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Old 11-24-2009, 09:54 AM   #1
sanush
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Default New SOLiD user

Hi
I am a new SOLiD user, and completed our first RNA-Seq experiment. I have a question regd the secondary analysis report. For the secondary analysis we uploaded whole human genome as reference and the % matched summary report for our 2 samples were around 25%. But according to ABI the run is considered successful only if the % match is above 30%. Can anyone comment on what the problem might be of low % match.

Thanks
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Old 11-25-2009, 08:35 AM   #2
pmiguel
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Quote:
Originally Posted by sanush View Post
Hi
I am a new SOLiD user, and completed our first RNA-Seq experiment. I have a question regd the secondary analysis report. For the secondary analysis we uploaded whole human genome as reference and the % matched summary report for our 2 samples were around 25%. But according to ABI the run is considered successful only if the % match is above 30%. Can anyone comment on what the problem might be of low % match.

Thanks
Could you give us a little more detail about your experiment? Like method of RNA isolation, tissue, method of ribosomal RNA depletion. Also the version of the SOLiD instrument/chemistry your results derive from. Read length.

Also, what version of analysis software are you using?

If you want generic possibilities they would be:

(1) Library construction issues: failure to deplete your RNA of ribosomal may or may not result in low % match--depends on your reference sequence. Could also be high % no insert or very short amplicons. Obviously, if your RNA contains high percentages of non-human (eg, of viral origin) sequence, those will not map.

(2) ePCR issues: high multiple-amplicon beads, or poor amplification will result in beads that give poor results. What do your satays look like? What are your %best beads?

etc. There is a whole laundry list of troubleshooting you can do.

However, 25% isn't that bad. It may not be great, but I don't see any reason it would not be usable for that reason alone.

--
Phillip
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Old 11-25-2009, 10:36 AM   #3
sanush
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Hi Phillip,
Thanks for the reply. Here are some details about the experiment. We had 2 samples 1. Melanocyte and 2. Wistar Melanoma cell line. The RNA was isolated using Trizol method and further purified using Qiagen RNeasy cleanup procedure. We checked the Total RNA quality(RIN >9.8) & Ribo-depleted RNA (rRNA Contamination < 2%) using Bionalayzer ......ribosomal depletion was using Ribominus kit from invitrogen.
The prepared library was quantified by qPCR, % post enriched beads recovery of 20%, our satays look good. Even WFA run was good, but the N2S after sequence run is around 45%.

What are your %best beads?
% best beads is around 55% and the % best+good beads is around 70%

Version of SOLiD instrument - 3
Read Length - 50bp

We are not sure if 25% is good or bad, but according to the training manual above 30% is considered a successful run.

Thanks for the comments
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Old 11-25-2009, 11:59 AM   #4
Chipper
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How did you align the reads? 25 % is low if you are using the WT pipeline but not if you are trying to align full length reads (due to reads spannig junctions or short amplicons).
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Old 11-27-2009, 07:25 AM   #5
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How did you align the reads? 25 % is low if you are using the WT pipeline but not if you are trying to align full length reads (due to reads spannig junctions or short amplicons).
Yes Sanush,
Which analysis program are your using? For this purpose you should use the AB WT analysis pipeline or whatever its successor is in BioScope (if you are v3.5-ready).

Whatever your answer, I would still recommend mapping your data to the ribosomal repeat unit. It should be in your genome sequence, of course, but I have been surprised to discover that this is not always the case. (For example, the draft honey bee genome on the Baylor web site does not have the ribosomal repeat unit in it.)

Ribominus will deplete intact ribosomal RNA, but if your RNA degrade before or during depletion, only the segments containing the hybridizing oligos will actually be pulled out. The remaining degraded ribosomal RNA will look no different than polyA on a labchip. What % of your RNA remained after ribominus? Did you do one or two cycles of ribominus?

--
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Old 12-01-2009, 07:19 AM   #6
sanush
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Thanks for the suggestions. We aligned using full length reads and not WT pipeline.

What % of your RNA remained after ribominus? Did you do one or two cycles of ribominus?

We started of with 5 ug total RNA and after one cylce Ribominus protocol Ribo depleted RNA concentrations were around 250ng.

I have one question, for RNA seq project what is the recommended number of segments in a Quad slide used for loading 1 sample. In our experiment we loaded one sample per segment in a quad slide and wondering whether we can improve % mapped reads by loading same sample in 2 segments. That way we can increase the number of tags and as well increase the % match.... or does it depend on the application

Thanks for your suggestion
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Old 12-01-2009, 08:10 AM   #7
pmiguel
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Originally Posted by sanush View Post
Thanks for the suggestions. We aligned using full length reads and not WT pipeline.
Ah, that explains it then. If you use the WT pipeline you should get above 30% because it is more sensitive than the plain genomic pipeline. I would recommend having WT mapping done for you. If possible try to have the new BioScope software used. Its new "Seeded Extension Mapping" algorithm is said to map 10-30% more reads than previous mapping methods. If not available to you yet, that is okay, the WT pipeline written for corona-lite uses a more sensitive algorithm similar to the one deployed in BioScope.

Quote:
Originally Posted by sanush View Post
What % of your RNA remained after ribominus? Did you do one or two cycles of ribominus?


We started of with 5 ug total RNA and after one cylce Ribominus protocol Ribo depleted RNA concentrations were around 250ng.
5% yield sounds reasonable. Still, if you only did one cycle of Ribominus, it is possible that you still have fairly high rRNA levels. Mapping your reads to the ribosomal repeat unit would be a good check for how effective Ribominus was.


Quote:
Originally Posted by sanush View Post
I have one question, for RNA seq project what is the recommended number of segments in a Quad slide used for loading 1 sample. In our experiment we loaded one sample per segment in a quad slide and wondering whether we can improve % mapped reads by loading same sample in 2 segments. That way we can increase the number of tags and as well increase the % match.... or does it depend on the application

Thanks for your suggestion
If your depletion method reduced rRNA amounts below 20% or so, then I would think a v3 or v3.5 quad (60 or 90 million raw reads, respectively) would be more than enough for most assays. Brian Coullahan, Lifetech Application Specialist, gave a talk at the 2009 SOLiD Summit in September. He recommended:

SREK 10-20 million mapped reads
SWTAK 10-100 million
SOLiD SAGE 2-5 million

But, yes, the number of mapped reads you will need is going to be dependent on the expression levels of your genes of interest with respect to everything else in your sample.

By the way, if you have more than a few samples, I strongly recommend using molecular barcoding. That way you can take something like 10 samples, each barcoded and run them all in a single flow cell. The advantage is that without the gasket using up slide real-estate, you get considerably more reads per sample. Roughly, 40% more reads/run are possible with a single spot flow cell than with a quad.

--
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