Question: Using Kaiju Taxonomic Classification for 16S Marker Gene Analysis
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gravatar for piercemanlangit
6 weeks ago by
piercemanlangit0 wrote:

Hi all,

I am new to doing metagenomic studies. My co-adviser introduced me to KBase for analysis and I followed the tutorial narrative protocol on " Genome Extraction from Shotgun Metagenome Sequence Data."

What I've only done was just pair the reads, remove the adapters and low-quality reads. After which, I had them run on Kaiju for taxonomic classification with resolution down to species level. I've read some journals doing annotation without assembly, but I have not read doing the same thing with 16S data.

I may have overlooked that the platform mainly supports shotgun metagenomics. I'm not confident and will plan to do a common pipeline such as the MiSeqSOP by Mothur. But are my results valid, even with low confidence/accuracy?

Thanks

kaiju next-gen metagenomics 16s • 142 views
ADD COMMENTlink written 6 weeks ago by piercemanlangit0
1

Hi piercemanlangit, I don't understand clearly whether what you sequenced is 16S amplicons or whole metagenome - most of the first two sections reads like you deal with whole metagenome sequencing data - then you suddenly switch to 16S.

The distinction is important. There are so much more options from WMGS, and the methods are naturally very different for 16S.

For example, a key element of Kaiju is "protein-level classification". In my opinion, this won't work so well with 16S rRNA ;-)

ADD REPLYlink modified 6 weeks ago • written 6 weeks ago by Carambakaracho1.4k

Hi Carambakaracho, I'm dealing with 16S amplicons (V3-V4). Kbase offers a point-and-click interface so it was easy to navigate through. The results were also congruent with our culture, so I thought it was no problem.

Does this mean the results are erroneous? I'm just considering if I could still use it.

Thanks!

ADD REPLYlink written 6 weeks ago by piercemanlangit0
1

Well, if the results confirm what you know, it might have worked somehow. That's the thing with classification, it always works. However, sometimes you'll find Anthrax in the subway or a platypus in northern Europe.

If this will be part of a publication and the referee understands something about 16S classification, that referee might dismiss the method.

ADD REPLYlink written 6 weeks ago by Carambakaracho1.4k
1

for reference, you mentioned a very popular software for 16S analysis, mothur, there's qiime, too. MEGAN provides a GUI interface, as far as I know. Recently, the FROGS pipeline gave me excellent results.

ADD REPLYlink written 6 weeks ago by Carambakaracho1.4k

Many thanks, Carambakaracho!

ADD REPLYlink written 6 weeks ago by piercemanlangit0

If you want to identify the sequences to species level then just blasting is maybe the best option. The identity and coverage will be your confidence/accuracy score. If you want to also identify them on a higher taxonomic rank if there is no significant BLAST hit you could use MEGAN, RDP classifier or SINTAX.

MEGAN has a UI, but most of the input for MEGAN needs to be generated with commandline tools.

ADD REPLYlink modified 6 weeks ago • written 6 weeks ago by gb830
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