Identifying CNV events
The expression values should be normalized using TPM or FPKM. Below is an example normalized gene expression input :
library(CaSpER)
data("scell_gbm")
data <- scell_gbm$data
loh <- scell_gbm$loh
loh.name.mapping <- scell_gbm$loh.name.mappingNormalized gene expression matrix rows represents genes either with ensembl gene id or hgnc gene symbol. Columns represent samples or cells.
Cytoband information can be downloaded from UCSC.
http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/cytoBand.txt.gzdata(hg19_cytoband)
kable(cytoband[1:5, ])| V1 | V2 | V3 | V4 |
|---|---|---|---|
| 1 | 1.00e+00 | 125000001 | p |
| 1 | 1.25e+08 | 249250622 | q |
| 2 | 1.00e+00 | 93300001 | p |
| 2 | 9.33e+07 | 243199374 | q |
| 3 | 1.00e+00 | 91000001 | p |
Centromere information can be downloaded from UCSC.
#curl -s "http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/cytoBand.txt.gz" | gunzip -c | grep acen | headAnnotation data which contains the positions of each gene along each chromosome in the genome can be generated using the following functions:
annotation <- generateAnnotation(id_type="hgnc_symbol", genes=rownames(data), ishg19=T, centromere)
data <- data[match( annotation$Gene,rownames(data)), ]Identifying CNV events:
The main functions you will need to use to identify CNV events are CreateCasperObject() and runCaSpER(casper_object).
1. Creating casper object
The casper object is required for performing CNV analysis on single-cell and bulk RNA-Seq. Casper object is created using following functions
object <- CreateCasperObject(raw.data=data,loh.name.mapping=loh.name.mapping,
sequencing.type="single-cell",
cnv.scale=3, loh.scale=3,
annotation=annotation, method="iterative", loh=loh,
control.sample.ids="REF", cytoband=cytoband)Above CaSpER object stores all information associated with the dataset, including expression data, smoothed expression data in n (default:3) different scales, original baf signal, smoothed baf signal, annotations, control sample ids.
2. Pairwise comparison of scales from BAF and expression signals
After creating the casper object we perform HMM on all scales of expression signal and integrate HMM segment states with Allele Frequency Shift Information. CaSpER algorithm outputs CNV calls using all the pairwise comparisons of expression signal and BAF signal decompositions.
final.objects <- runCaSpER(object, removeCentromere=T, cytoband=cytoband, method="iterative")3. Harmonization and Summarization of CNV calls from multiple scales and from multiple pairwise comparison of BAF and Expression Signals
The pairwise comparison and assignment of CNV calls generates a large number of per-scale information that must be summarized such that each position of the genome is assigned a final call about its CNV status, i.e., deletion/amplification/neutral. We use a consistency-based approach for harmonizing the pairwise comparisons: The events are put together and we assign the final CNV for a gene or large-scale event if the CNV calls are consistent among at least a certain number of pairwise scale comparisons. CaSpER harmonizes and summarizes the CNV calls by dividing them into large-scale, gene-based, and segment-based CNV calls
We assign a large-scale CNV call to every chromosome arm for each of the N×M pairwise scale comparisons. Next, for each chromosome arm, we ask whether the large-scale CNV call is consistent among at least \(\gamma\) of the N×M large-scale CNV calls. N denotes the index for the highest smoothing scale for expression signal. M denotes the index for the highest smoothing scale for baf signal. thr represents minimum percentage, 75% (at least 7 out of 9 scales), of consistent CNV calls (Out of N×M comparisons of expression scales and BAF scales) while assigning a final CNV (amp/del/neutral) call to a segment/gene/chromosome arm.
finalChrMat <- extractLargeScaleEvents (final.objects, thr=0.75) The segments-based summarization aims at generating a final set of CNV calls for a final set of segments that are computed by comparison of scales. We first compare the segments from different expression scales and generate the consistent set of segments. For each segment in the final set, if there are more than ?? (default=6) consistent CNV calls among N×M CNV calls, we assign the consistent CNV call to segment. When there is no consistency among the calls, we assign a neutral CNV state to segment.
gamma <- 7
all.segments <- do.call(rbind, lapply(final.objects, function(x) x@segments))
segment.summary <- extractSegmentSummary (final.objects)
loss <- segment.summary$all.summary.loss
gain <- segment.summary$all.summary.gain
loss.final <- loss[loss$count>=gamma, ]
gain.final <- gain[gain$count>=gamma, ]Calculate large scale event accuracy using snp genotyping array as gold standard.
genoMat <- tcga_gbm$genoMat
common <- intersect(rownames(finalChrMat), rownames(genoMat))
finalChrMat <- finalChrMat[match(common, rownames(finalChrMat)), ]
genoMat <- genoMat[match(common, rownames(genoMat)), ]
calcROC(chrMat=finalChrMat, chrMat2=genoMat)Similar to the large-scale summarization, we generate a matrix where rows are the samples (cells) and columns are the genes. The matrix entry of 0 corresponds to no alteration, 1 corresponds to amplification and -1 corresponds to deletion. If an alteration is consistent in more than ?? scale comparisons (out of N×M comparisons), we report that alteration event for that sample.
all.summary<- rbind(loss.final, gain.final)
colnames(all.summary) [2:4] <- c("Chromosome", "Start", "End")
rna <- GRanges(seqnames = Rle(gsub("q", "", gsub("p", "", all.summary$Chromosome))),
IRanges(all.summary$Start, all.summary$End))
ann.gr <- makeGRangesFromDataFrame(final.objects[[1]]@annotation.filt, keep.extra.columns = TRUE, seqnames.field="Chr")
hits <- findOverlaps(geno.rna, ann.gr)
genes <- splitByOverlap(ann.gr, geno.rna, "GeneSymbol")
genes.ann <- lapply(genes, function(x) x[!(x=="")])
all.genes <- unique(final.objects[[1]]@annotation.filt[,2])
all.samples <- unique(as.character(final.objects[[1]]@segments$ID))
rna.matrix <- gene.matrix(seg=all.summary, all.genes=all.genes, all.samples=all.samples, genes.ann=genes.ann)Calculate gene based TPR and FPR using genotyping array as gold standard
segments <- yale_meningioma$segments
segments$type<- segments$Type_Corrected
geno.gr <- GRanges(seqnames = Rle(gsub("q", "", gsub("p", "", segments$chr))), IRanges(segments$start, segments$end))
hits <- findOverlaps(geno.gr, ann.gr)
genes <- splitByOverlap(ann.gr, geno.gr, "GeneSymbol")
genes.ann <- lapply(genes, function(x) x[!(x=="")])
gt.matrix <- gene.matrix(seg=segments, all.samples=all.samples, all.genes=all.genes, genes.ann=genes.ann)
calcROC(chrMat=rna.matrix, chrMat2=gt.matrix)all.summary <- getDiffExprGenes(final.objects, sampleName="MGH31", chrs=c("5q", "14q"), event.type=c(1, -1))4.Visualization
In order to comprehensively visualize the CNV events, CaSpER first generates visuals that combine all the samples so that user can view the events jointly over all samples and co-occurrence and mutually exclusive patterns can be visually inspected. To characterize these patterns, CaSpER performs clustering of samples and clustering of detected CNVs and generates clustering plots. We summarize these visualizations below.
obj <- final.objects[[9]]
plotHeatmap(object=obj, fileName="heatmap.png",cnv.scale= 3, cluster_cols = F, cluster_rows = T, show_rownames = T, only_soi = T)plotLargeScaleEvent (object=obj, fileName="large.scale.events.png") plotSingleCellLargeScaleEventHeatmap(finalChrMat, sampleName="MGH31", chrs=c("5p", "14q"))## calculate significant mutual exclusive and co-occurent events
results <- extractMUAndCooccurence (finalChrMat, loh, loh.name.mapping)
## visualize mutual exclusive and co-occurent events
plotMUAndCooccurence (results)