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Old 10-12-2010, 02:40 AM   #1
Scoob
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Location: London

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Default Intron spikes in Nugen Ovation data?

We are seeing very large spikes in RNA seq reads in introns using the Nugen WT Ovation pico prep method. These peaks dominate the read profile even more than exonic reads. As a result reads mapping to "transcriptome" (ie cDNA) drops to below 30%. The peaks are very consistent between samples. Does anyone else see this and have an explanation for it or do we have a technical problem here?
I did wonder if its a result of the rather odd amplification method (polymerase run off) or perhaps "non-random" random priming?
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Old 10-15-2010, 04:04 PM   #2
kainsteven
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Default Ovation RNA-Seq System

As I've posted in prior threads, the Pico System from NuGEN (Ovation Pico WTA System) was not designed for sequencing applications, and was optimized for production of single-stranded cDNA used with microarrays.

The Ovation RNA-Seq System is based on the same core technology, but has been further optimized for sequencing. The first strand synthesis is in fact random priming, and amplification is initiated at the 3 end as well as randomly throughout the whole transcriptome. The final product is double-stranded cDNA ready for library construction.

Because the input material is total RNA, without poly(A) selection, we and others using the system observe intronic reads, but we have not observed these to "dominate the read profile" as you say.

Best,
Steve
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