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Old 05-08-2014, 07:35 AM   #1
create.share
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Default bacterial and viral genome sequencing with illumina myseq

Hola

I have a several bacteria which harbor lysogenic viruses (I can induce them with MitomycinC). On one hand side I want to sequence the host. On the other hand side I want to sequence the virus. The host (bacterial) DNA is easy to obtain, however, due to some particularities of the culture media, the virus DNA is very hard to get (at least in a asuficient quantity as required by the sequencing company). That beinng said, I want to do the following:
- sequence the host DNA from an uninduced culture (no viruses present, only bacterial DNA, of a size of 3-4 Mbp genome)
- in paralel, sequence the DNA from an induced culture (both bacterial and viral DNA are present, the viral DNA in 10-30 copies more than the bacterial DNA, but shorter, in between 10-100 kbps).

And of course, I donīt want to do it too expensive. Therefore, I have chosen a MiSeq Illumina package, which will give me about 2.2 mil reads, 300 nt, pair ended (thereore 2x300) per sample.

My question is:
- with this type of sequencing,would I be able to pull appart the viral DNA from the host DNA? My intention is to compare with the virus free bacterial sequences.
- do you thing it would work without sequencing the bacterial host separately, but rather sequencing the infected culture only and then pull appart the bacterial genome from the viral genome?
- with the chosen package - 2.2 mil 2x300 nt reads per sample, do you think I will have a good coverage of the host and viral genomes?

thank you
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Old 05-08-2014, 10:14 AM   #2
Brian Bushnell
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Quote:
Originally Posted by create.share View Post
- with this type of sequencing,would I be able to pull apart the viral DNA from the host DNA? My intention is to compare with the virus free bacterial sequences.
This is not difficult if you have assemblies of the bacteria and/or viruses, and very hard otherwise.
Quote:
- do you thing it would work without sequencing the bacterial host separately, but rather sequencing the infected culture only and then pull apart the bacterial genome from the viral genome?
I think sequencing the uninfected bacteria separately is essential if you don't already have references for them. You can then assemble them and map the reads from the infected bacteria to split into bacterial and viral read sets.
Quote:
- with the chosen package - 2.2 mil 2x300 nt reads per sample, do you think I will have a good coverage of the host and viral genomes?
Depends on how many bacteria you have, what percent of the DNA is viral, and how big the virus is. But you're generating around 1320Mbp, best case, which would give 330x coverage of a single 4Mbp bacteria. For one bacteria that should be fine, but if you have multiple bacteria at different concentrations, it's probably inadequate. And for recovering the viral DNA, well... I don't know, it depends on too many factors for me to predict.
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Old 05-09-2014, 01:26 AM   #3
create.share
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Quote:
Originally Posted by Brian Bushnell View Post
This is not difficult if you have assemblies of the bacteria and/or viruses, and very hard otherwise.

I think sequencing the uninfected bacteria separately is essential if you don't already have references for them. You can then assemble them and map the reads from the infected bacteria to split into bacterial and viral read sets.
Thanks for the answer. You're right, I should do the uninfected host separately then. Also, what I hope is that, in the infected sample, the virus will produce a sufficiently high number of copies which will result in an enrichment in reads belonging to the virus (I hope to use the read enrichment as one criterion for pulling the virus apart).

Quote:
Originally Posted by Brian Bushnell View Post
Depends on how many bacteria you have, what percent of the DNA is viral, and how big the virus is. But you're generating around 1320Mbp, best case, which would give 330x coverage of a single 4Mbp bacteria. For one bacteria that should be fine, but if you have multiple bacteria at different concentrations, it's probably inadequate. And for recovering the viral DNA, well... I don't know, it depends on too many factors for me to predict.
The 2.2 mil 2x300 nt reads is for one single bacteria, e.g. for the uninfected host.
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