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Hi, first, paired-seq is a really good protocol for joint profiling RNA and DNA(ATAC) on the same cell!

Now, I want to use the paired-seq protocol with slight customizing: changing Linker sequence slightly and only 3 BC rounds.

In my case, which script should I change?

I see that reachtool's combine function deals with BC and UMIs.
reachtools.h has a section for bc_library and trimming linkers:

If I change reachtools.h's trim() function, can I modify BCs and linker sequence?

thanks!

Hi Chenxu,

I notice the function of bam2Mtx in the reachtools package need a ref parameter (reachtools Bam2Mtx in.bam/in.sam [ref], ref is optional for RNA-seq but mandatory for ATAC-seq) However in the main.cpp, the ref parameter point to private files (such as mm10_1kb, does it mean a 1kb bed file for mm10 genome?) which are not available for custom users. Could you please provide a header in the instruction if you are convenient？ Thanks a lot!

Best,
Fangnong

Hi,

I am running into errors Cannot open ref file when executing the last step reachtools bam2Mtx CZ${s}_mm10.bam mm10_1kb in proc_DNA.sh.

It seems currently the ref file is hard-coded in main.cpp https://github.com/cxzhu/Paired-seq/blob/master/ReachTools/main.cpp#L120

else if(ref == "mm10_1k"){
     ref = "/projects/ps-renlab/chz272/genome_ref/mm10.bin1k.txt";

What would you suggest I do to get rid of this error and where can I find this file?

Thanks!

Hi, Dr. Zhu,
I'm processing the RNA part of Paired-seq data using Seurat following these codes

counts <- Read10X('./data/Adult_Cerebrail_Cortex/Adult_CTX_RNA/')
metadata <- read.csv('./data/Adult_Cerebrail_Cortex/Cell_embeddings.csv')
rownames(metadata) <- metadata$ID
metadata <- metadata[colnames(RNA),]
RNA$batch <- metadata$Rep
RNA$cluster <- metadata$Cluster
RNA$true <- as.character(factor(metadata$Cluster,labels = c('AS','MG','OC','Ex1','Ex2','Ex3','In1','In2','In3')))
RNA <- NormalizeData(RNA)
RNA <- FindVariableFeatures(RNA)
LabelPoints(plot = VariableFeaturePlot(RNA), points = head(VariableFeatures(RNA), 10), repel = TRUE)
RNA <- ScaleData(RNA)
RNA <- RunPCA(RNA)
RNA <- RunUMAP(RNA, dims = 1:20)
DimPlot(RNA, group.by = "true", label = TRUE) + NoLegend()

But I got this strange UMAP output of the RNA data

Similarly, I process the ATAC part of Paired-seq data using Signac following these codes

counts <- Read10X("./data/Adult_Cerebrail_Cortex/Adult_CTX_DNA/")
chrom_assay <- CreateChromatinAssay(counts,sep = c(":", "-"))
ATAC <- CreateSeuratObject(chrom_assay,assay = "ATAC")
ATAC <- RunTFIDF(ATAC)
ATAC <- FindTopFeatures(ATAC, min.cutoff = 'q0')
ATAC <- RunSVD(ATAC)
DepthCor(ATAC,n = 50)
ATAC <- RunUMAP(ATAC, dims = 2:30, reduction = 'lsi')
metadata <- read.csv('./data/Adult_Cerebrail_Cortex/Cell_embeddings.csv')
rownames(metadata) <- metadata$ID
metadata <- metadata[colnames(ATAC),]
ATAC$batch <- metadata$Rep
ATAC$cluster <- metadata$Cluster
ATAC$true <- as.character(factor(metadata$Cluster, labels = c('AS','MG','OC','Ex1','Ex2','Ex3','In1','In2','In3')))
DimPlot(object = ATAC, label = T,group.by = 'true') + NoLegend()

And I got this

Could you please give me some suggestions on how to deal with Pair-seq data in a proper way? Many thanks!

I download the raw read and try to get the valid RNA data. The 'proc_RNA.sh' can't run correctly. How can I get RNA data with correct barcode with the rawreads (SRR8980195).

Dear author,

Thanks for your awesome work. After reading your paper, I want to know more about the maker gene of the cerebral cortex cells data, but I cannot find this file on github. Maybe I miss it carelessly, and could you please tell me where it was placed or upload it?

Hi Chenxu,

I got high proportions of cell ids suppressed due to mismatch. Is it expected outcome or did I do something wrong?

Best regards,
Zhen

Hi Chenxu,

I downloaded the dataset from SRA and run reachtools combine2 on R1 and R2, but the full barcoded reads were reported at only 0.02%. Is that normal?

Best,
Zhen