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Old 11-13-2013, 09:34 AM   #1
bsp017
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Default Creating bacterial reference genomes

Dear all,

I would like to create two complete bacterial genomes from two recently described bacteria. I have draft genomes for both. My plan is to create a second library using the 454. Is it likely that I would be able to close the genomes using this strategy?

For the draft assembly I used the Illumina MiSeq 250bp paired end reads and got x60 coverage. I then assembled the genomes using CLC genomics workbench and got these assemblies:
1. Contigs 92, N50 144,715, GC content 56%, size 5.2 Mb
2. Contigs 327, N50 38,957, GC content 53%, size 5.4 Mb

Thanks
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Old 11-13-2013, 09:37 AM   #2
GenoMax
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If you can get access to a Pacific Biosciences sequencer then that would be best. There are commercial providers in case you do not have local access to one.

http://blog.pacificbiosciences.com/2...ighlights.html

Last edited by GenoMax; 11-13-2013 at 09:40 AM.
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Old 11-13-2013, 09:42 AM   #3
bsp017
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Thanks GenoMax,

I thought about PacBio but guessed the cost would be prohibitive and hoped the 454 would be able to get the job done for cheaper.
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Old 11-13-2013, 10:09 AM   #4
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You could try MiSeq with large inserts. It should definitely help with assembly..
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Old 11-13-2013, 10:13 AM   #5
GenoMax
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If you are interested in truly "finishing" the genomes it may be worth spending the extra effort/money on PacBio. You can inquire with a provider (http://www.pacificbiosciences.com/su...cing_provider/) to see what the cost may be. That should only take a few emails/calls.
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Old 11-13-2013, 11:59 AM   #6
LeightonP
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We didn't manage to close any of 25 bacterial genomes with 454 only. I haven't used PacBio, but I hear good things about it for closing.
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Old 11-13-2013, 03:15 PM   #7
nickloman
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Definitely PacBio, it's actually competitively priced with 454, perhaps cheaper. Try Duke University's quote generator: https://dugsim.net/
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Old 11-13-2013, 06:30 PM   #8
bckirkup
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Default Primer Walking

If you can scaffold pretty well, you can use Sanger to close the small stuff and then roll on to primer walking for anything too large for that. Genewiz and other companies offer services at a per megabase rate.

The problem with using short read to fill gaps in short read is that the same problems trip them both up.

If you can't scaffold well, you can use OpGen to get that bootstrapped, by the way. However, it isn't inexpensive.
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