RNA-seq basecalls?

lilian619 · 10-27-2010, 12:21 AM

I have been obtained the RNA-seq data of a sample on illumina. The % base calls plot of this lane is below, Is it reasonable? Or what is the problem with it? Why?

And，do you think it is important to optimize adapter oligo concentrations during ligation to the cDNA ends? Why?

mnkyboy · 10-27-2010, 03:54 PM

Looks like you sequenced larger than your insert and you are sequencing the adapters at the end that is why they are no longer random.

You have some other stuff going on the 5' end.

What kind of library prep are these and why are you sequencing so long?

lilian619 · 10-27-2010, 07:52 PM

thanks for your reply, mnkyboy.

this sample was RNA sample library, followed the protocol of illumina, and the size was about 300bp, sequenced by 151 cycles. You are right, mostly, the adapters may be sequenced.

we got the SBSv5 recently, just testing the quality and quantity of sequencing when using SBS v5.

Is that useful to seq as long as possible for RNA seq?

what might be the stuffs on the 5' end, in your opinion? In our previous RNA seq, eg, 1X100bp([URL="https://www.sugarsync.com/pf/D030688_6685421_71585"]https://www.sugarsync.com/pf/D030688_6685421_71585[/URL), is the plots for base calls reasonable? there had also some other stuff in it.

mnkyboy · 10-28-2010, 09:09 AM

For expression profiling for mRNA seq we have been pretty happy with 54 or 76 bp. We find for whole transcriptome we get better results with 76 compare to over 100 bp.

Illumina mRNA-seq libraries are default paired end libraries so you may find some use doing paired end mRNA-seq at 76 instead of longer reads.

It depends really on the question you are asking.

lilian619 · 10-28-2010, 07:47 PM

Thanks for your kindly reply. May I further ask some questions?

I have no idea why RNA seq is suitable for 54-76bp, since the exons might be more longer than 100bp. Or is the software dealing with RNA data is not suitable for longer reads?

lilian619 · 10-28-2010, 07:51 PM

Another question: Is RNA seq mostly used single read or paired end? why?

mnkyboy · 11-01-2010, 01:37 PM

It all goes back to what you want to ask of your RNA-seq data, which would determine your read length.

Illumina RNA seq using their mRNA-seq kit is usually read single read but the libraries are actually paired end. They are releasing a support mRNA paired end kit soon I believe. Their directional prep however is only single read. The paired end really helps with RNA structure, non-coding detection, and if you do not have a really good reference genome.

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10-27-2010, 03:54 PM	#2
mnkyboy Member Location: Seattle, WA Join Date: Mar 2009 Posts: 87	Looks like you sequenced larger than your insert and you are sequencing the adapters at the end that is why they are no longer random. You have some other stuff going on the 5' end. What kind of library prep are these and why are you sequencing so long?

10-27-2010, 07:52 PM	#3
lilian619 Junior Member Location: china Join Date: Oct 2010 Posts: 5	thanks for your reply, mnkyboy. this sample was RNA sample library, followed the protocol of illumina, and the size was about 300bp, sequenced by 151 cycles. You are right, mostly, the adapters may be sequenced. we got the SBSv5 recently, just testing the quality and quantity of sequencing when using SBS v5. Is that useful to seq as long as possible for RNA seq? what might be the stuffs on the 5' end, in your opinion? In our previous RNA seq, eg, 1X100bp([URL="https://www.sugarsync.com/pf/D030688_6685421_71585"]https://www.sugarsync.com/pf/D030688_6685421_71585[/URL), is the plots for base calls reasonable? there had also some other stuff in it.

10-28-2010, 09:09 AM	#4
mnkyboy Member Location: Seattle, WA Join Date: Mar 2009 Posts: 87	For expression profiling for mRNA seq we have been pretty happy with 54 or 76 bp. We find for whole transcriptome we get better results with 76 compare to over 100 bp. Illumina mRNA-seq libraries are default paired end libraries so you may find some use doing paired end mRNA-seq at 76 instead of longer reads. It depends really on the question you are asking.

10-28-2010, 07:47 PM	#5
lilian619 Junior Member Location: china Join Date: Oct 2010 Posts: 5	Thanks for your kindly reply. May I further ask some questions? I have no idea why RNA seq is suitable for 54-76bp, since the exons might be more longer than 100bp. Or is the software dealing with RNA data is not suitable for longer reads?

10-28-2010, 07:51 PM	#6
lilian619 Junior Member Location: china Join Date: Oct 2010 Posts: 5	Another question: Is RNA seq mostly used single read or paired end? why?

11-01-2010, 01:37 PM	#7
mnkyboy Member Location: Seattle, WA Join Date: Mar 2009 Posts: 87	It all goes back to what you want to ask of your RNA-seq data, which would determine your read length. Illumina RNA seq using their mRNA-seq kit is usually read single read but the libraries are actually paired end. They are releasing a support mRNA paired end kit soon I believe. Their directional prep however is only single read. The paired end really helps with RNA structure, non-coding detection, and if you do not have a really good reference genome.