Can I directly compare the regions defined by peaks from pair-end 50 and pair-end 100 ATAC-seq?
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11 weeks ago
Dan ▴ 60

My ATAC-seq samples have both pair-end 50 and pair-end 100 reads. After peak calling using macs2, can I directly compare the counts of the regions defined by these peaks from different read lengths?

library(Rsubread)
library(GenomicRanges)
library(DESeq2)

peaks <- dir("peaks/", pattern = "*.narrowPeak", 
             full.names = TRUE)

myPeaks <- lapply(peaks, ChIPQC:::GetGRanges, simple = TRUE)

myGRangesList<-GRangesList(myPeaks)   
reduced <- reduce(unlist(myGRangesList))
consensusIDs <- paste0("consensus_", seq(1, length(reduced)))
mcols(reduced) <- do.call(cbind, lapply(myGRangesList, function(x) (reduced %over% x) + 0))
reducedConsensus <- reduced
mcols(reducedConsensus) <- cbind(as.data.frame(mcols(reducedConsensus)), consensusIDs)

bamsToCount <- dir("bam/", full.names = TRUE, pattern = "*.\\.bam$")


regionsToCount <- data.frame(GeneID = paste("ID", seqnames(reducedConsensus), 
                                            start(reducedConsensus), end(reducedConsensus), sep = "_"), Chr = seqnames(reducedConsensus), 
                             Start = start(reducedConsensus), End = end(reducedConsensus), Strand = strand(reducedConsensus))


fcResults <- featureCounts(bamsToCount, annot.ext = regionsToCount, isPairedEnd = TRUE, 
                           countMultiMappingReads = FALSE, maxFragLength=1000)

myCounts <- fcResults$counts

colnames(myCounts) <- c("sample_1", "sample_2", "sample_3", "sample_4")



# [generate the metadata] to describe/group the samples
Group <- factor(c("Control", "Control", "Treat","Treat"))

metaData <- data.frame(Group, row.names = colnames(myCounts))
atacDDS <- DESeqDataSetFromMatrix(myCounts, metaData, ~Group, rowRanges = reducedConsensus)
atacDDS <- DESeq(atacDDS)

atac_Rlog <- rlog(atacDDS)

rawCounts <- counts(atacDDS, normalized = FALSE)

Thanks

ATAC-seq • 478 views
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Please define compare.

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I am sorry the question was not clear. I edited it. Could you tell me if the codes are reasonable or not? Thanks

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I see. Basically that is correct I think, so using a dedicated stats framework like DESeq2. The impact of read length is probably minor and limited to regions that are difficult to map. Still, to be sure you can trim the reads all down to 50bp and then repeat alignment.

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I used trim_galore --illumina --paired --fastqc to trim the adaptors, is it possible and how should I trim the reads to pair-end 50? Thanks a lot.

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trim_galore has parameters that can trim specific bp from either end: --clip_R1, --clip_R2, --three_prime_clip_R1, --three_prime_clip_R2, which parameter should I use for my situation? Thanks a lot.

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You will need to clip on 3'-end for both reads.

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I see. Thanks

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I always use seqtk for these kinds of things.

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Is seqtk better than trim_galore?

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I do not know, I do not use trim_galore. If trim_galore can do the job it is fine.

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