We are new to NGS and have a project that we need to set up. In general, our collaborators are looking for sequencing an RNA plant virus from small (25-30 mg) leaf samples that have been stored in a herbarium. The goal is to do some evolutionary calculations based on the sequences that are pulled out of the samples. We can isolate RNA from the sample but only get a 2.3-2.5 RIN with a yield of around 5 micrograms of total RNA from the extraction (this will include plant rRNA & mRNA, chloroplast & mitochondrial RNAs, as well as viral RNAs). Based on preliminary work, the RNA is intact enough to perform RT-PCR for fragments up to 1000 bp. I don't know the titer level of the virus in these plants.
Any advice on whether we can proceed with RNAseq for Illumina sequencing? What sequence length would be most appropriate? Would GAIIx or HiSeq2000 make a difference? Also, we are looking to pool samples together - any advice? Any other input would also be appreciated!
Thanks,
Nikki
Any advice on whether we can proceed with RNAseq for Illumina sequencing? What sequence length would be most appropriate? Would GAIIx or HiSeq2000 make a difference? Also, we are looking to pool samples together - any advice? Any other input would also be appreciated!
Thanks,
Nikki
Comment