[westerman]

Do you mean 450M reads as in 225M PE reads? I don't think counting a read on the same fragment twice is the right thing to do here, if that's in deed what you're doing.

That is what I meant -- 450M total reads, 225M PE reads. For 12 samples per lane that would give about 37M total reads or about 3700 Mbases per sample.

Considering assembly only and assuming that the samples are not overwhelmed with rRNA or other highly expressed transcripts, then there are about 14800 Mbases to work with -- if all 4 samples are merged together to create the assembly (which is the only way I would do it.) With the transcriptome being, what around 100M base pairs?, then we have 148x coverage. Even with rRNA and the highly expressed genes using up a lot of the bases the coverage should be high enough for a rough assembly. Perhaps not high enough to tease out very low expression transcripts but still good enough to get a handle on the transcriptome.

On a side note, I am finding this thread to be interesting.

[GenoMax]

That is what I meant -- 450M total reads, 225M PE reads.

That is illumina marketing speak

I prefer to think of it as 225M unique clusters. The libraries being referred to must be extra good quality libraries, something that is not guaranteed (there is beginner's luck, if applicable here).

We have given tjeff01 a ton of advice but Taylor is not going to do much with the data as indicated in the original post. Hopefully these suggestions will be passed on to the person who would be doing the data analysis.

Taylor: Do report back in this thread as to how the sequencing turned out. Good luck.

[tjeffe01]

Thanks for all the info everybody

Wow, this forum is amazing, thanks for answering everyone.

A couple updates:
- After talking to our colleague who actually arranged the sequencing we purchased 4 lanes, he purchased 12 in total. I apologize if I don't use the technical jargon properly. My background is in molecular genetics and biochemistry and while I understand how RNAseq and other next gen sequencing technology works I'm not very familiar with the technical side of the process or with the computer science side.

- In terms of candidate genes we have sequenced the EPSPS gene and found no polymorphisms between resistant or susceptible plants and haven't found any evidence of gene duplication or over expression. When it comes to glyphosate resistance, resistance tends to either be EPSPS mutation or some other mechanism unrelated to EPSPS (for example changes in translocation patterns or sequestration in the vacuole) that are responsible. Having ruled EPSPS mutation as a mechanisms we are attempting to use a reverse genetic approach to find anything that may be linked to our resistance trait.

- I've forwarded all of this information on to my supervisor and he has decided that he either needs to sit on this data for a few years until the technology advances enough to make it easier, or he needs to hire a bioinformatician.

- Unfortunately sequencing the genome is not really a possibility. Funding for plant genomes is cropping up (excuse the pun) but is heavily focused solely on crop plants. There just isn't funding available for sequencing a genome for a plant species that only effects a small portion of the U.S and Canada. In addition there's no guarantee that sequencing the genome would lead to a new control mechanism for resistant plants. Farmers and other researchers would rather just blast the plants with more roundup and other herbicides.

Thanks everyone, I'll update you as new data becomes available,
Taylor

[Melissa]

3) This data will not be in my MSc and I won't be doing much work with it beyond posting here; but I would like to include a short section in my thesis on the data we collect here. Is it possible to get average read lengths, fold number and other quality statistics about the data to include in my thesis, or does that not make any sense?
>>> I would advise against including the data in your thesis. It might not fit very well in your thesis. More importantly, it might invite questions from your examiners. Or worst, confuse them.

Another advice is forget everything you just learned from this post until you have finished writing your thesis.