Urgent question about sequence assembly

[Omneya]

Dear Members,

It is my pleasure to join this community.
I am a doctoral student in Japan.
I have a problem and I need help please. I was able to isolate to new unknown plasmids from halophilic bacteria and I was doing sequence of these plasmid by random DNA library construction. Now the sequence fragments of each plasmid doesnt show any overlap. I am nor sure what is the reason.
The purification was intensive to separate each plasmid but I would like to know if there is other possibilities for such a problem please.
I need your expert opinion as soon as possible.



Best Regards.
Omeya

[ECO]

Questions that may help others answer:

What technology did you sequence with?
What assembly tools did you use (and with what settings)?

[Omneya]

Thanks for your reply,
Answering your questions as follows:

What technology did you sequence with?
Normal sequence was done with ABI3730xl sequencer.

What assembly tools did you use (and with what settings)?
I compared with all results by DNA alignment software GENETIX,and could not find any overlap.

I hope someone can help me to solve the problem as soon as possible.
Thanks

[westerman]

The 3730 is not second generation technology and thus a lot of the tools that we discuss here will not be perfectly applicable to assembling 3730 reads although they can be made to work. On the other hand there are many proven tools and techniques to work with 3730 data. Our facility uses the tried and true 'phred/phrap/consed' triad of programs. However these programs are a pain to set up and learn. My suggestion: Go back to the facility that did the 3730 sequencing for you and ask what program they use. At the very least they should have a pipeline set up.

As for your problem, it would be nice to know what type of coverage you got. In other words the number of reads times the average length of the reads compared to the expected size of your plasmids. It is quite possible that you did not sequence in enough depth to be able to assemble anything. Once again your sequencing facility should be able to offer some advice.