Hi all -
Not to derail the conversation, but I wanted to point out some Galaxy
resources that may help when considering how to approach solution. These
may be knowns, but thought I'd put them out there just in case. See below.
Best!
Jen
Galaxy team
There are at least three public Galaxy instances that focus heavily on
Metagenomics. Maybe worth a look?
http://wiki.galaxyproject.org/PublicGalaxyServers
Just do a browser search on "metagenomics" to find on page. May be
others, but these are top 3.
The Tool Shed may or may not contain specialized tools from these
servers. Asking to have those tools made available via TS route is can
be done through direct contact. Other repos may have tools that fit or
could be tuned. Tool authors own tools - changes could potentially be
incorporated through direct contact. Or, as is open source, used as
baseline with attribution if that doesn't work out.
http://toolshed.g2.bx.psu.edu/
Making a Galaxy Trello ticket for new tools and discussing new tool
development on the [email protected] list may help you find other
Galaxy community developers working on similar projects. Tickets are not
just for the Galaxy core team, and even though the issue to solve is
scientific, a technical implementation seems to be where this is going
(new tool or existing tool tuning).
http://wiki.galaxyproject.org/Issues -> Inbox is where this would go.
Final home almost certainly Tool Shed (same for all tools), but
possibility of also including on Galaxy Main server also exists once
there are a valid repo and it is determined to be a good fit (resource,
etc.).
On 9/24/13 7:17 AM, Scott Tighe wrote:
Jing et al
Thank you for the offer to write some code to help advance the
metagenomics arena. It is certainly needed.
So the problem is well known with megablast and shotgun metagenomics
and without proper understanding and correct software will yield very
misleading and in many cases incorrect data. For those of us who wish
NOT to move to a protein level of comparison for specific reasons, we
are stuck.
*The Problem:*
If I megablast 50 million sequences from a HiSeq run, millions of rRNA
sequences will have a 99% match to all microbes rRNA genbank deposits.
Not surprizing since the rRNA is highly conserved. The difference
between E.coli and Shigella is 1 to 2 bases for the full 1540 bp 16s.
So 16s is not useful for Genus level, and certainly not Species
*So what happens:*
The returned matches will have many hits to whatever model organism is
in Genbank. For example E coli has 13000 entries for rRNA and
Sphearotilus has 3 entries for rRNA. If the blasted sequence matches
both, the results will mislead the investigator to think they have
13000 hits to E coli, EVEN if the microbe is Sphearotilus.
*The cure?:*
If there was a way to filter/ remove all hits ? Let say, for example,
that a result has a first match (say E. coli) at >99% a second match
(say Pseudomanas) at >99% and a third , forth and fifth match >99 for
three other organisms. This sequence _must_ be discarded because it is
a conserve sequence.
Basically conserved sequence is the enemy and invalidates the entire
result.
*
**Another problem:*
If you have a reference sample with 19 non-model microbes, and you
run that by HiSeq Shotgun for metagenomics and then megablast, what do
you think you get? If E coli is not in the reference sample, how many
hits do you think you get? Yes, 10,000 of thousands. So without
removing conserved sequences, your data is wrong and you are much
better served by culturing and running a Biolog metabolic panel and
comparing to the sequence result.
So where do we start? I have some shotgun metagenomics data from the
reference sample which included the 19 microbes. That was data from a
MiSeq.
Scott
Scott Tighe
Senior Core Laboratory Research Staff
Advanced Genome Technologies Core
University of Vermont
Vermont Cancer Center
149 Beaumont ave
Health Science Research Facility 303/305
Burlington Vermont 05405
802-656-2557
On 9/20/2013 9:17 PM, Jing Yu wrote:
Hi Scott,
I can do some perl programming, such as local/remote blasting. Can
you specify your problem a little bit clearer, so that maybe I can
write a program to do just that?
Regards,
Jing
Gerald
16s is basically useless for identification to genus. Since I
started sequencing 16s in 1992, I have come to realize that without
sequencing the full 1540 bases, it is generally misleading, and even
than, it is not accurate enough to nail genus on more than 1/2 the
cases. However, what is your feeling on ITS and gyrase, They seem
to be far more discriminating but those databases have been
decommissioned sometime ago.
The desirable thing would be that Galaxy or NCBI add a "filter
conserved genes" [ ie any hit with a second choice greater than 3%
distance]. Something such as that.
If you (or others) are aware of such a thing, I'd love the here
about it.
Sincerely
Scott
___________________________________________________________
The Galaxy User list should be used for the discussion of
Galaxy analysis and other features on the public server
at usegalaxy.org. Please keep all replies on the list by
using "reply all" in your mail client. For discussion of
local Galaxy instances and the Galaxy source code, please
use the Galaxy Development list:
http://lists.bx.psu.edu/listinfo/galaxy-dev
To manage your subscriptions to this and other Galaxy lists,
please use the interface at:
http://lists.bx.psu.edu/
To search Galaxy mailing lists use the unified search at:
http://galaxyproject.org/search/mailinglists/
--
Jennifer Hillman-Jackson
http://galaxyproject.org
___________________________________________________________
The Galaxy User list should be used for the discussion of
Galaxy analysis and other features on the public server
at usegalaxy.org. Please keep all replies on the list by
using "reply all" in your mail client. For discussion of
local Galaxy instances and the Galaxy source code, please
use the Galaxy Development list:
http://lists.bx.psu.edu/listinfo/galaxy-dev
To manage your subscriptions to this and other Galaxy lists,
please use the interface at:
http://lists.bx.psu.edu/
To search Galaxy mailing lists use the unified search at:
http://galaxyproject.org/search/mailinglists/
