In my opinion, when the analysis is made by the NGS, the similarity of the 16S rDNA in environmental bacterias gives place to a bigger diversity than the real one.
Yes, this is at the moment becoming the standard approach to assess diversity in microbial communities. Many labs are now switching to 454 or Illumina reads, using barcoded primers. There is (currently) no way of knowing true diversity of an unknown sample, and no matter what method one chooses, there will be a biassed view of the true diversity. The choice of extraction method, primers, PCR mastermix ingredients, and PCR cycling conditions all could introduce some bias. But as long as one compared samples using the same amplification method, one could still say something about beta diversity between samples.
I hope this helps!
Yes, this is at the moment becoming the standard approach to assess diversity in microbial communities. Many labs are now switching to 454 or Illumina reads, using barcoded primers. There is (currently) no way of knowing true diversity of an unknown sample, and no matter what method one chooses, there will be a biassed view of the true diversity. The choice of extraction method, primers, PCR mastermix ingredients, and PCR cycling conditions all could introduce some bias. But as long as one compared samples using the same amplification method, one could still say something about beta diversity between samples.
I hope this helps!
This is an old question from the late 80's early 90's. Around 90% of bacteria are uncultivated. The only way to access them is via molecular (cultivation independent) techniques. The problem there is that you might amplify/characterize residual DNA/RNA (NA that are not from a functional cell, but still present). One method is used to avoid that, is a photosensitive dye that binds to the NA, but cannot pass into intact cells. Normally it is working relatively good, but with some limitations like every method. The interesting question to my eyes is how the "rare biosphere" is real, or sequencing artifact. One of the things I would like to do into the future is to sequence one amplicon library in extremely deep sequencing depth (>600 Mbp) in FLX. Nevertheless, what it is reality at the moment is that we just open the "box" and we are not sure yet of what is in there. So, I am not really sure that it is giving "higher" diversity than the real one... In the mean time, if you are interested in NGS for 16S rDNA, check this http://f1000.com/prime/717951509
It (NGS) will give a snapshot of microbial diversity present in a particular place. It can be bias for exaggeration of some groups of microbes, but it will not exaggerate the diversity. I still believe that there are certain group of microbes which we are not able detect by using such advance techniques. Yes, the diversity analysis done by NGS explored higher microbial diversity in comparison to cultivation methods. We need to explore more beyond NGS (in present) to understand and to get real picture of microbial diversity.
Culture techniques assume around 10% or less of the community represented. Molecular (culture independent) techniques are not a panacea, just a different way of viewing microbial communities. So really, culture methods (plates, BIOLOG) are valid ways of examining spatial and temporal community structure differences, they just can be more laborious and lack the definitive resolution of sequencing work. I think Bik's answer, that the important thing is using a consistent method to make comparisons between samples valid, is the key.
When a colleague was using qPCR with 16S rDNA universal eubacteria primers and presented an estimate of total bacteria numbers in his samples from that, with some assumptions for copy number per cell, I figured he was getting about 10% of the numbers that should have been there!
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0040467
Illumina with 250 bp max, is not really method of choice...FLX is at the moment the "Ferrari" of Amplicon seq...And soon will be even longer (XL+)...We still waiting for Roche to release it (since a year now!)
I agree with Michael, FLX is the best way because read length matters in this case. It gives the opportunity to match more than one hypervariable region, making it less biased. It is not the only bias, however, and there is still the debate about the rare biosphere. However, in my opinion it is still the best approach, especially for comparative analysis.
A nice paper about the effect of the hypervariable region and other parameters used with respect to the complete 16S: http://dx.doi.org/10.1371%2Fjournal.pcbi.1000844
454-based sequencing provides a reasonable method for determining the makeup of complex microbiological communities. We have found that the biggest source of error is the DNA extraction methodology as what works well for gram-negatives does not work as well for gram-positives and vice versa. Therefore, it is important to utilize a compound sample extraction protocol that includes multiple sizes of glass (zirconia) beads, as well as organic extraction.
Elizabeth got it covered: consistency of methods is the best option (i'd even add the bioinformatics pipeline to her list). There are, however, many examples in the literature of comparisons between sequencing methods: the results suggest there is pretty good agreement between what you get on FLX based sequencing and Sanger, for example.
One of the most important issues is actually the region of the 16S being used in relation to its length. A good analysis of this is covered here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2094085/).
On a personal experience note, when FLX was first released (and we are talking about the original FLX, not the current Titanium chemistry) we had just finished a large sample set using GS20 (100 bp), and we actually actually compared the results sequencing the same amplicon (bsf8-bsr357, read from r357) using the 100 bp reads and the 250 from FLX with the same results.
Of course the matter of estimating the true alpha diversity is an entirely separate issue (what defines an uncultured microbial species is good starting point on that question...).
Just one more point about the read length, the illumina paired end reads can give you a total of 500bp if you assemble each pair, and you don't have to deal with the any pyrosequencing error (if that is a concern).
On of the main concerns about PCR step, is about its exponential nature.
In this paper is reported how PCR will bring to an exponential over-estimation of naturally over-represented bacteria while on the other hand, rare biosphere bocomes more and more rare.
Gonzalez JM, Portillo MC, Belda-Ferre P, Mira A. Amplification by PCR artificially reduces the proportion of the rare biosphere in microbial communities. PLoS One. 2012;7(1):e29973. Epub 2012 Jan 11.
http://www.ncbi.nlm.nih.gov/pubmed/22253843
Anyway I use 454 for microbial population studies and I think is still the best option. Just as suggestion, when is possible try to reduce as much as possible the cycles of PCR reaction. For 454 amplicon sequencing is not really necessary to obtain an overload of PCR product from which finaly only few microliters are really used for sequencing. Anyway there are project where this is not possible and you need the whole PCR product. Pooling several PCRs products obtained with few cicles could be a solution but this is just my thoretical speculation.
Metagenomic charcaterization using next-gen sequencing of the PCR products obtained on variable regions of the 16S gene is frequently used to study microbial popolations.
We used this technique to characterize various communities usign FLX-sequencing of amplicons. We used software like MOTHUR to determine the number of taxonomic units and it is also our opinion that the extimated bacterial diversity is higher than the real one. We are not able to determine if this overestimation could be due to sequencing errors but we are still checking this hypothesis.
As Elisabeth said 454 is the way things are going.
I would add to this you also need to culutre at each sample site. We take a combined approach of using 454 and plating out a given community. Shade et. al. in Culturing captures members of the soil rare biosphere (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3466410/) point out some of the advantages to culturing in the field. In additional we use combined bioinformatic and thermodynamic models to understand communities
So given time and money (I know, I know) a combined approach of multiply media types (general environmental to specifically targeted media), 454, and possibly DGGE would give you a range of choices of analyzing a microbial community.
For amplicons, the method of choice is FLX. Size is long enough and cost is cheaper from a clone library with 100 clones (based on the prices we are paying at the moment for the consumables). Illumina has some advantages regarding chemistry (i.e. not so many problems from homopolymers as in FLX and Torrent), but is really lacking size. Pair end of 500bp it is the upper extreme, much more expensive and more difficult for amplicon libraries. Torrent is a combination of the negatives of both (for amplicons only), small size and homopolymers problem. Regarding bioinformatics: are getting easier and easier...QIIME, Muthor, Greengens, Seqman..etc are available and easy to use (a rookie needs less than a week to go into these). The problem is the interpretation steps afterwards and the parameters modifications (I am not sure if we will ever agree on a universal accepted way ;-) ). Fingerprinting techniques (DGGE, tRFLP,SSCP etc) are giving only relatively abundance and no OTU infos, therefore, are good only for screening and not concluding.
When estimating the diversity from PCR amplicons (besides de sequencing technology used afterwards), please consider to use modified PCR protocols, such as a reaconditioning step (http://aem.asm.org/content/71/12/8966.abstract), in order to reduce the accumulation of PCR artefacts.
When direct pyrosequencing (without previous PCR) is used to estimate diversity, and even abundance of a given taxon in a sample, the size and degree of conservation of the rRNA fragment used for classification should also be taken into consideration. Also the divergence and redundancy of rRNA sequences in genomes with multiple rrn operons (http://jb.asm.org/content/186/9/2629.abstract).
It is possible to analyze bacterial diversity with NGS sequencing of 16S amplicons. You can use 454 or illumina. With illumina you can design an insert size that ovelaps the paired reads and to assemble only the reads of each pair. Then you get a size around 150 or 200 that could be sufficient to determine bacterial taxa. It is important the selection of the variable region to be amplified. The selection of the primers is also very important because universal primers have bias. Many times it is needed to combine several primers to amplyfy for examle lactic baceria or other kind of bacteria that are difficult to amplify with universal primers.
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