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
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Originally posted by greigite View PostNot 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.
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.
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Originally posted by NextGenSeq View PostNote 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.
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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.
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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.Tags: None
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