MSTS has been developed to analyze NGS data obtained from MNase-Seq experiments and provide utilities to perform simple statistics and to plot results.
MSTS is developed by the BioinfoBIOGER plateform (N.Lapalu, A.Simon) at INRAE-BIOGER. Please do not hesitate to contact us (nicolas.lapalu at inrae dot fr) if you have any comments or questions.
- Installation
- Protocol to analyze MNase-Seq data
- References
Python 3.X (tested with 3.7)
Packages:
- numpy
- scipy
- scikit-learn
- fastcluster (http:https://danifold.net/fastcluster.html)
- matplotlib
- pysam
- pyBigWig
To install required packages download the requirements and launch:
pip install -r requirements.txt
pip install https://github.com/nlapalu/MSTS/archive/master.zip
- samtools
- wigToBigWig Kent's tools
- bedToBigBed Kent's tools
The tool of your choice can be used to map sequencing reads as long as the output is in bam format. Make sure your mapping file is sorted by position and indexed before using MSTS. To sort and index bam files run:
samtools sort mapping.bam > mapping.sorted.bam
samtools index mapping.sorted.bam
Then use the MSTS converter to process bam files:
MSTS_converter.py mapping.sorted.bam -m single-expanded -p mapping -g assembly.genome --wig --size
MSTS_converter.py mapping.sorted.bam -m fragment-middle -w 20 -p mapping -g assembly.genome --wig --size --bed
wigToBigWig mapping.wig assembly.genome mapping.bw
One of the first things to do for primary data exploration is to plot a phasogram derived from your data. This graph highlights general nucleosome arrangement on the genome and the average spacing between two nucleosomes.
MSTS_phasogram.py mapping.bw -w 1000 -o mapping.phasogram.png -t "phasogram - mapping" -v 2 --flush --regression > mapping.phaso
In this example, the phasogram reveals an average distance between two nucleosome summits of nearly 164 bp, with a standard deviation of 2.3 bp. Thus, considering a size of 147 bp for the length of nucleosome-bound DNA, an average spacing of 18 nucleotides between nucleosomes can be proposed. We recommend window of 1kb as one-fits starting point. Beyond this value, the signal could become less reliable with some datasets.
In case of needing to restrict the analysis to particular genome compartments, e.g., with specific characteristics such as high AT content or exon only, you can limit the phasogram analysis to chosen regions. An example of analysis restricted to genes is provided below. We can observe that standard deviation is reduced, indicating spacing between nucleosome is less variable within genes than when the entire genome is considered.
MSTS_phasogram.py mapping.bw -w 1000 -o mapping.phasogram.png -t "phasogram - mapping" -v 2 --flush --regression -b genes.bb > mapping.phaso
We also propose a "phasogram like" graph, that show the cumulative signal on specific features. The "context" option limits the graph to the desired bases, no spanning feature or base outside of the feature. For example, if you wish to analyze 1kb before and after the Transcription Start Sites (TSS), but some adjacent TSS are closer than 1kb, we remove bases spanning the new TSS to avoid bias. The number of bases taken into account to draw the signal are plotted as an histogram on the graph. We provide another histogram in the upper area with adjacent features of the same type. This option is usefull for small genomes with short intergenic regions.
We also propose to plot "phasogram-like" graphs that show cumulative signal on specific features. The "context" option limits the data to be considered for plotting to the nucleotides concerned by the feature option without hitting any nearby feature. For example, if you wish to analyze 1 kb upstream and 1 kb downstream Transcription Start Sites (TSS), the "context" option will perform the analysis using up to 1 kb on each side, cutting out any eventual adjacent mRNA found closer than 1kb to avoid bias. The number of nucleotides actually taken into account to draw the signal is then plotted as a histogram (grey area) on the graph. Another histogram is also provided in the upper area representing the number of adjacent features of the same type at each position of the studied window. This option is particularly useful for small genomes with short intergenic regions. The context ifilter works on any type of feature defined with the "featureType" option.
Analysis of TSS:
MSTS_feature_phasogram.py mapping.bw genes.gff3 -v 2 -o featurephasogram.TSS.png -t "TSS, with context, with smoothing" -p start -ft mRNA --context --GaussianSmoothing
Analysis of TTS:
MSTS_feature_phasogram.py mapping.bw genes.gff3 -v 2 -o featurephasogram.TTS.png -t "TTS, with context, with smoothing" -p end -ft mRNA --context --GaussianSmoothing
You can perform the same analysis on all the feature types available in your gff file.
If you have RNA-Seq data and MNase-Seq data, you could draw phasograms on genes (or other relevant feature) grouped by expression levels. To do so, you have to defined a list of features to which the analysis will be limited.
If you do not have counts on your features, you can generate TPM count file from RNA-Seq mapped data (sorted and indexed bam) and annotation fil :
MSTS_count_TPM.py RNA_mapping.sorted.bam genes.gff3 -v 2 > counts.tpm
Export list of transcripts IDs for each defined Expression Level intervals, e.g [x>50,50>=x>5,5>=x>1,x<=1].
tail -n+2 counts.tpm | awk -F"\t" '{if($5 > 50){print $1}}' > 50.tpm
tail -n+2 counts.tpm | awk -F"\t" '{if($5 <= 50 && $5 > 5){print $1}}' > 50-5.tpm
tail -n+2 counts.tpm | awk -F"\t" '{if($5 <= 5 && $5 > 1){print $1}}' > 5-1.tpm
tail -n+2 counts.tpm | awk -F"\t" '{if($5 < 1){print $1}}' > 1.tpm
Generate phasograms restricting the analysis to genes on each list
MSTS_feature_phasogram.py mapping.bw genes.gff3 -v 2 -o myfeaturestartphasogram50.png -t "phasogram on transcript, start as pivot, TPM > 50" -ft mRNA -l 50.tpm --context --GaussianSmoothing --flush > 50.tpm.phaso
MSTS_feature_phasogram.py mapping.bw genes.gff3 -v 2 -o myfeaturestartphasogram50-5.png -t "phasogram on transcript, start as pivot, 50>TPM>5" -ft mRNA -l 50-5.tpm --context --GaussianSmoothing --flush > 50-5.tpm.phaso
MSTS_feature_phasogram.py mapping.bw genes.gff3 -v 2 -o myfeaturestartphasogram5-1.png -t "phasogram on transcript, start as pivot, 5>TPM>1" -ft mRNA -l 5-1.tpm --context --GaussianSmoothing --flush > 5-1.tpm.phaso
MSTS_feature_phasogram.py mapping.bw genes.gff3 -v 2 -o myfeaturestartphasogram1.png -t "phasogram on transcript, start as pivot, TPM < 1" -ft mRNA -l 1.tpm --context --GaussianSmoothing --flush > 1.tpm.phaso
The '--flush' option, allows exporting the to be used to merge all phasograms on a single figure as below, with MSTS_merge_phasograms.py. Here you will find a piece of code to generate the "merged phasogram".
For most of available tools, the nucleosome detection and positioning are based on a simple greedy approach that consists for each sequence in:
- descending sort of occupancy values for each position
- defining nucleosome positions from the highest occupancy value to the lowest with a constraint of 147bp.
- limiting peak detection to a min/max coverage
More sophiscated tool, defined TBB (Zhou et al. 2016), that allow several positions for the same nucleosome, instead of a signal consensus. For MSTS, we decided to keep the conventional protocol to define an average position for a given nucleosome, allowing overlaps between nucleosomes for cases of 'fuzzy' regions (30ba-long overlap max by default). Then, MSTS perform a clustering and classification to provide an indication of the confidence level in nucleosome positioning. We defined 4 categories of positioned nucleosomes: "very-well", "well", "fuzzy" and "bad".
Typical command to run:
MSTS_detect_nucleosomes.py mapping.bw -p detection --bed --wig
Example of the output below:
seq start end mean stdev peak cluster positioning
SEQ01 13 159 8.0 4.25544868814 12.0 2 well
SEQ01 1982 2128 8.02040816327 2.78105688263 10.0 4 fuzzy
SEQ01 2355 2501 8.99319727891 1.9743132747 11.0 6 fuzzy
SEQ01 3249 3395 15.6666666667 4.47568386191 22.0 3 fuzzy
SEQ01 3387 3533 19.3605442177 6.35347200407 31.0 4 fuzzy
SEQ01 3504 3650 16.9183673469 4.30710566845 20.0 6 fuzzy
SEQ01 3670 3816 28.0476190476 11.419242224 41.0 4 fuzzy
SEQ01 3840 3986 12.3333333333 4.32101860839 17.0 4 fuzzy
SEQ01 4015 4161 10.0272108844 3.94165885558 17.0 4 fuzzy
SEQ01 4230 4376 20.7278911565 4.53190927482 27.0 6 fuzzy
SEQ01 4457 4603 29.4897959184 10.7477998678 47.0 4 fuzzy
SEQ01 4624 4770 42.4693877551 10.7540886845 54.0 3 fuzzy
SEQ01 4781 4927 35.0068027211 6.49698684597 44.0 3 fuzzy
SEQ01 4973 5119 39.2448979592 14.868398592 60.0 0 fuzzy
SEQ01 5103 5249 24.8707482993 7.21041584924 32.0 3 fuzzy
SEQ01 5269 5415 18.4489795918 3.76916178897 16.0 3 fuzzy
SEQ01 5387 5533 28.2517006803 7.58161357856 41.0 3 fuzzy
SEQ01 5557 5703 29.1972789116 13.2596464466 49.0 2 well
SEQ01 5707 5853 32.8843537415 11.6487814357 48.0 4 fuzzy
SEQ01 5904 6050 53.3333333333 21.8162809245 91.0 4 fuzzy
SEQ01 6071 6217 44.4557823129 9.44099563275 60.0 3 fuzzy
SEQ01 6277 6423 37.8979591837 12.0106158791 57.0 4 fuzzy
SEQ01 6473 6619 45.2380952381 20.1052784667 75.0 2 well
SEQ01 6634 6780 40.1020408163 24.3066393589 79.0 2 well
SEQ01 6817 6963 55.768707483 30.0518979733 103.0 2 well
...
The mean is computed with occupancy value for all positions of the defined nucleosome (147 bp in length).
For further analysis or visualization, the --bed option exports positioned nucleosomes per cluster and the --wig option exports the smoothed signal used to classify occupancy values. We also export the clustering as a cartoon to easily visualize the average profile of each cluster.
If you find few very-well and, well positioned nucleosomes and a remarkably high number of fuzzy nuclesomes, you can try the --refine option, that will relaunch a clustering on the fuzzy class with a relaxed classification. See below the example of the upper analysis re-done with a refined clustering.
We propose a simple approach to analyze differences between 2 conditions. We only analyze positions detected as potential dyad of nucleosomes, with a possible filter on the reliability of each type of nucleosome positioning (default=fuzzy). We use a Poisson distribution with a lambda equals to the normalized peak occupancy for each condition. Then we use a test ratio for two Poisson rates followed by a Benjamini-Hochberg correction for multiple tests. The default normalization method is quantile, but a global scaling (scaling factor defined with the average of number reads in all replicats) is also available. In case of multiple replicates, we provide a dendogram of hierarchical clustering to control the homogeneity of replicates. In the same manner, the p-value plot allows you to validate the expected results. The user must provide a design file with link to all required files.
design file:
#condition label nucfile bigwigfile
c1 43 43.nucleosomes.txt 43.mapping.bw
c1 44 44.nucleosomes.txt 44.mapping.bw
c1 45 45.nucleosomes.txt 45.mapping.bw
c2 46 46.nucleosomes.txt 46.mapping.bw
c2 47 47.nucleosomes.txt 47.mapping.bw
c2 48 48.nucleosomes.txt 48.mapping.bw
MSTS_nucleosome_difference.py design.txt -v 2 -p C1vsC2 -f 2.0 -a 0.05 --bed --merge
result file:
We only export positions with potential difference in signal with defined thresholds (default: FC=2.0 and alpha=0.05).
#ref pos mean1 CV1 mean2 CV2 FC pval paj
SEQ10 708598 16.2 21.9 2.7 48.7 0.16 7.12e-08 7.45e-06
SEQ14 253307 13.9 22.5 2.7 31.9 0.19 1.64e-06 8.95e-05
SEQ18 129813 14.2 40.1 2.9 10.9 0.20 2.25e-06 1.14e-04
SEQ18 125906 29.6 55.5 6.1 21.4 0.20 8.39e-12 4.10e-09
SEQ12 30014 10.7 37.7 2.2 74.1 0.21 4.28e-05 1.07e-03
SEQ18 125912 30.2 69.4 6.3 7.6 0.21 7.18e-12 3.60e-09
SEQ05 2586933 10.6 20.9 2.3 52.2 0.21 5.81e-05 1.35e-03
SEQ16 436879 72.4 26.0 15.7 22.7 0.22 1.10e-25 5.24e-22
SEQ01 3531964 50.8 22.2 11.1 5.0 0.22 2.14e-18 5.00e-15
SEQ18 125909 30.1 63.3 6.7 16.2 0.22 2.66e-11 1.09e-08
...
companion plots
hierarchical clustering:
p-values plot:
The di-nucleotide pattern AT/GC with a frequency of 10 bp for nucleosome fixation site has been largely described and proposed to be used as data quality control (S.Hu et al. 2017). You can perform such analysis on your mapping output converted in bigBed file with MSTS_converter.py
# convert your mapping bed file to bigBed and run MSTS_dinuc_frequency
bedToBigBed mapping.bed assembly.genome mapping.bb
MSTS_dinuc_frequency.py assembly.fasta mapping.bb
You can also control your nucleosome detection and classification with this tool. We expect a better signal for well positioned compared to badly/loosely positioned nucleosomes. To do this, run MSTS_detect_nucleosomes.py with --bed option and convert them to bigBed. We present below dinucleotide frequency profiles analyzed for 4 types of clusters (bad, fuzzy, well, very-well):
badly positioned nucleosomes:
MSTS_dinuc_frequency.py genome.fasta detect.k1-bad.cluster.sorted.bb --pAutocorMix --pFreqNormMix -p detect_dinuc_bad -ami -65 -amx 65
fuzzily positioned nucleosomes:
MSTS_dinuc_frequency.py genome.fasta detect.k2-fuzzy.cluster.sorted.bb --pAutocorMix --pFreqNormMix -p detect_dinuc_fuzzy -ami -65 -amx 65
well positioned nucleosomes:
MSTS_dinuc_frequency.py genome.fasta detect.k3-well.cluster.sorted.bb --pAutocorMix --pFreqNormMix -p detect_dinuc_well -ami -65 -amx 65
very-well positioned nucleosomes:
MSTS_dinuc_frequency.py genome.fasta detect.k4-very-well.cluster.sorted.bb --pAutocorMix --pFreqNormMix -p detect_dinuc_very-well -ami -65 -amx 65
- MSTS_converter.py
- MSTS_phasogram.py
- MSTS_feature_phasogram.py
- MSTS_count_TPM.py
- MSTS_dinuc_frequency.py
- MSTS_detect_nucleosomes.py
- MSTS_nucleosome_difference.py
- S. Hu, X. Chen, J. Liao, Y. Chen, C. Zhao, and Y. Zhang, “CAM: A quality control pipeline for MNase-seq data,” PLoS One, vol. 12, no. 8, p. e0182771, Aug. 2017.
- X. Zhou, A. W. Blocker, E. M. Airoldi, and E. K. O’Shea, “A computational approach to map nucleosome positions and alternative chromatin states with base pair resolution.,” Elife, vol. 5, Sep. 2016.