Hi Scott,

My first thought is:

1. Remove rDNA sequences (and/or other well known highly-conserved sequences to 
reduce the workload in step 2).
2. Blast, then remove sequences with > (say 99%) match to > (say 5) genus. 
(Optional if step 1 is already good enough)

For step 1:
    Build a fasta file of the chosen highly conserved sequences, and use it as 
a feed to blast against your MiSeq result.
    Remove positive hits.
For step 2:
    Blast remaining MiSeq sequences against NCBI (or whatever) database.
    Remove if it hits more than n genus.

On 24 Sep 2013, at 22:17, Scott Tighe <scott.ti...@uvm.edu> 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

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