10x genomics

mitsaki · 06-15-2017, 07:41 AM

Hi everyone,

I am kind of new to rna seq and mostly to 10x genomics. I got some data from a 10x genomics and in contrast to Hiseq/Nextseq etc, I have 4 types of files.

I have R1,R2,R3 and I1 files. I am a bit confused about what is what. So R1 has reads of 114 length so it should be my sequence. The R2 files have sequences of 14bp (is that the 10x barcode ?)
R3 contain sequences of 10 bp (UMI ?) and I1 should be sample barcodes

Could someone help me clear this out ?

GenoMax · 06-15-2017, 07:52 AM

You would find it pretty hard to work with these files in a standalone manner (I am not sure why you have an R3 file). I assume this is single cell RNAseq data? You should ask your sequence provider to process this data with 10x Cellranger software (unless you are willing and capable of installing/running this software yourself). You can find information about CellRanger here.

mitsaki · 06-15-2017, 08:17 AM

Thank you for your reply. Yes it is RNAseq data. The R3 file is the one that mostly confuses me too. We are the sequence provider and we have cell ranger. But we want to run some tests for a specific experiment standalone

So, I thought that I could treat R1 as single end and run star. It seems to work but the rest of the files made me confused, because I do not actually use them

GenoMax · 06-15-2017, 01:24 PM

How was this run set up (on sequencer)? It looks like the UMI's were treated like a separate read and must have ended up in one of the files.

I assume you used the mkfastq protocol to generate the files? What was the --use-bases-mask used? And the <RunInfo> section from RunInfo.xml?

nucacidhunter · 06-15-2017, 02:35 PM

Your explanation of files is correct and the data set must be for v1 chemistry which is obsolute. If you are planning runs and want to evaluate the performance or check your software set up you should download data for v2 chemistry which has different library structure and sequencing configuration.

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06-15-2017, 07:41 AM	#1
mitsaki Junior Member Location: Netherlands Join Date: Nov 2013 Posts: 2	10x genomics Hi everyone, I am kind of new to rna seq and mostly to 10x genomics. I got some data from a 10x genomics and in contrast to Hiseq/Nextseq etc, I have 4 types of files. I have R1,R2,R3 and I1 files. I am a bit confused about what is what. So R1 has reads of 114 length so it should be my sequence. The R2 files have sequences of 14bp (is that the 10x barcode ?) R3 contain sequences of 10 bp (UMI ?) and I1 should be sample barcodes Could someone help me clear this out ?

06-15-2017, 01:24 PM	#4
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,088	How was this run set up (on sequencer)? It looks like the UMI's were treated like a separate read and must have ended up in one of the files. I assume you used the mkfastq protocol to generate the files? What was the --use-bases-mask used? And the <RunInfo> section from RunInfo.xml? Last edited by GenoMax; 06-15-2017 at 01:30 PM.

06-15-2017, 07:52 AM	#2
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,088	You would find it pretty hard to work with these files in a standalone manner (I am not sure why you have an R3 file). I assume this is single cell RNAseq data? You should ask your sequence provider to process this data with 10x Cellranger software (unless you are willing and capable of installing/running this software yourself). You can find information about CellRanger here.

06-15-2017, 08:17 AM	#3
mitsaki Junior Member Location: Netherlands Join Date: Nov 2013 Posts: 2	Thank you for your reply. Yes it is RNAseq data. The R3 file is the one that mostly confuses me too. We are the sequence provider and we have cell ranger. But we want to run some tests for a specific experiment standalone So, I thought that I could treat R1 as single end and run star. It seems to work but the rest of the files made me confused, because I do not actually use them

06-15-2017, 02:35 PM	#5
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	Your explanation of files is correct and the data set must be for v1 chemistry which is obsolute. If you are planning runs and want to evaluate the performance or check your software set up you should download data for v2 chemistry which has different library structure and sequencing configuration.