I made bacterial RNA-seq libraries with ScriptSeq v2 and did a small sequencing test (MiSeq Nano, Single Read, 50 bp).
ScriptSeq v2 prepares directional libraries, as per the manual: "The sequence produced by the Read 1 Sequencing Primer is that of the sense strand of the original fragmented RNA molecule."
After trimming and QC, I aligned the reads against the reference genome using BowTie.
When I visualize the resulting SAM files on a genome viewer (IGV), I see reads overlapping annotated CDS features going in _both_ directions. This is definitely _not_ what I expected, since the library should be directional.
Since I am new to this, I am hoping to understand might be going on. Is there something wrong with my library?
Is it possible that there was some genomic DNA contamination in my RNA preps (although I did treat with DNase and I didn't see a large peak on Bioanalyzer) which then became fragmented and incorporated as a sequence-able molecule in the library?
What are the possible reasons for seeing bidirectional reads from what was supposed to be a directional library?
ScriptSeq v2 prepares directional libraries, as per the manual: "The sequence produced by the Read 1 Sequencing Primer is that of the sense strand of the original fragmented RNA molecule."
After trimming and QC, I aligned the reads against the reference genome using BowTie.
When I visualize the resulting SAM files on a genome viewer (IGV), I see reads overlapping annotated CDS features going in _both_ directions. This is definitely _not_ what I expected, since the library should be directional.
Since I am new to this, I am hoping to understand might be going on. Is there something wrong with my library?
Is it possible that there was some genomic DNA contamination in my RNA preps (although I did treat with DNase and I didn't see a large peak on Bioanalyzer) which then became fragmented and incorporated as a sequence-able molecule in the library?
What are the possible reasons for seeing bidirectional reads from what was supposed to be a directional library?
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