question about 10X sc-RNA-seq
1
0
Entering edit mode
15 months ago
fuhaolll2 ▴ 30

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?

RNA-Seq • 1.2k views
ADD COMMENT
1
Entering edit mode

10x does have 5' scRNAseq.

ADD REPLY
0
Entering edit mode

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?

ADD REPLY
0
Entering edit mode

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.

ADD REPLY
0
Entering edit mode

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

ADD REPLY
0
Entering edit mode

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

ADD REPLY
1
Entering edit mode
15 months ago
nlehmann ▴ 120

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

ADD COMMENT
0
Entering edit mode

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?

ADD REPLY

Login before adding your answer.

Traffic: 2152 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6