I am working on a de novo assembly for an organism with a fairly small genome (~200 Mb), and for which we have already generated paired end (2x100) Illumina reads which assembled gives an N50 >30kb and about 50-100X coverage.
We want to now generate a large insert library, probably for PacBio, to generate about 10-15X coverage fro scaffolding.
The problem is these are very small organisms, and we cannot attain very many- so all of the DNA we have if we pooled it all would be 1-2 ug. PacBio facilities I have looked into list a requirement of >5 ug of input DNA for insert sizes >10 kb... So my questions are:
Hi Tyler,
I am not quite sure if you need PacBio for linking the contigs. You might be better off with "mate-pair" (jumping) library(s) of different sizes (5, 10, 15kb) that can help your bioinformatics for linking the contigs.
Of course you may use PBio with broader range and less than 5ug. As customer you may request that, providing that you will not get any warranties from the facility that will do it.
just my2c
Hi Petre,
Thanks for your input. We had considered generating some mate-pair libraries, but still with the large insert-sizes we will need a lot of input DNA. And with the cost of PacBio coming down, the thought was that we might as well have full-length sequences rather than paired short reads. Since the project has the funding to accommodate the increased cost.
I have done illumina mate-pair with less than 1 ug. Your complexity is 1/15 of human genome so you should be OK with less DNA. Anyhow, I think that if you are missing some DNA fragments it is for a reason.... so PBio might help, however I am not sure if it will give you all the needed information. Do you expect too many (long) repeats?
Good Luck
No, actually the genome is of an internal parasite and is expected (based on related genomes) to have reduced repeat composition- something in the range of 20%.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods