question about 10X sc-RNA-seq

Question

[fuhaolll2]

Hi, i know 10X sc-RNA-seq is 3'only ,and 10X' reads sequence from 3' about 100bp. So should most of these reads cover with the last exon or 3'UTR?

[GenoMax]

10x does have 5' scRNAseq.

[fuhaolll2]

If my sample is 10X 3' sc-RNA-seq , should most of these reads cover with the last exon or 3'UTR? My sample is 10X 5' sc-RNA-seq , should most of these reads cover with the first exon or 5'UTR?

[igor]

You can view the BAM file in a genome browser like IGV and then know for sure which part of the gene you are sequencing.

[fuhaolll2]

This results in final 10x libraries that either represent the 3' end of the transcript (as the 10x Barcode is adjacent to the polyA tail on the 3' end of the transcript) or the 5' end of the transcript (as the the 10x Barcode is adjacent to the TSO and the 5' end of the transcript). But,in IGV ,i can see reads randomly at the transcript

[igor]

You may need to check a few different genes. For some, the pattern is not very clear.

Answer 1

[nlehmann]

Hi,

But, in IGV, I can see reads randomly at the transcript

I encountered the same issue some time ago, and it's actually normal to have reads all along the transcripts for 10x single cell data. If you check this question in 10x support forum, you will get that "the fragmentation produces molecules of varying sizes", thus leading to different coverage along the trancripts. So you don't have data that is "3' only", but "3' biased". Try geneBody_coverage.py from RSeQC to have a better idea of the 3' biais on the genome-scale. You can also refer to this preprint - check figure 2 - to go further.

Nathalie

[fuhaolll2]

Hi Nathalie, your answer is useful . However, i see "After amplifying the cDNA, molecules are randomly fragmented under conditions that favor 300-400 bp length fragments." in 10X support .Is it means that reserved reads are about 300-400bp along the 3' ,and 300-400bp along the 3' can only cover the last exon or 3'UTR for many genes?