MesKit: Analyze and Visualize Multi-region Whole-exome Sequencing Data
2021-03-27
1. Introduction
Intra-tumor heterogeneity (ITH) is now thought to be a key factor that results in the therapeutic failures and drug resistance, which have arose increasing attention in cancer research. Here, we present an R package, MesKit, for characterizing cancer genomic ITH and inferring the history of tumor evolutionary. MesKit provides a wide range of analyses including ITH evaluation, enrichment, signature, clone evolution analysis via implementation of well-established computational and statistical methods. The source code and documents are freely available through Github (https://github.com/Niinleslie/MesKit). We also developed a shiny application to provide easier analysis and visualization.
1.1 Citation
In R console, enter citation("MesKit").
MesKit: A Tool Kit for Dissecting Cancer Evolution of Multi-region Tumor Biopsies through Somatic Alterations (Under revision)
2. Prepare input Data
To analyze with MesKit, you need to provide:
- A MAF file of multi-region samples from patients. (
*.maf / *.maf.gz). Required - A clinical file contains the clinical data of tumor samples from each patient. Required
- Cancer cell fraction (CCF) data of somatic mutations. Optional but recommended
- A segmentation file. Optional
- The GISTIC outputs. Optional
Note: Tumor_Sample_Barcode should be consistent in all input files, respectively.
2.1 MAF files
Mutation Annotation Format (MAF) files are tab-delimited text files with aggregated mutations information from VCF Files. The input MAF file could be gz compressed, and allowed values of Variant_Classificationcolumn can be found at Mutation Annotation Format Page.
The following fields are required to be contained in the MAF files with MesKit.
Mandatory fields:
Hugo_Symbol, Chromosome, Start_Position, End_Position, Variant_Classification, Variant_Type, Reference_Allele, Tumor_Seq_Allele2, Ref_allele_depth, Alt_allele_depth, VAF, Tumor_Sample_Barcode
Note:
- The
Tumor_Sample_Barcodeof each sample should be unique. - The
VAF(variant allele frequencie) can be on the scale 0-1 or 0-100.
Example MAF file
## Hugo_Symbol Chromosome Start_Position End_Position Variant_Classification
## 1 KLHL17 1 899515 899515 Silent
## 2 MFSD6 2 191301342 191301342 Missense_Mutation
## 3 KIAA0319 6 24596837 24596837 Missense_Mutation
## Variant_Type Reference_Allele Tumor_Seq_Allele2 Ref_allele_depth
## 1 SNP C T 85
## 2 SNP G A 53
## 3 SNP C T 6
## Alt_allele_depth VAF Tumor_Sample_Barcode
## 1 1 0.012 V402_P_1
## 2 0 0.000 V750_P_2
## 3 0 0.000 V750_BM_32.2 Clinical data files
Clinical data file contains clinical information about each patient and their tumor samples, and mandatory fields are Tumor_Sample_Barcode, Tumor_ID, Patient_ID, and Tumor_Sample_Label.
Example clinical data file
## Tumor_Sample_Barcode Tumor_ID Patient_ID Tumor_Sample_Label
## 1 V402_P_1 P V402 P_1
## 2 V402_P_2 P V402 P_2
## 3 V402_P_3 P V402 P_3
## 4 V402_P_4 P V402 P_4
## 5 V402_BM_1 BM V402 BM_12.3 CCF files
By default, there are six mandatory fields in input CCF file: Patient_ID, Tumor_Sample_Barcode, Chromosome, Start_Position, CCF and CCF_Std/CCF_CI_High (required when identifying clonal/subclonal mutations). The Chromosome field of your MAF file and CCF file should be in the same format (both in number or both start with “chr”). Notably, Reference_Allele and Tumor_Seq_Allele2 are also required if you want include contains INDELs in the CCF file.
Example CCF file
## Patient_ID Tumor_Sample_Barcode Chromosome Start_Position CCF CCF_Std
## 1 V402 V402_P_1 1 899515 0.031 0.126
## 2 V402 V402_P_1 1 982996 0.031 0.117
## 3 V402 V402_P_1 1 2452742 0.125 0.239
## 4 V402 V402_P_1 1 6203883 0.422 0.750
## 5 V402 V402_P_1 1 11106655 0.094 0.3242.4 Segmentation files
The segmentation file is a tab-delimited file with the following columns:
Patient_ID- patient IDTumor_Sample_Barcode- tumor sample barcode of samplesChromosome- chromosome name or IDStart_Position- genomic start position of segments (1-indexed)End_Position- genomic end position of segments (1-indexed)SegmentMean/CopyNumber- segment mean value or absolute integer copy numberMinor_CN- copy number of minor allele OptionalMajor_CN- copy number of major allele OptionalTumor_Sample_Label- the specific label of each tumor sample. Optional
Note: Positions are in base pair units.
Example Segmentation file
## Patient_ID Tumor_Sample_Barcode Chromosome Start_Position End_Position
## 1 V402 V402_P_1 1 1 1650882
## 2 V402 V402_P_1 1 1650883 33159352
## 3 V402 V402_P_1 1 33159353 33610373
## 4 V402 V402_P_1 1 33610374 88509894
## 5 V402 V402_P_1 1 88509895 89462108
## CopyNumber Minor_CN Major_CN Tumor_Sample_Label
## 1 2 1 1 P_1
## 2 1 0 1 P_1
## 3 3 0 3 P_1
## 4 2 1 1 P_1
## 5 2 0 2 P_13. Installation
Via Bioconductor
4. Start with the Maf object
readMaf function creates Maf/MafList objects by reading MAF files, clinical files and cancer cell fraction (CCF) data (optional but recommended). Parameter refBuild is used to set reference genome version for Homo sapiens reference ("hg18", "hg19" or "hg38"). You should set use.indel.ccf = TRUE when your ccfFile contains INDELs apart from SNVs.
maf.File <- system.file("extdata/", "CRC_HZ.maf", package = "MesKit")
ccf.File <- system.file("extdata/", "CRC_HZ.ccf.tsv", package = "MesKit")
clin.File <- system.file("extdata", "CRC_HZ.clin.txt", package = "MesKit")
# Maf object with CCF information
maf <- readMaf(mafFile = maf.File,
ccfFile = ccf.File,
clinicalFile = clin.File,
refBuild = "hg19") 5. Mutational landscape
5.1 Mutational profile
In order to explore the genomic alterations during cancer progression with multi-region sequencing approach, we provided classifyMut function to categorize mutations. The classification is based on shared pattern or clonal status (CCF data is required) of mutations, which can be specified by class option. Additionally, classByTumor can be used to reveal the mutational profile within tumors.
# Driver genes of CRC collected from [IntOGen] (https://www.intogen.org/search) (v.2020.2)
driverGene.file <- system.file("extdata/", "IntOGen-DriverGenes_COREAD.tsv", package = "MesKit")
driverGene <- as.character(read.table(driverGene.file)$V1)
mut.class <- classifyMut(maf, class = "SP", patient.id = 'V402')
head(mut.class)plotMutProfile function can visualize the mutational profile of tumor samples.
5.2 CNA profile
The plotCNA function can characterize the CNA landscape across samples based on copy number data from segmentation algorithms. Besides, MesKit provides options to integrate GISTIC2 results, which can be obtained from http://gdac.broadinstitute.org. Please make sure the genome version based on these results is consistent with refBuild of the Maf/MafList object .
# Read segment file
segCN <- system.file("extdata", "CRC_HZ.seg.txt", package = "MesKit")
# Read gistic output files
all.lesions <- system.file("extdata", "COREAD_all_lesions.conf_99.txt", package = "MesKit")
amp.genes <- system.file("extdata", "COREAD_amp_genes.conf_99.txt", package = "MesKit")
del.genes <- system.file("extdata", "COREAD_del_genes.conf_99.txt", package = "MesKit")
seg <- readSegment(segFile = segCN, gisticAllLesionsFile = all.lesions,
gisticAmpGenesFile = amp.genes, gisticDelGenesFile = del.genes)## --Processing COREAD_amp_genes.conf_99.txt
## --Processing COREAD_del_genes.conf_99.txt
## --Processing COREAD_all_lesions.conf_99.txt
6. Measurement of intra-tumor heterogeneity
6.1 Within tumors
6.1.1 MATH score
The mathScore function estimates ITH per sample using mutant-allele tumor heterogeneity (MATH) approach 1. Typically, the higher the MATH score is, more heterogeneous a tumor sample is 2 3. For MRS, this function can estimate the MATH score within tumor based on the merged VAF when withTumor = TRUE.
## $V402
## Patient_ID Tumor_Sample_Barcode MATH_Score
## 1 V402 V402_BM_1 123.550
## 2 V402 V402_BM_2 105.900
## 3 V402 V402_BM_3 101.664
## 4 V402 V402_BM_4 108.724
## 5 V402 V402_P_1 80.191
## 6 V402 V402_P_2 99.897
## 7 V402 V402_P_3 100.515
## 8 V402 V402_P_4 92.3376.1.2 AUC of CCF
The ccfAUC function calculates the area under the curve (AUC) of the cumulative density function based on the CCFs per tumor. Tumors with higher AUC values are considered to be more heterogeneous 4.
## [1] "AUC.value" "CCF.density.plot"
6.1.3 Mutation clustering
For a single region/tumor, mutCluster function clusters mutations in one dimension by VAFs or CCFs based on a Gaussian finite mixture model (using mclust). This function only focuses on heterozygous mutations within copy-number neutral and loss of heterozygosity (LOH)-free regions when clustering VAFs, as copy number aberrations will alter the fraction of reads bearing mutations. Note that, “low VAF/CCF” populations might be a mixture of subclones mutations represent admixed polyphyletic lineages 14.
mut.cluster <- mutCluster(maf, patient.id = 'V402',
use.ccf = TRUE, use.tumorSampleLabel = TRUE)
clusterPlots <- mut.cluster$cluster.plot
cowplot::plot_grid(plotlist = clusterPlots[1:6])
6.2 Between regions/tumors
To quantify the genetic divergence of ITH between regions or tumors, we introduced two classical metrics derived from population genetics, which were Wright’s fixation index (Fst) 5 6 7 and Nei’s genetic distance 8.
6.2.1 Fixation index
# calculate the Fst of brain metastasis from V402
calFst(maf, patient.id = 'V402', plot = TRUE, use.tumorSampleLabel = TRUE,
withinTumor = TRUE, number.cex = 10)[["V402_BM"]]## $Fst.avg
## [1] 0.03600727
##
## $Fst.pair
## BM_1 BM_2 BM_3 BM_4
## BM_1 1.00000000 0.03825504 0.05133115 0.02173604
## BM_2 0.03825504 1.00000000 0.04722441 0.02393010
## BM_3 0.05133115 0.04722441 1.00000000 0.03356684
## BM_4 0.02173604 0.02393010 0.03356684 1.00000000
##
## $Fst.plot
6.2.2 Nei’s genetic distance
# calculate the Nei's genetic distance of brain metastasis from V402
calNeiDist(maf, patient.id = 'V402', use.tumorSampleLabel = TRUE,
withinTumor = TRUE, number.cex = 10)[["V402_BM"]]## $Nei.dist.avg
## [1] 0.01008835
##
## $Nei.dist
## BM_1 BM_2 BM_3 BM_4
## BM_1 1.000000000 0.007271303 0.01751193 0.004240568
## BM_2 0.007271303 1.000000000 0.01573008 0.004302810
## BM_3 0.017511933 0.015730084 1.00000000 0.011473404
## BM_4 0.004240568 0.004302810 0.01147340 1.000000000
##
## $Nei.plot
7. Metastatic routes inference
Metastasis remains poorly understood despite its critical clinical significance, and the understanding of metastasis process offers supplementary information for clinical treatments.
7.1 Pairwise CCF comparison
Distinct patterns of monoclonal versus polyclonal seeding based on the CCFs of somatic mutations between sample/tumor pairs. compareCCF function returns a result list of pairwise CCF of mutations, which are identified across samples from a single patient. Recently, this method has been widely used to deduce the potential metastatic route between different paired tumor lesions 9 10.
ccf.list <- compareCCF(maf, pairByTumor = TRUE, min.ccf = 0.02,
use.adjVAF = TRUE, use.indel = FALSE)
V402_P_BM <- ccf.list$V402$`P-BM`
# visualize via smoothScatter R package
graphics::smoothScatter(matrix(c(V402_P_BM[, 3], V402_P_BM[, 4]),ncol = 2),
xlim = c(0, 1), ylim = c(0, 1),
colramp = colorRampPalette(c("white", RColorBrewer::brewer.pal(9, "BuPu"))),
xlab = "P", ylab = "BM")
## show driver genes
gene.idx <- which(V402_P_BM$Hugo_Symbol %in% driverGene)
points(V402_P_BM[gene.idx, 3:4], cex = 0.6, col = 2, pch = 2)
text(V402_P_BM[gene.idx, 3:4], cex = 0.7, pos = 1,
V402_P_BM$Hugo_Symbol[gene.idx])
title("V402 JSI = 0.341", cex.main = 1.5)
7.2 Jaccard similarity index
The Jaccard similarity index (JSI) can be used to calculate mutational similarity between regions, which is defined as the ratio of shared variants to all variants for sample pairs 11. Users can distinguish monoclonal versus polyclonal seeding in metastases (including lymph node metastases and distant metastases) via calJSI function, and higher JSI values indicate the higher possibility of polyclonal seeding 12.
JSI.res <- calJSI(maf, patient.id = 'V402', pairByTumor = TRUE, min.ccf = 0.02,
use.adjVAF = TRUE, use.indel = FALSE, use.tumorSampleLabel = TRUE)
names(JSI.res)## [1] "JSI.multi" "JSI.pair"## [1] 0.3410853## P BM
## P 1.0000000 0.3410853
## BM 0.3410853 1.00000007.3 Neutral evolution
The subclonal mutant allele frequencies of a tumor follow a simple power-law distribution predicted by neutral growth 13. Users can evaluate whether a tumor follows neutral evolution or not under strong selection via the testNeutral function. Tumors with R2 >= R2.threshold (Default: 0.98) are considered to follow neutral evolution. Besides, this function can also generate the model fitting plot of each sample if the argument plot is set toTRUE. Please note that this analysis has been superseded by mobster 14.
neutralResult <- testNeutral(maf, min.mut.count = 10, patient.id = 'V402', use.tumorSampleLabel = TRUE)
neutralResult$neutrality.metrics
8. Phylogenetic tree analysis
8.1 Phylogenetic tree construction
With MesKit, phylogenetic tree construction for each individual is based on the binary present/absence matrix of mutations across all tumor regions.
Based on the Maf object, getPhyloTree function reconstructs phylogenetic tree in different methods, including “NJ” (Neibor-Joining) , “MP” (maximum parsimony), “ML” (maximum likelihood), “FASTME.ols” and “FASTME.bal”, which can be set by controlling the method parameter. The phylogenetic tree would be stored in a phyloTree/phyloTreeList object, and it can be further used for functional exploration, mutational signature analysis and tree visualization.
8.2 Compare Phylogenetic trees
Comparison between phylogenetic trees can reveal consensus patterns of tumor evolution. The compareeTree function computes distances between phylogenetic trees constructed through different methods, and it returns a vector containing four distances by treedist from phangorn R package. See treedist for details.
tree.NJ <- getPhyloTree(maf, patient.id = 'V402', method = "NJ")
tree.MP <- getPhyloTree(maf, patient.id = 'V402', method = "MP")
# compare phylogenetic trees constructed by two approaches
compareTree(tree.NJ, tree.MP, plot = TRUE, use.tumorSampleLabel = TRUE)## 4 clades are common between two trees.## $compare.dist
## Symmetric.difference KF-branch distance Path difference
## 4.000000 115.559715 5.291503
## Weighted path difference
## 715.926657
##
## $compare.plot
8.3 Functional exploration (custom module)
Users can conduct functional exploration based on phyloTree objects. Below is an example showing how to perform KEGG enrichment analysis via ClusterProfiler by extracting genes in certain categories from a phylotree object.
library(org.Hs.eg.db)
library(clusterProfiler)
# Pathway enrichment analysis
V402.branches <- getMutBranches(phyloTree)
# pathway enrichment for private mutated genes of the primary tumor in patient V402
V402_Public <- V402.branches[V402.branches$Mutation_Type == "Private_P", ]
geneIDs = suppressMessages(bitr(V402_Public$Hugo_Symbol, fromType="SYMBOL",
toType=c("ENTREZID"), OrgDb="org.Hs.eg.db"))
KEGG_V402_Private_P = enrichKEGG(
gene = geneIDs$ENTREZID,
organism = 'hsa',
keyType = 'kegg',
pvalueCutoff = 0.05,
)
dotplot(KEGG_V402_Private_P)
8.4 Mutational characteristics analysis
The sequence context of the base substitutions can be retrieved from the corresponding reference genome to construct a mutation matrix with counts for all 96 trinucleotide changes using “mut_matrix”. Subsequently, the 6 base substitution type spectrum can be plotted with “plot_spectrum” , which can be divided into several sample groups.
mutTrunkBranch function calculates the fraction of branch/trunk mutations occurring in each of the six types of base substitution types (C>A, C>G, C>T, T>A, T>C, T>G) and conducts two-sided Fisher’s exact tests. For C>T mutations, it can be further classified as C>T at CpG sites or other sites by setting CT = TRUE. This function provides option plot to show the distribution of branch/trunk mutations. Substitutions types with significant difference between trunk and branch mutations are marked with Asterisks.
## [1] "mutTrunkBranch.res" "mutTrunkBranch.plot"
triMatrix function generates a mutation count matrix of 96 trinucleotides based on somatic SNVs per sample, which can be later fed into the fitSignatures function to estimates the optimal contributions of known signatures to reconstruct a mutational profile. Besides, the signature matrix can be specified ("cosmic_v2", "exome_cosmic_v3" and "nature2013") or provided by users via signaturesRef parameter. plotMutSigProfile function can be utilized to visualize both original mutational profile and reconstructed mutational profile.
trimatrix_V402 <- triMatrix(phyloTree, level = 5)
# Visualize the 96 trinucleodide mutational profile
plotMutSigProfile(trimatrix_V402)[[1]]
# reconstruct mutational profile of V402 using COSMIC V2 signatures
fit_V402 <- fitSignatures(trimatrix_V402, signaturesRef = "cosmic_v2")
# Compare the reconstructed mutational profile with the original mutational profile
plotMutSigProfile(fit_V402)[[1]]
# Below plot shows cosine similarities between the mutational profile of each group and COSMIC signatures
library(ComplexHeatmap)
ComplexHeatmap::Heatmap(fit_V402$V402$cosine.similarity, name = "Cosine similarity")
9. Phylogenetic tree visualization
The plotPhyloTree functionan of MesKit implemented an auto-layout algorithm to visualize rooted phylogenetic trees with annotations. The branches can be either colored according to classification of mutations or putative known signatures by branchCol parameter. Argument show.bootstrap is provided to show the support values of internal nodes. Additionally, a phylogenetic tree along with a heatmap of mutation profile (via mutHeatmap functionan) enable a better depiction of mutational patterns in tumor phylogeny.
# A phylogenetic tree along with binary and CCF heatmap of mutations
phylotree_V402 <- plotPhyloTree(phyloTree, use.tumorSampleLabel = TRUE)
binary_heatmap_V402 <- mutHeatmap(maf, min.ccf = 0.04, use.ccf = FALSE, patient.id = "V402", use.tumorSampleLabel = TRUE)
CCF_heatmap_V402 <- mutHeatmap(maf, use.ccf = TRUE, patient.id = "V402",
min.ccf = 0.04, use.tumorSampleLabel = TRUE)
cowplot::plot_grid(phylotree_V402, binary_heatmap_V402,
CCF_heatmap_V402, nrow = 1, rel_widths = c(1.5, 1, 1))
10. Shiny APP with video tutorial
The guidance video for MesKit Shiny APP can be found at http://meskit.renlab.org/video.html.
11. References
Mroz, E. A., & Rocco, J. W. (2013). MATH, a novel measure of intratumor genetic heterogeneity, is high in poor-outcome classes of head and neck squamous cell carcinoma. Oral oncology, 49(3), 211–215. https://doi.org/10.1016/j.oraloncology.2012.09.007
Mroz, E. A., Tward, A. D., Pickering, C. R., Myers, J. N., Ferris, R. L., & Rocco, J. W. (2013). High intratumor genetic heterogeneity is related to worse outcome in patients with head and neck squamous cell carcinoma. Cancer, 119(16), 3034–3042. https://doi.org/10.1002/cncr.28150
Mroz, E. A., Tward, A. D., Hammon, R. J., Ren, Y., & Rocco, J. W. (2015). Intra-tumor genetic heterogeneity and mortality in head and neck cancer: analysis of data from the Cancer Genome Atlas. PLoS medicine, 12(2), e1001786. https://doi.org/10.1371/journal.pmed.1001786
Charoentong, P., Finotello, F., Angelova, M., Mayer, C., Efremova, M., Rieder, D., Hackl, H., & Trajanoski, Z. (2017). Pan-cancer Immunogenomic Analyses Reveal Genotype-Immunophenotype Relationships and Predictors of Response to Checkpoint Blockade. Cell reports, 18(1), 248–262. https://doi.org/10.1016/j.celrep.2016.12.019
Weir, B. S., & Cockerham, C. C. (1984). ESTIMATING F-STATISTICS FOR THE ANALYSIS OF POPULATION STRUCTURE. Evolution; international journal of organic evolution, 38(6), 1358–1370. https://doi.org/10.1111/j.1558-5646.1984.tb05657.x
Bhatia, G., Patterson, N., Sankararaman, S., & Price, A. L. (2013). Estimating and interpreting FST: the impact of rare variants. Genome research, 23(9), 1514–1521. https://doi.org/10.1101/gr.154831.113
Holsinger, K. E., & Weir, B. S. (2009). Genetics in geographically structured populations: defining, estimating and interpreting F(ST). Nature reviews. Genetics, 10(9), 639–650. https://doi.org/10.1038/nrg2611
Puente, X. S., Pinyol, M., Quesada, V., Conde, L., Ordóñez, G. R., Villamor, N., Escaramis, G., Jares, P., Beà, S., González-Díaz, M., Bassaganyas, L., Baumann, T., Juan, M., López-Guerra, M., Colomer, D., Tubío, J. M., López, C., Navarro, A., Tornador, C., Aymerich, M., … Campo, E. (2011). Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia. Nature, 475(7354), 101–105. https://doi.org/10.1038/nature10113
Hu, Z., Ding, J., Ma, Z., Sun, R., Seoane, J. A., Scott Shaffer, J., Suarez, C. J., Berghoff, A. S., Cremolini, C., Falcone, A., Loupakis, F., Birner, P., Preusser, M., Lenz, H. J., & Curtis, C. (2019). Quantitative evidence for early metastatic seeding in colorectal cancer. Nature genetics, 51(7), 1113–1122. https://doi.org/10.1038/s41588-019-0423-x
Xue, R., Chen, L., Zhang, C., Fujita, M., Li, R., Yan, S. M., Ong, C. K., Liao, X., Gao, Q., Sasagawa, S., Li, Y., Wang, J., Guo, H., Huang, Q. T., Zhong, Q., Tan, J., Qi, L., Gong, W., Hong, Z., Li, M., … Zhang, N. (2019). Genomic and Transcriptomic Profiling of Combined Hepatocellular and Intrahepatic Cholangiocarcinoma Reveals Distinct Molecular Subtypes. Cancer cell, 35(6), 932–947.e8. https://doi.org/10.1016/j.ccell.2019.04.007
Makohon-Moore, A. P., Zhang, M., Reiter, J. G., Bozic, I., Allen, B., Kundu, D., Chatterjee, K., Wong, F., Jiao, Y., Kohutek, Z. A., Hong, J., Attiyeh, M., Javier, B., Wood, L. D., Hruban, R. H., Nowak, M. A., Papadopoulos, N., Kinzler, K. W., Vogelstein, B., & Iacobuzio-Donahue, C. A. (2017). Limited heterogeneity of known driver gene mutations among the metastases of individual patients with pancreatic cancer. Nature genetics, 49(3), 358–366. https://doi.org/10.1038/ng.3764
Hu, Z., Li, Z., Ma, Z., & Curtis, C. (2020). Multi-cancer analysis of clonality and the timing of systemic spread in paired primary tumors and metastases. Nature genetics, 52(7), 701–708. https://doi.org/10.1038/s41588-020-0628-z
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12. Session Info
## R version 4.0.4 (2021-02-15)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 18.04.5 LTS
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## attached base packages:
## [1] grid parallel stats4 stats graphics grDevices utils
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## other attached packages:
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## [3] BSgenome_1.58.0 rtracklayer_1.50.0
## [5] Biostrings_2.58.0 XVector_0.30.0
## [7] GenomicRanges_1.42.0 GenomeInfoDb_1.26.4
## [9] clusterProfiler_3.18.1 org.Hs.eg.db_3.12.0
## [11] AnnotationDbi_1.52.0 IRanges_2.24.1
## [13] S4Vectors_0.28.1 Biobase_2.50.0
## [15] BiocGenerics_0.36.0 MesKit_1.0.1
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## [29] jsonlite_1.7.2 scatterpie_0.1.5
## [31] phangorn_2.6.2 ape_5.4-1
## [33] glue_1.4.2 polyclip_1.10-0
## [35] gtable_0.3.0 zlibbioc_1.36.0
## [37] GetoptLong_1.0.5 DelayedArray_0.16.3
## [39] shape_1.4.5 scales_1.1.1
## [41] DOSE_3.16.0 DBI_1.1.1
## [43] Rcpp_1.0.6 viridisLite_0.3.0
## [45] clue_0.3-58 bit_4.0.4
## [47] mclust_5.4.7 fgsea_1.16.0
## [49] RColorBrewer_1.1-2 ellipsis_0.3.1
## [51] pkgconfig_2.0.3 XML_3.99-0.6
## [53] farver_2.1.0 sass_0.3.1
## [55] utf8_1.2.1 tidyselect_1.1.0
## [57] labeling_0.4.2 rlang_0.4.10
## [59] reshape2_1.4.4 munsell_0.5.0
## [61] tools_4.0.4 cachem_1.0.4
## [63] downloader_0.4 generics_0.1.0
## [65] RSQLite_2.2.5 ggridges_0.5.3
## [67] evaluate_0.14 stringr_1.4.0
## [69] fastmap_1.1.0 yaml_2.2.1
## [71] knitr_1.31 bit64_4.0.5
## [73] tidygraph_1.2.0 purrr_0.3.4
## [75] ggraph_2.0.5 nlme_3.1-152
## [77] pracma_2.3.3 DO.db_2.9
## [79] debugme_1.1.0 compiler_4.0.4
## [81] png_0.1-7 tibble_3.1.0
## [83] tweenr_1.0.2 bslib_0.2.4
## [85] stringi_1.5.3 highr_0.8
## [87] lattice_0.20-41 Matrix_1.3-2
## [89] vctrs_0.3.6 pillar_1.5.1
## [91] lifecycle_1.0.0 BiocManager_1.30.10
## [93] jquerylib_0.1.3 GlobalOptions_0.1.2
## [95] data.table_1.14.0 cowplot_1.1.1
## [97] bitops_1.0-6 qvalue_2.22.0
## [99] R6_2.5.0 KernSmooth_2.23-18
## [101] gridExtra_2.3 codetools_0.2-18
## [103] MASS_7.3-53.1 assertthat_0.2.1
## [105] SummarizedExperiment_1.20.0 rjson_0.2.20
## [107] GenomicAlignments_1.26.0 Rsamtools_2.6.0
## [109] GenomeInfoDbData_1.2.4 mgcv_1.8-34
## [111] quadprog_1.5-8 tidyr_1.1.3
## [113] rmarkdown_2.7 rvcheck_0.1.8
## [115] MatrixGenerics_1.2.1 Cairo_1.5-12.2
## [117] ggforce_0.3.3