Why don't you extract chloroplasts and then DNA from them afterwards. A single SMRT Cell (PacBio) is enough to sequence the DNA thna. Just do an 1k bp insert size run and use the CCS reads generated by the SMRT portal to assemble the chloroplast genome. Depended on you sequencing provider it will costs you between 1000-2000$.

Hi!

You have 2 choice.

1: Illumina sequencing of total DNA. You will have enough read for a good chloroplast coverage.

2: You can separate the chloroplast DNA of the total DNA by ultracentrifugation with CsCl at 50 000 rpm/24h. After, you can make a 454 and/or illumina sequencing.

For the price, it may vary (1500-3000$)

If you habe any question, feel free to ask!

Hi!

If you can isolate the chloroplasts (see the previous answer), I would suggest Illumina MiSeq paired-end sequencing (2x 250bp or 2x 300bp) as a chloroplast genome does usually not contain long repetitive elements. Newbler for the assembly and Consed for in silico finishing.

Concerning the costs: The library prep costs ~100$ per library if you do it yourself. I am not sure what you pay for ~20MByte (~200x coverage) on the free market as we have a machine in house. But I would guess no mor than 500$ for that, as it can be multiplexed.

Hi,

You do not need to isolate chloroplasts. Whole DNA extraction from young leaves and low coverage Illumina paied-end seq provide high coverage of chloroplast DNA. You can use related species for assembly. It should not be expensive.

This ref can help: http://www.frontiersin.org/Journal/10.3389/fpls.2012.00243/full

Thank you for all your useful responses. Seems that there are 2 ways of dealing with this, exactly as A. Vincent commented: (a) total genome sequencing and later separation of chloroplast sequences "in silicio"; (b) isolation of chloroplasts and sequencing (multiplexed) of their smaller genome.

There are some relatively easy protocols of chloroplast isolation, like Sigma Chloroplast isolation kit (http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/cpisobul.pdf), so it seems that (b) option could be cheaper in sequencing part (as Ch. Rückert mentioned).

Hi Viacheslav

the ease of chloroplast isolation totally depends on your species and tissue. It can be tricky ...

Previously, people have used PCR-based strategies, using universal primers for example those listed in my chloroplast primer database at: http://bfw.ac.at/rz/bfwcms2.web?dok=4977 . Nowadays, I would only do this for tricky cases, or for finishing any gaps. Though PCR plus next gen sequencing (Illumina) also works.

Hi

I just recently finished assembly of chloroplast genome. I used Illumina sequencing to seqeunce the whole genome. My coverage is only one times which was enough for getting the whole chloroplast. It cost me about 7000-8000$ which was not very bad. I've got lots of data to play with it.

Hi Viacheslav,

I am from Science Exchange. Since you've decided to isolate chloroplasts yourself followed followed by sequencing (as per the suggestion of Dr Rückert), you can access several core facilities through our platform, who can do the the sequencing service for you. Please check them out using the following link.

https://www.scienceexchange.com/services/illumina-ngs

Let me know if it helps. Good Luck.

Best,

Atif