Hello.
I am trying to find out which library type to run for a single stranded RNA seq run. the manual says
I am not sure which library type to use (fr-firststrand or fr-secondstrand), what should I do?
"One possible way to figure out the correct library-type is to run TopHat with a small subset of the reads (e.g., 1M) as follows.
run TopHat with fr-firststrand and count the number of junctions in junctions.bed (one of the output files from TopHat)
run TopHat with fr-secondstrand and count the number of junctions in junctions.bed
Since the splice junction finding algorithm of TopHat makes use of library-type information (if provided), one of the two TopHat runs would result in many more splice junctions than the other one. You can then use the library type that gives more junctions. If this is not the case TopHat might not work well with your sequencing protocol. Please let us know more details about your protocol so we can add support for new library types."
For 10 samples, I have ran the first strand library type and completed the alignment producing the alignment report.
Now I am running the second library type for a single sample and counting the number junctions in the junctions.bed file (when it copmletes).
My question is this
1) Say for this single sample A, if the second library type has more junctions then the second library type is the correct library type. But does the manual mean to say that it is the correct library type FOR ALL samples, or FOR JUST THIS ONE?
2) If for this single sample, if the alignment for the second library type comes out to have less junctions in the junctions.bed output file, does this mean that the second library type is the incorrect library type FOR ALL samples, or for just this one?
I am trying to find out which library type to run for a single stranded RNA seq run. the manual says
I am not sure which library type to use (fr-firststrand or fr-secondstrand), what should I do?
"One possible way to figure out the correct library-type is to run TopHat with a small subset of the reads (e.g., 1M) as follows.
run TopHat with fr-firststrand and count the number of junctions in junctions.bed (one of the output files from TopHat)
run TopHat with fr-secondstrand and count the number of junctions in junctions.bed
Since the splice junction finding algorithm of TopHat makes use of library-type information (if provided), one of the two TopHat runs would result in many more splice junctions than the other one. You can then use the library type that gives more junctions. If this is not the case TopHat might not work well with your sequencing protocol. Please let us know more details about your protocol so we can add support for new library types."
For 10 samples, I have ran the first strand library type and completed the alignment producing the alignment report.
Now I am running the second library type for a single sample and counting the number junctions in the junctions.bed file (when it copmletes).
My question is this
1) Say for this single sample A, if the second library type has more junctions then the second library type is the correct library type. But does the manual mean to say that it is the correct library type FOR ALL samples, or FOR JUST THIS ONE?
2) If for this single sample, if the alignment for the second library type comes out to have less junctions in the junctions.bed output file, does this mean that the second library type is the incorrect library type FOR ALL samples, or for just this one?
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