Hi All -
I am new to the forum, but I am hoping that you might be able too help me parse a question of library preparation. I am about to embark on an RNA seq project on an Illumina HiSeq2000 to compare behavior responsive transcripts in the brains of three insect species, all of which should have annotated genomes by the time we have data back, but the genomes aren't out yet. It is going to take a lot of libraries from brains (200+) to narrow down the transcripts specific to the behavior we are interested in, which means this is going to be a very expensive and time consuming project. Right now, we are trying to figure out which protocol to use to make our libraries. The options we have considered are 1) TruSeq, 2) ScriptSeq v2, and 3) the updated protocol from the recently published Wang et al paper. At this point, each method has its advantages: TruSeq is well described and well worked out, 2) ScriptSeq is strand-specific and supposedly takes about half the time to make each library, and 3) the Wang protocol looks pretty inexpensive and is again strand-specific. As a side note, we have been interested in the strand-specificity because we have thought that it might help us describe our genomes better, giving us better quality data for cross-species comparisons.
Has anyone thought through the strand-specific vs non-specific issue for annotating non-model systems? Is the state of the art using strand-specific protocols, or does it not really matter for most users? Does anyone have a strong opinion about any of these library preparation methods?
Thanks a lot for any help!
I am new to the forum, but I am hoping that you might be able too help me parse a question of library preparation. I am about to embark on an RNA seq project on an Illumina HiSeq2000 to compare behavior responsive transcripts in the brains of three insect species, all of which should have annotated genomes by the time we have data back, but the genomes aren't out yet. It is going to take a lot of libraries from brains (200+) to narrow down the transcripts specific to the behavior we are interested in, which means this is going to be a very expensive and time consuming project. Right now, we are trying to figure out which protocol to use to make our libraries. The options we have considered are 1) TruSeq, 2) ScriptSeq v2, and 3) the updated protocol from the recently published Wang et al paper. At this point, each method has its advantages: TruSeq is well described and well worked out, 2) ScriptSeq is strand-specific and supposedly takes about half the time to make each library, and 3) the Wang protocol looks pretty inexpensive and is again strand-specific. As a side note, we have been interested in the strand-specificity because we have thought that it might help us describe our genomes better, giving us better quality data for cross-species comparisons.
Has anyone thought through the strand-specific vs non-specific issue for annotating non-model systems? Is the state of the art using strand-specific protocols, or does it not really matter for most users? Does anyone have a strong opinion about any of these library preparation methods?
Thanks a lot for any help!
Comment