/ng.3805/RRBS_180118.gethaplo.mhl.mhbs1.0.useSampleID.txt.gz`

Usage info

```
preprocess.sh [rrbs.matrix.gz] [wgbs.matrix.gz] [rrbs.meta] [wgbs.meta] [output Rdata name]

```

Run example

```
./scripts/preprocess.sh data/ng.3805/RRBS_180118.gethaplo.mhl.mhbs1.0.useSampleID.txt.gz data/ng.3805/WGBS_180118.gethaplo.mhl.mhbs1.0.useSampleID.txt.gz data/ng.3805/RRBS.getHaplo.sampleInfo.txt data/ng.3805/WGBS.getHaplo.sampleInfo.txt wgbs_rrbs_clean.Rdata

```

The output is an R data file named `wgbs_rrbs_clean.Rdata` and several PDF files showing the data quality.

### Tumor load estimation

We generated a set of simulation files by merging a subset of normal plasma data with cancer tissue data. We have both primary tumor tissue data for lung cancer and colon cancer. Since the large files cannot be hosted on github, we randomly sampled (with replacement) the datasets and provided them in the BAMfiles folder. Note that read IDs can contain either mapped reads or all reads. We performed the analysis on all reads so that the mappability of sampled fragments also reflect the mappability of 'non-cancer' versus 'cancer' fragments.

The script needs to be modified from within (using a text editor) in order to run user-provided read IDs and BAM files. The following lines as is will perform the analysis on the example read IDs and BAM files.

```bash
numsimulation=20 # number of simulations to perform
n1=1000000 # number of reads per simulation 
 
cctfastq="BAMfiles/CCT.readIDs.txt" # path to the read IDs for colon tumor samples
lctfastq="BAMfiles/LCT.readIDs.txt" # path to the read IDs for lung tumor samples
ncpfastq="BAMfiles/NCP.readIDs.txt" # path to the read IDs for normal plasma training subset

cctbam="BAMfiles/CCT.bam" # path to the merged BAM for colon tumor samples
lctbam="BAMfiles/LCT.bam" # path to the merged BAM for lung tumor samples
ncpbam="BAMfiles/NCP.bam" # path to the merged BAM for normal plasma training subset

cpgs="allcpg/hg19.fa.allcpgs.txt.gz" # path to the CpG positions file

```
Run example

```
 ./scripts/simulate-cghap.sh

```

There should be many simulated files generated including two list files.

1. `cct.hapinfo.list_20` 
2. `lct.hapinfo.list_20`

The should also be three directories which contain simulated data for each sample type.
1. `CCT_simulation` 
2. `LCT_simulation`
3. `NCP_simulation`

Next, we ran features selection and tumor load estimation using the simulated data files. The script for tumor load estimation have the following usage.

```
tumor_load_estimation.R [rdata file] [list of normal plasma training samples]

```

Run example

```
Rscript /src/tumor_load_estimation.R data/ng.3805/wgbs_rrbs_clean.Rdata data/ng.3805/subset_normal_plasma_training 20

```

The output files are as follows.

1. The Rdata which saves all the outputs : `Tumor_load_estimation.Rdata`
2. Boxplots similar to Figure 4d from Guo et al. 2017 which shows the estimated tumor proportions for the plasma samples: `estimated_proportions.pdf`
3. Boxplots similar to Figure 4a,b from Guo et al. 2017 which shows the differential MHL levels in tumor marker regions for different sample types: `mhl_boxplot.pdf`
4. Heatmaps similar to Figure 4a,b from Guo et al. 2017 which shows the MHL levels in normal plasma, cancer plasma, and cancer tissues in the tumor marker regions: `heatmap.pdf`
5. A table of the standard curve values generated from simulated data: `standard_curves_values.txt`
6. A list of lung cancer markers used in the analysis: `lung_cancer_markers.txt`
7. A list of colon cancer markers used in the analysis: `colon_cancer_markers.txt`

### Plasma prediction

To perform plasma prediction, we asked if plasma samples from healthy individuals, lung cancer patients, and colon cancer patients can be identified from their methylation haplotype load profiles. Thus, a random sample of plasma was used to train a random forest model (R `caret` and R `randomForest` package).

Run example

```
Rscript src/find_tissues_markers.R
Rscript src/plasma_prediction.R

```
Example of output for one randomly selected train/test split for Normal vs Cancer:

```
[1] "Normal vs Cancer RF features: 132"
randomForest 4.6-12
Type rfNews() to see new features/changes/bug fixes.

Attaching package: ‘randomForest’

The following object is masked from ‘package:ggplot2’:

margin

Confusion Matrix and Statistics

Reference
Prediction Cancer Normal
    Cancer     11      5
    Normal      2     11

Accuracy : 0.7586
                 95% CI : (0.5646, 0.897)
    No Information Rate : 0.5517
    P-Value [Acc > NIR] : 0.01799

Kappa : 0.5224
 Mcnemar's Test P-Value : 0.44969

Sensitivity : 0.8462
            Specificity : 0.6875
         Pos Pred Value : 0.6875
         Neg Pred Value : 0.8462
             Prevalence : 0.4483
         Detection Rate : 0.3793
   Detection Prevalence : 0.5517
      Balanced Accuracy : 0.7668

'Positive' Class : Cancer

[1] "AUC=0.829326923076923"
```

For Colon vs Lung
```

[1] "Colon vs Lung RF features: 69"
Confusion Matrix and Statistics

Reference
Prediction Colon Lung
     Colon     5    2
     Lung      2    4

Accuracy : 0.6923
                 95% CI : (0.3857, 0.9091)
    No Information Rate : 0.5385
    P-Value [Acc > NIR] : 0.2033

Kappa : 0.381
 Mcnemar's Test P-Value : 1.0000

Sensitivity : 0.7143
            Specificity : 0.6667
         Pos Pred Value : 0.7143
         Neg Pred Value : 0.6667
             Prevalence : 0.5385
         Detection Rate : 0.3846
   Detection Prevalence : 0.5385
      Balanced Accuracy : 0.6905

'Positive' Class : Colon

[1] "AUC=0.761904761904762"
```

MONOD2

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