[nanos]

Hi,

I recently did my first ScriptSeq mRNA libraries.

I am now analyzing the data and I see huge amounts of antisense reads mapping to the genes. I already did strand specific mRNAseq with a different method and there I did not see that much antisense.

It would be nice to know if somebody also experienced this problem and if there is a explanation for this.

Thank you

[arabidopsis]

I produced some libraries with ScriptSeq kit from 200 ng of rRNA-depleted total RNAs. After 10 PCR cycles, recommended by the protocol I still have very low amount of final product. DNA is barely detected by spectrophotometry (around 20 ng/ul) with great variablity between separate measures. What is the kit's expected yield of dsDNA?
I made some qPCRs on those libraries that worked fine, showing that there is some DNA in the libraries. But still, is it normal to have about 300-500 ng of DNA at the end of preparation, or something has gone wrong?

[upendra_35]

I produced some libraries with ScriptSeq kit from 200 ng of rRNA-depleted total RNAs. After 10 PCR cycles, recommended by the protocol I still have very low amount of final product. DNA is barely detected by spectrophotometry (around 20 ng/ul) with great variablity between separate measures. What is the kit's expected yield of dsDNA?
I made some qPCRs on those libraries that worked fine, showing that there is some DNA in the libraries. But still, is it normal to have about 300-500 ng of DNA at the end of preparation, or something has gone wrong?

I don't think there is any problem with the library making. It is quite normal to see the libraries in the range of 20-30ng/ul. I have successfully sequenced my scriptseq libraries that are in this range. So don't worry and send them for sequencing. If at all there is any problem you will hear from the sequencing guys. If you are too worried about the concentrations try increasing your PCR by 2 more cyles. Good luck.

[pzumbo]

epicentre vs illumina

We tested the technical reproducibility of scriptSeq on poly-adenylated human templates.
Additionally, we compared the results to illumina's standard mRNA-seq (rev D).

Our results may be of general interest (attached).

Also here,

http://physiology.med.cornell.edu/faculty/mason/lab/new/styled-6/styled-7/index.html

[upendra_35]

We tested the technical reproducibility of scriptSeq on poly-adenylated human templates.
Additionally, we compared the results to illumina's standard mRNA-seq (rev D).

Our results may be of general interest (attached).

Also here,

http://physiology.med.cornell.edu/fa...d-7/index.html

Hi pzumbo, the information was so useful to evaluate the kit. Personally what do you think of the kit? Also i was wondering how did you manage to calculate the adapter contamination in your Scriptseq libraries in the absence of 3' and 5' adapter sequences?

Thanks for your reply in advance.

[dsidote]

We are a core facility and we use the ScriptSeq kits for all of our Illumina RNA-Seq libraries. The results are just as good, if not better because of the strand information, than the Illumina kits.

[upendra_35]

We are a core facility and we use the ScriptSeq kits for all of our Illumina RNA-Seq libraries. The results are just as good, if not better because of the strand information, than the Illumina kits.

Good to know that. Also could you please provide me the sequence of 5' and 3' adapter of Script seq so that i can see how much adapter contamination is there in my script seq libraries.

[sszelinger]

Hi pzumbo,
I have been studying your pdf comparison of Illumina and Scriptseq.
There are a few thing I wanted to ask for clarification, because the pdf suggests that there is a bias in the Scriptseq kit compared to the Illumina.
1. You mention that you used polyA selection. That is curious, since the Scriptseq only uses random hexamers as far as I know.
2. The reads mapping to erccs is striking in the mapping statistics graph. However, if you actually did random hexamer capture of RNA than, maybe the erccs have large homology to the various hexamers used in the Srciptseq kit? What do you think?
3. Alternatively, did you spike in the ercc mixture after the library was prepared or you prepared the libraries with ercc spiked in at the beginning of the sample prep? If spike in was not equimolarly, that could explain the bias in mapping statisitics. What do you think?

I am thinking of using the Scriptseq kit for barcoded RNAseq of 25 people on the Hiseq2000, and your pdf made me think twice about using it.
Thanks for your help.
Szabi

[JakobHedegaard]

70 and 135 bp peak in ScriptSeq libraries

Hi,
I have prepared some ScriptSeq libraries from 500 ng samples of total-RNA - RiboZero followed by index ScriptSeq and minielute purification (following the protocols). I get the expected smear with a peak around 250 bp (see the image), but what are the 70 and 135 bp peaks in ScriptSeq libraries?
/Jakob

[epibio]

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