Interpretation of Agilent Tapestation

[weeseda]

Hello all,

I recently submitted several RNA extractions to our sequencing core for illumina tru-seq library prep. Attached is an electropherogram they sent of one of the samples saying that the samples looked fine (all looked very similar) and were going to go ahead with the library prep.

I’m not too familiar with the tapestation, but from what I know from bioanalyzer I would say the sample looks degraded and needs to be re-extracted before moving forward with the library prep. When I asked the core about this they responded “We are more familiar with Human/mouse RNA which have peaks at both 18s and 28s. We aren’t exactly sure what your samples should look like, but your samples don’t look degraded”.

I can only figure that they are determining no degradation based on the RIN numbers which are all around 8-9. As my samples are also metazoans (snails), I would also expect the 18s and 28s peaks…

Any insight would be greatly appreciated.

[kwaraska]

Are you having them do ribo reduction?

This trace looks as though they have already done ribosomal reduction in preparation for sequencing. Is that possible?

[weeseda]

Thanks for the help!

No, I am pretty sure that at this point the core has not done anything to the samples with the exception of nanodrpping then and running them on the tapestation for quality control purposes.

[sara_ahmed]

We have sequenced a few snails and they don't show a 28S peak so your sample is probably similar to that. Most of the material on the trace is in the 18S peak and doesn't look degraded. If it was degraded you would see a spread of smaller fragments (similar to a smear on a gel). Looks like your sample is indeed good quality and ready for some libraries!

Sara Ahmed, PhD | Director of Sequencing | Cofactor Genomics
3141 Olive St. | St. Louis, MO 63103 | tel. (314) 531-4647

[weeseda]

Thanks Sara Ahmed- its strange that snails don't show the 28S peak...

Guess I'll have the core proceed with the library preps and just keep my fingers crossed.

Thanks again

[mcampo86]

Hello all,

I recently submitted several RNA extractions to our sequencing core for illumina tru-seq library prep. Attached is an electropherogram they sent of one of the samples saying that the samples looked fine (all looked very similar) and were going to go ahead with the library prep.

I’m not too familiar with the tapestation, but from what I know from bioanalyzer I would say the sample looks degraded and needs to be re-extracted before moving forward with the library prep. When I asked the core about this they responded “We are more familiar with Human/mouse RNA which have peaks at both 18s and 28s. We aren’t exactly sure what your samples should look like, but your samples don’t look degraded”.

I can only figure that they are determining no degradation based on the RIN numbers which are all around 8-9. As my samples are also metazoans (snails), I would also expect the 18s and 28s peaks…

Any insight would be greatly appreciated.

I'd stick with the bioanalyzer. It takes longer, but the quality of data is better. If youre used to looking at data from the bioanalyzer, and run the same set on the tapestation... you will get different values that are about 50% lower on the tapestation. Ive done quite a few validation runs comparing the two and agilent has no answer for why theyre different.

[kerplunk412]

Although I have no experience with snail RNA, I have worked with a lot of RNA and used the Tapestation and I agree that this RNA does not look degraded. Like sara_ahmed said, if it was degraded you would see a smear of smaller fragments, and often what appears to be a "peak" ~100 nts on a Tapestation or Bioanalyzer trace, which from my experience is probably just where the dye used in those systems stops binding well to the RNA. This is observation is based on running the same sample on a PAGE gel and seeing a lot of smaller material that was not indicated on the Bioanalyzer.

I also agree with mcampo86 that the Bioanalyzer gives much better data quality than the Tapestation. The peaks in Bioanalyzer traces are much sharper, which can be helpful for complex samples. The Bioanalyzer software also allows you to "play" with your data in more ways, although it isn't extremely user friendly.

[northpaw]

You all should check out the Fragment Analyzer from Advanced Analytical.

Illumina uses the Fragment Analyzer (FA) now. They evaluated all systems on the market to improve reliability, throughput, and dynamic range of analysis for their workflows. This of course included the TapeStation. Illumina doesn't recommend the TapeStation, they have recommended the BA as you know for years. Its great that FINALLY Illumina has offered a recommended alternative to the chip based systems. They may even start a trade in program where you get to blow the thing up! Pretty funny

Do a google search for the Illumina Automation Partners webpage. Scroll down and you will find the Advanced Analytical Fragment Analyzer (FA).

Illumina R&D has integrated the FA into multiple divisions including Consumables Product Development, Diagnostics, and Oncology. They are even starting to release new sample prep kits with the FA as a recommended QC platform.

The latest was the TruSeq RNA Access kit. They have a tech note for degraded RNA sample analysis that describes how they use custom software settings on the FA to streamline analysis of a new critical QC metric for RNA samples they call DV200.

You can find this in a google search on the Illumina website:

[PDF]Evaluating RNA Quality from FFPE Samples - Illumina
res.illumina.com/documents/.../technotes/technote-truseq-rna-access.pdf
Technical Note: RNA Sequencing. Introduction. The TruSeq® RNA Access Kit provides an exon-capture, RNA-Seq approach for difficult samples such as RNA …

If you work with FFPE or any samples prone to degradation by digestive enzymes, you should definitely check out this new kit from Illumina. Its pretty amazing.

[nucacidhunter]

Choice of instrument for nucleic acids analysis also depends on work scale. For a lab that has around 12 samples at a time a Bioanalyzer Chip or other similar instrument may be a good option. For lower scale a Tapestation may be a better choice. FA might be more suitable for labs which process higher number of samples.

[ddpuskas]

You should check out the new Nucleic Acid QC instrument by PerkinElmer - the LabChip GX Touch - they have a low throughput and a high throughput version for DNA/RNA QC.