Custom barcoded RNA-seq

nasobema · 07-30-2010, 07:20 AM

Hi,

we are planning on setting up our own custom method for RNA-sequencing with barcodes on an Illimina GAII. I've already found some hints in this forum and elsewhere but still have some questions:

- My plan is to the use Illumina Small RNA adapters and corresponding PCR primers (like they are displayed at http://seqanswers.com/forums/showthr...pters+sequence) with and additional barcode of 4-6 nucleotides added to the 5' RNA Adapter.
Does anyone have experience with that and wants to share it?

- After RT, do I need a second strand synthesis at all? If I get it right, a PCR on the ss-cDNA should do the job and generate a ready-to-use cDNA library with adapters and all.

Any help is highly wellcome.

NextGenSeq · 07-30-2010, 09:28 AM

Note it is a very common problem to get uneven distribution of coverage in bar-coded libraries even using the Illumina adaptors. Thus if you are planning on expression analysis I doubt it will be valid.

greigite · 08-02-2010, 01:26 PM

Quote:

Originally Posted by NextGenSeq

Note it is a very common problem to get uneven distribution of coverage in bar-coded libraries even using the Illumina adaptors. Thus if you are planning on expression analysis I doubt it will be valid.

Not necessarily- expression analysis packages like edgeR and DESeq have library size correction factors built in to the statistics. If one library is severely under-represented though it will reduce the statistical power of your analysis.

nasobema · 08-03-2010, 12:30 AM

Quote:

Originally Posted by greigite

Not necessarily- expression analysis packages like edgeR and DESeq have library size correction factors built in to the statistics. If one library is severely under-represented though it will reduce the statistical power of your analysis.

Expression analysis is indeed one of the applications that we planned.
If the unequal distribution of barcodes is genome wide, I wouldn't expect severe problems because normalization would be simple. But if there's unequal distribution within the genome (e.g. gene A shows higher coverage than gene B for barcode 1 but lower coverage for the same sample using another barcode) it's more difficult.
A probable solution could be to use two barcodes for each sample and take average values or to limit the number of barcodes used to keep overall coverage high.

I should add that we are working with bacterial cDNA, thus the reference genome size is not very large, which increases the coverage.

link1 · 08-04-2010, 08:46 PM

we tried barcoding the 5'RNA adapter and found significant bias in our small RNA preps. it turns out that RNA Lig I prefers some bases over others. You will need to barcode the 3' adenylated adapter because it utilizes a truncated version of RNA Ligase 2, which does not exhibit as much base preference.
You can design and make these yourself or purchase a set that has already been designed and optimized to eliminate bias. Initially I tried making these myself. Now I purchase the set below. I've done several runs now with multiplexed samples with good results.

http://www.biooscientific.com/Detail...tGenSequencing

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07-30-2010, 07:20 AM	#1
nasobema Member Location: Germany Join Date: Jul 2010 Posts: 14	Custom barcoded RNA-seq Hi, we are planning on setting up our own custom method for RNA-sequencing with barcodes on an Illimina GAII. I've already found some hints in this forum and elsewhere but still have some questions: - My plan is to the use Illumina Small RNA adapters and corresponding PCR primers (like they are displayed at http://seqanswers.com/forums/showthr...pters+sequence) with and additional barcode of 4-6 nucleotides added to the 5' RNA Adapter. Does anyone have experience with that and wants to share it? - After RT, do I need a second strand synthesis at all? If I get it right, a PCR on the ss-cDNA should do the job and generate a ready-to-use cDNA library with adapters and all. Any help is highly wellcome.

08-04-2010, 08:46 PM	#5
link1 Member Location: US Join Date: May 2010 Posts: 21	barcoding small RNAs we tried barcoding the 5'RNA adapter and found significant bias in our small RNA preps. it turns out that RNA Lig I prefers some bases over others. You will need to barcode the 3' adenylated adapter because it utilizes a truncated version of RNA Ligase 2, which does not exhibit as much base preference. You can design and make these yourself or purchase a set that has already been designed and optimized to eliminate bias. Initially I tried making these myself. Now I purchase the set below. I've done several runs now with multiplexed samples with good results. http://www.biooscientific.com/Detail...tGenSequencing

07-30-2010, 09:28 AM	#2
NextGenSeq Senior Member Location: USA Join Date: Apr 2009 Posts: 482	Note it is a very common problem to get uneven distribution of coverage in bar-coded libraries even using the Illumina adaptors. Thus if you are planning on expression analysis I doubt it will be valid.