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Millefy: Genome browser-like visualization of single-cell RNA-seq dataset

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Use with 10x data and detecting loss of chromosomes

ccruizm opened this issue · comments

commented

Good day,

I would like to know whether I can use your tool to check coverage in 10x genomics data. My aim is to check the coverage in some specific chromosomes per cluster. I have tumor samples and, as many others do, I assign malignant cells to those that have CNV (based on transcriptomics data). I want to visualize and confirm that some clusters lack genes on those specific chromosomes (loss). Is it possible to use Millefy for this purpose? can I get a plot similar to the one used for the example, but instead of depicting two conditions, divide the gene coverage per cell cluster?

Thanks in advance!

commented

Thank you for asking!

Before answering the question, let me clarify the following points for "10x genomics data":

  1. The data is BAM?
  2. The data contains information for all cells in one file?
commented

Hello @yuifu

Yes, the data is in BAM format and all cells are in the file, including other reads that are not used in the analysis but BAM files can be filtered for only those cells I want to analyze.

commented

@ccruizm Thanks!

Two options:

If you split a BAM file into BAM files for different cells (barcodes) using some tools (e.g. https://github.com/walaj/bxtools#split), you can use Millefy right now.
(For example, you have 3000 cells (barcode) in a BAM files, you will get 3000 BAM files after splitting.)

Currently, Millefy doesn't have native support for 10x genomics type BAM file (a BAM file with many different barcodes). So, you have to wait some time for me to make a new module to parse such file.

commented

Thanks for your reply @yuifu.

I will give it a try. Support for 10x BAM files will be a great tool! Most of the current experiments in scRNA are performed on that platform :)

commented

Hi, Thanks for the nice tool, but I also have some hard time figuring out how to launch it on individual cells. I've figured out that I need to split 10x bam into induvidual bam files, but what next? Should I also convert them into bigwig?

As far as I understand millefy can take bam as input, but so far I faild even to run the example dataset starting from bam:

bwfiles = Sys.glob(file.path(system.file("extdata/bam", package="millefy"), "*.bam"))
.
.
and set " isBw" to FALSE
scTrackBw <- list(path_bam_files = bwfiles, groups = groups, group_colors = color_labels, max_value = max_value, isBw=FALSE)

the plotting then through an error (
also If I'll have several K of cells how my "groups" variable suppose to look like? Is there a naming convention for individual bam files?

So if you can give me a hand on making the plot like in your original paper that would be super helpful!

Best, Eugene

commented

@EugeneEA Eugene

could you find a way how to use multiple BAM files from two groups?
thanks,

commented

@EugeneEA Eugene

could you find a way how to use multiple BAM files from two groups? thanks,

Hi, it was some time ago, so I do not remember it for sure, but I think I converted bam to BigWig via these two commands

bedtools genomecov -bg -ibam hHPCS_"$p".bam > hHPCS_"$p".bam.bedgraph
bedGraphToBigWig "$filename" ../chrom.sizes "$filename".bw

best, Eugene