Pacbio is usefull for plants with big genome?

[ebtekar64]

Hello everyone I wonder if anyone can help me

Can I use Pacbio to getting whole chloroplast sequence for a plant with ~21Gb genome size? Just with a library and run on a smrtcell.

Thanks

[Bukowski]

It's not clear here, are you trying to sequence the chloroplast or the entire genome?

[ebtekar64]

Sorry I stump you,

I am going to sequence the chloroplast genome. There are a few paper that they have used Pacbio to sequence the chloroplast genome like "An evaluation of the PacBio RS platform for sequencing and de novo assembly of a chloroplast genome" but still I don't know this technology is useful for a plant with big genome?

[ehatas]

Hi! We find in most cases it's possible to get a complete chloroplast assembly from plant samples with 1-2 SMRT Cells of data, using HGAP. We recently had success with a single cell of data from wheat (17 Gb). You would obviously want to keep the ratio of chloroplast DNA high by avoiding sample prep techniques that enrich for nuclear DNA. If you'd like more info, please shoot me an email ehatas(at)pacificbiosciences(dot)com

[Bukowski]

Surely you could use a targeted capture approach?

http://www.ncbi.nlm.nih.gov/pubmed/24690362

This would enrich for the chloroplast, and then the PacBio would give you just what you want from the platform - long reads (although if ehatas is correct, then you don't even need to enrich, presumably some judicious size selection could work just fine)

[gconcepcion]

Isolating a sample of chloroplasts is fairly easy and what I would recommend as the best way to go.

However, as ehatas alluded to, chloroplast DNA is generally grossly over represented in typical plant cell DNA extractions, and seeing as though a typical chloroplast genome is only 120-200Kb, 1-2 SMRTCells of data should be more than sufficient to close a chloroplast genome.

But isolating chloroplasts is probably the best way to go, and really not that difficult:

http://biology.clc.uc.edu/fankhauser/labs/cell_biology/chloroplasts/chloroplasts.htm

[ebtekar64]

Thanks gconcepcion

You're right, isolation chloroplast is easy but extract only chloroplast is too hard. For example I tried for Artemisia many protocol and couldn't have isolated chloroplast DNA. If we can isolate only Chloroplast DNA we will able to run many samples (~70) and sequence just with one smartcell.

[SylvainL]

I don't know in Artemisia but I think the number of copy of chloroplast genome is really higher (80X) than the nuclear one... Even a non fully pure chloroplast prep should work...