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I have two bam files from single-cell RNA sequencing mapped to the reference genome using CellRanger, I can view them in IGV and I have a particular region where the pattern of reads mapped to the reference genome are different between the two bam files but when I try the following code to plot trackplots with coverage, the resulting plot is not consistent with what I see in IGV. I am not very familiar with this, aren't I supposed to normalize the data as well? please help me.

txdb <-TxDb.Hsapiens.UCSC.hg38.knownGene
txTr <- GeneRegionTrack(txdb, chromosome = "chr1", 
                        start = 153561336,  end = 153561564)
dTrack <- DataTrack(range = bam.file, genome = "hg38", type = "l", 
                     name = "Coverage", window = -1, 
                     chromosome = "chr1")

dTrack0 <- DataTrack(range = bam.file.0, genome = "hg38", type = "l", 
                     name = "Coverage", window = -1, 
                     chromosome = "chr1", col="red")

plotTracks(c(dTrack,dTrack0, txTr), from = 153561357, to = 153561585)

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  • $\begingroup$ Welcome @A4747 its useful to describe what you are dealing with, e.g. humans, a model system and any further project details. Essentially the answer will be project specific. Anyway provided an appropriate summary paper. $\endgroup$
    – M__
    Jun 6, 2023 at 22:58

1 Answer 1

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I would recommend reading the following study by Zhao et al (2021).

The traditional simplest measure is CPM otherwise known as TPM. This is counts/transcripts per million. FPKM (fragments per kilobase of transcript per million fragments mapped) is used for metagenomics. I don't know its application in your context ... I don't know your context.

The discussions for the pros- and cons- of FPKM and TPM/CPM are discussed therein.

The authors in the study favour normalisation - in their context - not in general. The authors use the coefficient of variation, and intraclass correlation coefficient. I would see this as a form of standardisation. It's worth keeping an open mind because its a pivotal step in the calculation but all too easily overlooked area of investigation.

From RamRS's comments that may have changed already. In general statistics standardisation is a pivotal step, usually followed by the removal of outliers.

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  • $\begingroup$ Unfortunately, neither TPM nor CPM can be used in any context to compare two samples. FPKM is obsolete IMO $\endgroup$
    – Ram RS
    Jun 6, 2023 at 23:17
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    $\begingroup$ FPKM is used in metagenomics (a lot, lot more than one species present). Recovering a full length gene/transcript rarely happens. $\endgroup$
    – M__
    Jun 6, 2023 at 23:27
  • $\begingroup$ Thank you, I did not know that. $\endgroup$
    – Ram RS
    Jun 7, 2023 at 13:11
  • $\begingroup$ @M__ Thanks for your response. I agree that in this case TPM normalization may be required when displaying them in a trackplot. I think those types of normalization may not be accurate when performing differential expression analysis through DESeq. However, my particular problem is with the fact that what I see in IGV ends up being different from what I see when I plot the bam file in R through the script written above. $\endgroup$
    – A4747
    Jun 9, 2023 at 17:25

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