small library size (HiSeq)

Robby · 11-22-2011, 01:20 AM

Hello everyone,

we often have the the problem, that we have libraries with small size (ie small RNA), but we don't have enough samples to sequence a complete flowcell. So we were wondering, if it is possible to sequence the libraries with small size on a 100bp HiSeq run and use afterwards just the first 30-40bp. Are there any problems with that (sequencing or cBot) or would a small library size on one lane influence for example other lanes? Has anyone experience with that or any recommendations? I would be happy for all advice.
Thanks Robby

simonandrews · 11-23-2011, 01:02 AM

I can't see why that would be a problem (if you're prepared to pay the extra cost of a longer run). You'll just end up sequencing a load of adapter sequence, and plenty of people have done that (mostly unintentionally!) before.

You shouldn't have any effect on the other lanes as long as your lane hasn't been specified as the control lane for the run.

Robby · 11-23-2011, 02:09 AM

OK, thanks for your information.

We know, that we have more costs for that lane due to the longer reads, but it is still cheaper for us than a complete run with some empty lanes.

11-23-2011, 08:57 AM

As long as it's the minority of the flowcell, this should work. You run the risk of saturating the imager when you start sequencing the through to the adapter. Low complexity would be my concern.

simonandrews · 11-23-2011, 09:06 AM

If the low complexity comes after the sequence you care about then it shouldn't matter too much. If your inserts are variably sized then it won't matter at all. The only problem you might face is that the sequences might get kicked by the QC filter if the later base calls get too rubbish, but you could just choose to ignore the filter and take the raw calls in that case.

In any event it shouldn't affect anything outside the current lane.

Robby · 11-23-2011, 09:07 AM

@SeqAA: What do you mean with "risk of saturating the imager"? We would sequence 2-3 complete lanes with small RNA.In the other lanes we would sequence "normal" libraries. If we are behind the second adapter sequence, we will of course receive just N's. But do you think, that that influences the first bp? Or do you mean that this influences the other lanes? So for which part would you expect low complexity?

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
converting Ion torrent library for sequencing on HiSeq	cwachtel	Introductions	4	04-19-2016 04:07 PM
Sequencing a Low diversity library on the HiSeq	Simcom	Illumina/Solexa	38	09-26-2012 06:01 AM
HiSeq multiplexing for strand specific RNA library prep	ngon	Sample Prep / Library Generation	2	10-18-2011 09:32 AM
Library prep can you get both mRNA and small RNAs in the same library?	willgordon	Sample Prep / Library Generation	4	07-20-2011 12:31 PM
Minimal fragment size for GAIIx or HiSeq??	Unununium	Illumina/Solexa	3	05-02-2011 04:45 AM

11-22-2011, 01:20 AM	#1
Robby Member Location: Germany Join Date: Mar 2011 Posts: 68	small library size (HiSeq) Hello everyone, we often have the the problem, that we have libraries with small size (ie small RNA), but we don't have enough samples to sequence a complete flowcell. So we were wondering, if it is possible to sequence the libraries with small size on a 100bp HiSeq run and use afterwards just the first 30-40bp. Are there any problems with that (sequencing or cBot) or would a small library size on one lane influence for example other lanes? Has anyone experience with that or any recommendations? I would be happy for all advice. Thanks Robby

11-23-2011, 01:02 AM	#2
simonandrews Simon Andrews Location: Babraham Inst, Cambridge, UK Join Date: May 2009 Posts: 871	I can't see why that would be a problem (if you're prepared to pay the extra cost of a longer run). You'll just end up sequencing a load of adapter sequence, and plenty of people have done that (mostly unintentionally!) before. You shouldn't have any effect on the other lanes as long as your lane hasn't been specified as the control lane for the run.

11-23-2011, 02:09 AM	#3
Robby Member Location: Germany Join Date: Mar 2011 Posts: 68	OK, thanks for your information. We know, that we have more costs for that lane due to the longer reads, but it is still cheaper for us than a complete run with some empty lanes.

11-23-2011, 08:57 AM	#4
SeqAA Guest Posts: n/a	As long as it's the minority of the flowcell, this should work. You run the risk of saturating the imager when you start sequencing the through to the adapter. Low complexity would be my concern.

11-23-2011, 09:06 AM	#5
simonandrews Simon Andrews Location: Babraham Inst, Cambridge, UK Join Date: May 2009 Posts: 871	If the low complexity comes after the sequence you care about then it shouldn't matter too much. If your inserts are variably sized then it won't matter at all. The only problem you might face is that the sequences might get kicked by the QC filter if the later base calls get too rubbish, but you could just choose to ignore the filter and take the raw calls in that case. In any event it shouldn't affect anything outside the current lane.

11-23-2011, 09:07 AM	#6
Robby Member Location: Germany Join Date: Mar 2011 Posts: 68	@SeqAA: What do you mean with "risk of saturating the imager"? We would sequence 2-3 complete lanes with small RNA.In the other lanes we would sequence "normal" libraries. If we are behind the second adapter sequence, we will of course receive just N's. But do you think, that that influences the first bp? Or do you mean that this influences the other lanes? So for which part would you expect low complexity?