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Old 08-11-2015, 08:03 AM   #1
cngl
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Default minimum amount of DNA for analysis

What is the minimum amount of DNA for pyrosequencing analysis of bacterial samples?
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Old 08-11-2015, 10:58 AM   #2
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What is the minimum amount of DNA for pyrosequencing analysis of bacterial samples?
The 454 is basically obsolete. I've heard that Roche plans to discontinue producing reagents for it in the near future. I would suggest looking for Illumina sequencing cores.

"Analysis of bacterial samples" could mean any one of several assays. Do you want full genome sequences? ITS variable loop sequencing? Something else?

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Old 08-11-2015, 11:42 AM   #3
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I want to analyze the microorganisms residing in micro biome of an infected tooth. The problem is that, especially in persistent infections small amounts of bacteria are present in tooth. But the firm who will perform pyrosequencing required DNA concentrations higher than 10-15ng/ul and we could not reach that levels. That is why i want to know the minimum DNA concentration required.
In endodontics, which is a branch of dentistry, most of the analysis were conducted by pyrosequencing since 2009, Illumina has gained popularity in our field recently. So you suggest Illumina. Our research centre performs Illumina Miseq, you think i should convert the project and use Illumina Miseq?

Thank you for your answer.
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Old 08-11-2015, 12:08 PM   #4
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Originally Posted by cngl View Post
I want to analyze the microorganisms residing in micro biome of an infected tooth. The problem is that, especially in persistent infections small amounts of bacteria are present in tooth. But the firm who will perform pyrosequencing required DNA concentrations higher than 10-15ng/ul and we could not reach that levels. That is why i want to know the minimum DNA concentration required.
In endodontics, which is a branch of dentistry, most of the analysis were conducted by pyrosequencing since 2009, Illumina has gained popularity in our field recently. So you suggest Illumina. Our research centre performs Illumina Miseq, you think i should convert the project and use Illumina Miseq?

Thank you for your answer.
Yes, this is what I think you should do. See here. Roche is shutting down 454 next year. I presume this means they will not longer manufacture reagents for 454s.

As far as amount of DNA needed -- if your assay just involves amplifying 16S rRNA variable loops, then the assay should require very little DNA since it involves doing PCR.

If you want to do a full "meta-genomics" project -- where you obtain the sequence of the genomes of the bacteria in your sample, then you would need more.

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Old 08-11-2015, 12:56 PM   #5
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Here i quote the only one Illumina Hiseq study used in endodontics: "The samples were characterized by profiling the microbial community
on the basis of the 16S ribosomal RNA gene by using the Illumina
HiSeq2000 sequencing combinatorial sequence-tagged
polymerase chain reaction products. Forward (50-CAACGCGARG
AACCTTACC-30) and reverse (50-ACAACACGAG CTGACGAC-30)
primers were used to amplify the bacterial-specific V6 hypervariable
region of the 16S ribosomal RNA gene" and the results are "All root canals specimens displayed highly polymicrobial communities
in all 3 patient groups. One sample contained 58 (mean = 6.5)
phyla of bacteria. The most numerous were Firmicutes and Bacteroidetes,
but Actinobacteria, Fusobacteria, Proteobacteria, Spirochaetes,
Tenericutes, and Synergistetes were also present in most of
the patients" Isn't this study a metagenomics study, i've read journals used this term for these studies. Like this study, i aim to learn which bacteria reside in an infected root canal system. What is the concentration required for a study like this? I have access to use Illumina Miseq, would it be proper?

Thanks again.
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Old 08-11-2015, 08:36 PM   #6
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Metagenomics has been used to mean either sequencing 16S or whole genomes of a complex bacterial community. There are a substantial number of people who prefer that metagenomics be used just for whole genome approaches, though.

I think you are in good shape if you have access to a MiSeq. It is the predominant sequencer for 16S studies. I would look up references for 16S studies first to see which primers and protocols are current, then check if your case of low input has any special variations.
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Old 08-12-2015, 06:33 AM   #7
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Yes, a MiSeq should work fine for this purpose.
The idea is that bacterial genomes are generally in the 1-4 million base range, but they all have 1-10 (or more?) copies of their rRNA genes. By focusing on a "hypervariable loop" (V-loop) you anchor PCR primers in a highly conserved region of the 16S rRNA gene but the sequence between those priming sites is variable among species. I don't know exactly what size your V6 PCR product would be, but if it is 200bp, then you are only sequencing a diagnostic 200/2,000,000 = 1/20,000th of the genome.

I would focus first on how many bacterial genomes are represented by 1 ng of DNA. 1 ng of DNA is roughly 1 million bacterial genomes of a length of 1 million bp. So if your sample is pure bacterial DNA, then you probably have >100,000 bacterial genomes in it. But I'm guessing that the bacteria represent only a portion of the total DNA prep.

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