What is the minimum amount of DNA for pyrosequencing analysis of bacterial samples?
Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
X
-
Originally posted by cngl View PostWhat is the minimum amount of DNA for pyrosequencing analysis of bacterial samples?
"Analysis of bacterial samples" could mean any one of several assays. Do you want full genome sequences? ITS variable loop sequencing? Something else?
--
Phillip
-
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.
Comment
-
Originally posted by cngl View PostI 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.
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.
--
Phillip
Comment
-
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 5–8 (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.
Comment
-
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.Providing nextRAD genotyping and PacBio sequencing services. http://snpsaurus.com
Comment
-
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.
--
Phillip
Comment
Latest Articles
Collapse
-
by seqadmin
Non-coding RNAs (ncRNAs) do not code for proteins but play important roles in numerous cellular processes including gene silencing, developmental pathways, and more. There are numerous types including microRNA (miRNA), long ncRNA (lncRNA), circular RNA (circRNA), and more. In this article, we discuss innovative ncRNA research and explore recent technological advancements that improve the study of ncRNAs.
Nobel Prize for MicroRNA Discovery
This week,...-
Channel: Articles
10-07-2024, 08:07 AM -
-
by seqadmin
Metagenomics has improved the way researchers study microorganisms across diverse environments. Historically, studying microorganisms relied on culturing them in the lab, a method that limits the investigation of many species since most are unculturable1. Metagenomics overcomes these issues by allowing the study of microorganisms regardless of their ability to be cultured or the environments they inhabit. Over time, the field has evolved, especially with the advent...-
Channel: Articles
09-23-2024, 06:35 AM -
ad_right_rmr
Collapse
News
Collapse
Topics | Statistics | Last Post | ||
---|---|---|---|---|
Started by seqadmin, 10-11-2024, 06:55 AM
|
0 responses
11 views
0 likes
|
Last Post
by seqadmin
10-11-2024, 06:55 AM
|
||
Started by seqadmin, 10-02-2024, 04:51 AM
|
0 responses
110 views
0 likes
|
Last Post
by seqadmin
10-02-2024, 04:51 AM
|
||
Started by seqadmin, 10-01-2024, 07:10 AM
|
0 responses
114 views
0 likes
|
Last Post
by seqadmin
10-01-2024, 07:10 AM
|
||
Started by seqadmin, 09-30-2024, 08:33 AM
|
1 response
121 views
0 likes
|
Last Post
by EmiTom
10-07-2024, 06:46 AM
|
Comment