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Analysis of Directional mRNA-seq data / Illumina
We are developing a library preparation for directional mRNA-seq. All sequences will read 5' -> 3' direction. Our protocol isolates mRNA, fragments the messages and then these are re-purified with oligodT - resulting in a library of short 3' fragments for sequencing. This eliminates the need to adjust for transcript length when doing quantitative expression analysis. We're using bowtie to align the raw reads to a (cDNA) reference transcriptome (minus introns and intergenic regions). BUT - approximately 12% of the loci in this organism (Arabidopsis) have overlapping 3' tails (Watson/Crick strands) leading to ambiguities when using BowTie ( which looks for alignments on both strands). Basically we're looking for a short read alignment algorithm that would take our directional data and align it ONLY with transcripts in the 5' -> 3' direction. Illumina is developing a directional mRNA-seq library prep method similar to the one we are using (without the purification of the 3' ends). I wonder if anyone has experience analyzing directional mRNA-seq data?
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Since your libraries are directional, you can use that information to resolve the ambiguity of overlapping 3' ends. The tag will be in the same 5'->3' direction as one of the two genes (conventionally indicated as +).
Harold -
As you are already using Bowtie, have a look at its manual again, especially at the description of the option '--norc'. It causes Bowtie to do precisely this.Originally posted by jmtepp View PostComment
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strand specific RNA-seq mapping
Does anyone know how do mapping for strand specific RNA-seq data? which tools can work well? what the parameter of tools for strand specific infomation of RNA-seq data?
Looking forward to your answer.
Thank you very much! Best wishes!Comment
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Thank you for your reply! Do you know BWA has option that can treat reads in a strand specific ?Originally posted by jujubix View PostComment
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Mappind strand specific RNAseq bacteria pair-end reads
Just following up on this thread, which mapper will be most ideal for mapping strand specific RNAseq bacteria pair-end reads,
I know tophat has the library type option but its mostly ideal for eukaryotic genomes.
Any thoughts will be appreciated.
ThanxComment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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