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Old 01-07-2019, 07:33 AM   #1
iuliachiciudean
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Default PacBio, SMART, assembly problem of de novo small genome

Hey,

I am very new here and I don't have mach experience with bioinfo and sequencing.

I need your help because i recently send the DNA of one bacteria to be sequence by PacBio technology and i asked the sequencing company that I want a de novo genome assembly.

At the beginning they send a report in witch they imply that my DNA was contaminated before sequencing. As i told them that priory to send them the DNA I dis an 16S sequencing with another company and all was good, they replay that is their mistake and they will re-assembly the genome again.

Now I just got their new report and is a mess. (I am attaching the report).

I don't even know what to ask from them anymore. Insted of one circular genome I have one non-circular chromosome and 7!!!!! plasmids.

Any of your advise will be very welcomed.Results 1.jpg

Results 2 (1).jpg
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Old 01-07-2019, 06:03 PM   #2
luc
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Nothing about the read metrics raises warning flags - they could certainly have generated longer library molecules though since they did not multiplex the library.

Is the genome size about what you expected?

I would try additional assemblies with subsets of the data for examples only the longest reads. 50x coverage would be plenty. I would also try Canu as an alternative assembler.
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Old 01-08-2019, 01:07 AM   #3
iuliachiciudean
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Thx for your suggestion.

The genome size should be bigger, around 5.5 Mb.

I'm thinking that some of the "plasmids" are actually part of the chromosome. Or is also possible to have 2 chromosomes. This future can be found in this bacteria genus.
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Old 01-08-2019, 04:23 AM   #4
Bukowski
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Quote:
Originally Posted by iuliachiciudean View Post
Thx for your suggestion.

The genome size should be bigger, around 5.5 Mb.

I'm thinking that some of the "plasmids" are actually part of the chromosome. Or is also possible to have 2 chromosomes. This future can be found in this bacteria genus.
Judging from the total size of the contigs that would be my suspicion, because it looks like you have the whole 5.5Mb there in some form - just not in a single contig (which you can't guarantee, even with PacBio, all the time).
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Old 01-08-2019, 04:42 AM   #5
iuliachiciudean
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Quote:
Originally Posted by Bukowski View Post
Judging from the total size of the contigs that would be my suspicion, because it looks like you have the whole 5.5Mb there in some form - just not in a single contig (which you can't guarantee, even with PacBio, all the time).
So do you think would be possible to solve this only by doing a new assembly with different parameters (which ones?)?

Or should we do more sequencing?
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Old 01-08-2019, 09:08 PM   #6
SNPsaurus
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I think the confusion is that they are calling all the contigs except for the largest "plasmid" which would be premature to do so without other evidence, especially if they are called as non-circular. Other than that, it is an OK PacBio assembly. I'd say a majority of the time a bacterial genome assembles as a single contig but a significant minority is fragmented into multiple contigs.

I'd blast the resulting contigs. Usually it is obvious if it is a plasmid from the blast results. If you really really want a better assembly then it might be worth to re-sequence now that newer kits have been released with longer read lengths. The short fragment size may have been due to DNA quality, though.

I would try Canu assembly with a higher expected coverage since you have the read depth to do so. The only other thing I can think of is that some libraries have high rates of palindromic reads due to un-repaired ends folding back and acting as a barbell adapter. This can mess up assembly. The newest version of the SMRT assembler chooses a single subread from each well to avoid some of the problems with the palindromes (I think) so I'd check if they assembled with the System 6 update. We filter palindromes before Canu to avoid the problem.
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