16SRNA sequencing to determine differences between bacteria species
Koz
I want to do a fairly simple test and determine specific species of
bacteria that exist on different surfaces.
The agar plate/phenotype test does not give me enough precise data, so
I was wondering if there is a relatively inexpensive way to sequence
microorganisms from a collection of swabs?
Pretty new to this and have very limited access to basic lab equipment
so supposedly I would need to send this out.
Any suggestions?
Alexander
Nathan McCorkle
How to perform bacterial ID (I am trying to determine a Bacillus species)
http://groups.google.com/group/diybio/browse_thread/thread/449334a66c22444a
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Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics
Nathan McCorkle
On Oct 6, 12:00 am, Nathan McCorkle <nmz...@gmail.com> wrote:
> I was thinking that colony PCR is really quite easy, are there
> universal primers for 16S? I could get some in the mail in a few days
> for not more than $5-$15... not sure if I really need that level
> though, since I think this Prof is a "very very fair" grader.
JGI typically uses universal primers 27F/1391R for bacterial 16S rRNA:
http://my.jgi.doe.gov/general/protocols/SOP_16S18S_rRNA_PCR_Library_C...
The Wikipedia page actually has a table with universal primers as
well:
http://en.wikipedia.org/wiki/16S_ribosomal_RNA#Universal_Primers
16S is such a common target in microbiology, that chances are somebody
at your lab may have some primers available that you could use. Ask
around among the grad students and postdocs...
Derek
instead you are trying to identify species distribution from a swab
then my understanding is that the pcr step is largely the same but
that you'll have such a mix of species that you can no longer depend
on a simple sanger sequencing. I believe multi-tag pyrosequencing in a
454 run is the current preferred approach in determining species
distribution, but at $4k a run that's definitely not what you're
looking for.
If I'm wrong and there's a simpler way to do species distribution from
swab samples please let me know!
Otherwise, you may be best off in attempting to isolate colonies
inoculated from your swabs onto petri dishes and sanger sequencing
individual colonies at $10 a pop or so. That won't give you
distributions but will at least distinctly identify some individual
bacteria.
--Derek
On Dec 1, 8:16 pm, Nathan McCorkle <nmz...@gmail.com> wrote:
> (Specifically Patrik's response):
> On Oct 6, 12:00 am, Nathan McCorkle <nmz...@gmail.com> wrote:
>
> > I was thinking that colony PCR is really quite easy, are there
> > universal primers for 16S? I could get some in the mail in a few days
> > for not more than $5-$15... not sure if I really need that level
> > though, since I think this Prof is a "very very fair" grader.
>
> JGI typically uses universal primers 27F/1391R for bacterial 16S rRNA:http://my.jgi.doe.gov/general/protocols/SOP_16S18S_rRNA_PCR_Library_C...
> The Wikipedia page actually has a table with universal primers as
> well:http://en.wikipedia.org/wiki/16S_ribosomal_RNA#Universal_Primers
> 16S is such a common target in microbiology, that chances are somebody
> at your lab may have some primers available that you could use. Ask
> around among the grad students and postdocs...
>
>
>
>
>
>
>
>
>
> On Thu, Dec 1, 2011 at 11:15 PM, Nathan McCorkle <nmz...@gmail.com> wrote:
> > See this recent thread, you'll need lab access and tools to perform PCR:
>
> > How to perform bacterial ID (I am trying to determine a Bacillus species)
> >http://groups.google.com/group/diybio/browse_thread/thread/449334a66c...
>
> > On Thu, Dec 1, 2011 at 9:58 PM, Koz <kozovsk...@gmail.com> wrote:
> >> Hi ,
>
> >> I want to do a fairly simple test and determine specific species of
> >> bacteria that exist on different surfaces.
> >> The agar plate/phenotype test does not give me enough precise data, so
> >> I was wondering if there is a relatively inexpensive way to sequence
> >> microorganisms from a collection of swabs?
> >> Pretty new to this and have very limited access to basic lab equipment
> >> so supposedly I would need to send this out.
> >> Any suggestions?
>
> >> Alexander
>
> >> --
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Koz
Derek to your point,
I will start with some 20 or so TSA plates and see what grows per each
swab and then attempt to isolate.
Any suggestions on culturing? Should I split each petri in 4?
Alexander
Derek
plate just to keep them isolated.
Pretty close to a lab protocol at Washington U that a bit of google-fu
turned up, btw: www.nslc.wustl.edu/elgin/genomics/bio3055/idunknbacteria06.pdf
--Derek
Pat
bacteria out in pure culture before you try to amplify the 16s
region.
Right now I'm working with a endophytic plant bacteria that is
extremely hard to culture I ran a 16s PCR on the tissue and needless
to say I had quite a few bands on the gel. From my gel it does seem
there is a band in the same region as the control. I'm planning to cut
it out and having it sequenced. Long story short not everything is
culturable and it maybe neat to also determine if things are present
that you can't readily culture.
Koz
I'll share results as soon as I get something interesting :)
Alexander
On Dec 1, 11:16 pm, Nathan McCorkle <nmz...@gmail.com> wrote:
> (Specifically Patrik's response):
> On Oct 6, 12:00 am, Nathan McCorkle <nmz...@gmail.com> wrote:
>
> > I was thinking that colony PCR is really quite easy, are there
> > universal primers for 16S? I could get some in the mail in a few days
> > for not more than $5-$15... not sure if I really need that level
> > though, since I think this Prof is a "very very fair" grader.
>
> JGI typically uses universal primers 27F/1391R for bacterial 16S rRNA:http://my.jgi.doe.gov/general/protocols/SOP_16S18S_rRNA_PCR_Library_C...
> The Wikipedia page actually has a table with universal primers as
> well:http://en.wikipedia.org/wiki/16S_ribosomal_RNA#Universal_Primers
> 16S is such a common target in microbiology, that chances are somebody
> at your lab may have some primers available that you could use. Ask
> around among the grad students and postdocs...
>
>
>
>
>
>
>
>
>
> On Thu, Dec 1, 2011 at 11:15 PM, Nathan McCorkle <nmz...@gmail.com> wrote:
> > See this recent thread, you'll need lab access and tools to perform PCR:
>
> > How to perform bacterial ID (I am trying to determine a Bacillus species)
> >http://groups.google.com/group/diybio/browse_thread/thread/449334a66c...
>
> > On Thu, Dec 1, 2011 at 9:58 PM, Koz <kozovsk...@gmail.com> wrote:
> >> Hi ,
>
> >> I want to do a fairly simple test and determine specific species of
> >> bacteria that exist on different surfaces.
> >> The agar plate/phenotype test does not give me enough precise data, so
> >> I was wondering if there is a relatively inexpensive way to sequence
> >> microorganisms from a collection of swabs?
> >> Pretty new to this and have very limited access to basic lab equipment
> >> so supposedly I would need to send this out.
> >> Any suggestions?
>
> >> Alexander
>
> >> --
> >> You received this message because you are subscribed to the Google Groups "DIYbio" group.
> >> To post to this group, send email to diy...@googlegroups.com.
> >> To unsubscribe from this group, send email to diybio+un...@googlegroups.com.
> >> For more options, visit this group athttp://groups.google.com/group/diybio?hl=en.
helicase
why culture-independent bacterial community work has become
commonplace), there are other options short of doing next-gen 16S
amplicon seq.
As you're probably aware, three passages of subculturing is the
typical standard for isolation.
Some options if you can't obtain a pure culture / you're interested in
the unculturable portion of your community at hand:
1. You could make a 16S clone library. PCR amplify 16S and then clone
your PCR product. Sequence as many or few clones as necessary to
answer your question. Advantages: you can look at nearly full-length
16S sequences. Disadvantages: to elucidate diversity, you may have to
sequence a lot of clones. Not the case if you just want to ID the most
common members of a community.
2. Alternatively, Denaturing Gradient Gel Electrophoresis (DGGE)
involves PCR-amplifying a 16S fragment, adding a GC clamp in the
process. The PCR products (16S fragments with clamp) are run on an
arcylimide gel with a urea-formamide gradient. The PCR products are
separated by sequence as they denature in a sequence-specific manner
through the increasing gradient along the length of the gel. The gel
is stained with EtBr. Banding patterns can be used for comparison
directly (pretty crude), and bands can be excised from the gel, re-
amplified via PCR, and sequenced. Advantages: gives a (somewhat crude)
community profile without cloning. Disadvantages: limited sensitivity
- members constituting <5-10% of the community will likely be missed.
Also, you need to use acrylimide, which is a neurotoxin. The gradient
needs to be optimized for your project. And you need a way of casting
the gradient gel, and running a vertical gel at an elevated
temperature (say 60°C). It's all stuff that could be homegrown (the
gel running rigs are basically glorified fish tanks with a heater and
power supply) but would require work. And of course protection from
and disposal of the hazardous reagents.
Cheers,
Anthony
Patrik
> 1. You could make a 16S clone library. PCR amplify 16S and then clone
> your PCR product. Sequence as many or few clones as necessary to
> answer your question. Advantages: you can look at nearly full-length
> 16S sequences. Disadvantages: to elucidate diversity, you may have to
> sequence a lot of clones. Not the case if you just want to ID the most
> common members of a community.
You could also normalize the 16S library, so you can identify a wider
diversity of community members with a limited number of clones. You
lose information about relative abundance, but this may be a
reasonable DIY approach, if you can't afford to sequence too many
clones.
Russell Durrett
full of diverse 16S rRNA sequences on a PacBio RS. It sequences by
'watching' one polymerase incorporate radiolabeled nucleotides into
one of the 16S PCR amplicons (of course it watches tens of thousands
of them at a time, I'm currently getting 20,000 16S reads passing
filter at Q20 per sample) - so you can read all the different
sequences in your heterogeneous PCR product without having to culture
it.
So you have a price reference, I run the PacBio at Cornell Med and
we're currently charging just over $1000 / sample for a library prep
and sequencing [maybe a bit out of DIYbio range, but if you get a
grant or something it's an option..]
Koz
So much explore.
A bunch of TSA plates are coming tomorrow, so I will apparently have
the 6th grade experiment and deal with the unculturable by researching
a bit more on these other suggested options.
First glance, sounds like the 454 is the way to go for deep sequencing
and actually achieving my particular result, since I need both density
and type as fairly precise data sets.
I'll have a look around and see if there are any options for
collaboration or upcoming projects I could get involved in, in order
to get this achieved.
Alexander
Nathan McCorkle
so there may be grants floating around, or workable with sequencing
cores.
> For more options, visit this group at http://groups.google.com/group/diybio?hl=en.
Cathal Garvey
> If you're going the NGS route, you could sequence an environmental PCR
> full of diverse 16S rRNA sequences on a PacBio RS. It sequences by
> 'watching' one polymerase incorporate radiolabeled nucleotides into
> one of the 16S PCR amplicons (of course it watches tens of thousands
Surely not radiolabelled? They don't decay on demand, how can it "see"
individual molecules' sequence by radiolabelling? Or is it just
gathering info on each random decay and painting an aggregate picture?
--
www.indiebiotech.com
twitter.com/onetruecathal
joindiaspora.com/u/cathalgarvey
PGP Public Key: http://bit.ly/CathalGKey
helicase
is based on detecting incorporation of single fluorescently-labeled
nucleotides, with the subsequent cleavage of the fluorochrome and
extension by another fluorescently-labeled nucleotide.
Koz
programming program.
The 6th grade was a just a callout to some memories of doing similar
stuff when I was much younger :)
Russell Durrett
with working on too many projects at once :\ ). PacBio even developed
their own labeled nucleotides labeled on the termini of the
triphosphate, so the label is cleaved as soon as the base is
incorporated with no designated cleaving step. So, basically what you
see from this continuous processive incorporation are peaks of
fluorescence while the base is being incorporated.
Patrik
complex 16S samples on a single run, and still get thousands of
pyrotag reads per sample. Probably the best application for 454,
actually.
It would be *really* cool if we could come up with a decent DIY
approach for metagenomic 16S sequencing - even if it's just
identifying the right sequencing service.
Mac Cowell
PCR is used again to add the adapters necessary for sequencing to the ends of all the indexed DNA in a single reaction with the pooled metasample.
Then the metasample is ready for the rest of the standard 454 prep workflow (emulsion PCR + beads etc).
So if you do the math, minus the cost of labor for the aforementioned steps, sharing a single lane on a 454 with 96 other users spreads the costs out to around the $100 / sample range.
So I bet we could do this as a community- we just need 95 other interested people and confidence in our library generation protocol. And a 454 sequencer.
Mac
231.313.9062 // @100ideas // sent from my rotary phone
Patrik
> I believe the 454 supports sample multiplexing for up to 96 samples "out of the box"
(was?) doing in practice. It's the only thing they still keep a 454
around for. All the others are getting phased out.
> So I bet we could do this as a community- we just need 95 other interested people and confidence in our library generation protocol. And a 454 sequencer.
The "and a 454 sequencer" bit may the biggest hurdle, but we could
potentially farm that out somewhere. We'd just need some middleman to
collecting samples, multiplex them together, send out for sequencing,
and split the results out afterwards. If only there existed a little
startup company that specialized in low-cost, high tech biotechnology
tools and providing small-scale sequencing solutions. ;-)