bacterial RNA sequencing and rRNA removal

dca · 12-08-2010, 11:23 AM

Hello All -
I know there are a variety of posts on RNA sequencing and rRNA removal. However, I am specifically interested in bacterial transcriptomics and would appreciate advice on 1) sample prep and 2) platform (FLX vs Illumina vs SOLiD)
1) Bacterial mRNA (mostly) isn't poly-adenylated, so positive selection and PCR-suppression approaches won't work. So, what is the best way to get rid of rRNA and keep the good stuff?
Do the kits (RiboZero? Ribominus? others?) work?
A recent Nature Methods paper (http://www.nature.com/nmeth/journal/...nmeth.1507.pdf) suggested a combination of subtractive rRNA hybridization plus exonuclease methods. What is the preferred/real world approach?
I've heard good things about DSN for Illumina - but can't find an equivalent method that is compatible with library prep for the FLX. Suggestions?
Has anyone tried this published method (Biotechniques. 2010 February ; 48(2): 139–144. doi:10.2144/000113350) for positive selection of cDNA via hybridization to genomic DNA ?

2) The sequencing depth of Illumina and SOLiD make them attractive for RNA-seq... and probably essential for eukaryotic work. However, I have access to a FLX and bacterial genomes are significantly smaller. Do the length of FLX reads compensate for the (potential) lack of depth?
What about relative costs? Are the new Illumina library prep kits really faster/cheaper/better?

Thanks for any/all advice!

dca

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12-08-2010, 11:23 AM	#1
dca Junior Member Location: Canada Join Date: Apr 2010 Posts: 4	bacterial RNA sequencing and rRNA removal Hello All - I know there are a variety of posts on RNA sequencing and rRNA removal. However, I am specifically interested in bacterial transcriptomics and would appreciate advice on 1) sample prep and 2) platform (FLX vs Illumina vs SOLiD) 1) Bacterial mRNA (mostly) isn't poly-adenylated, so positive selection and PCR-suppression approaches won't work. So, what is the best way to get rid of rRNA and keep the good stuff? Do the kits (RiboZero? Ribominus? others?) work? A recent Nature Methods paper (http://www.nature.com/nmeth/journal/...nmeth.1507.pdf) suggested a combination of subtractive rRNA hybridization plus exonuclease methods. What is the preferred/real world approach? I've heard good things about DSN for Illumina - but can't find an equivalent method that is compatible with library prep for the FLX. Suggestions? Has anyone tried this published method (Biotechniques. 2010 February ; 48(2): 139–144. doi:10.2144/000113350) for positive selection of cDNA via hybridization to genomic DNA ? 2) The sequencing depth of Illumina and SOLiD make them attractive for RNA-seq... and probably essential for eukaryotic work. However, I have access to a FLX and bacterial genomes are significantly smaller. Do the length of FLX reads compensate for the (potential) lack of depth? What about relative costs? Are the new Illumina library prep kits really faster/cheaper/better? Thanks for any/all advice! dca