Chloroplast and mitochondria sequences

[kristin oosthuizen]

Hi,

I was just wondering what

k__Bacteria;p__Cyanobacteria;c__Chloroplast;o__Streptophyta;f__;g__;s__ and k__Bacteria;p__Proteobacteria;c__Alphaproteobacteria;o__Rickettsiales;f__mitochondria;g__Sarcandra;s__grandifolia means. How does this classification work. Does it mean that certain sequences were originally clustered under classes in a phylum and upon curation of that phylum, it was discovered that these sequences are rather chloroplast, and not true bacterial sequences. Then they were clustered as a new group (class) named chloroplast? Same with mitochondria? I'm just trying to make sense of how chloroplast can be a class and mitochondria a family. Kind regards, Kristin

[Colin Brislawn]

Hello Kristin,

Two major steps will influence "how chloroplast can be a class and mitochondria a family"; OTU picking and taxonomy assignment. Can you tell me more about how you clustered reads into OTUs and how you assigned taxonomy to each OTU?

Thanks!

Colin

[kristin oosthuizen]

Hi Colin,

Yes sure. Well first I need to say that I did amplify the V3V4 region of the 16S rRNA from total DNA extraced from plants, and I did determine that my primers cross-reacted significantly with host chloroplast (I did a bit of BLAST and read mapping analyses). So the way I see it, is that it definitely is chloroplast and mitochondria. I'm just trying to explain in my write-up how the classification works.

So first I used UPARSE to, merge, filter, dereplicate and cluster my reads into OTUs (97% similarity) and then filtered out chimeras. Then I assigined taxonomy with QIIME assign_taxonomoy.py against the greengenes 97_otus.fasta and 97_taxonomy_otu.txt files. I tried this both with RDP and BLAST methods.

So now I get those classifications and I'm thinking that obviously greengenes don't have plant chloroplast sequences, and that my sequences hit cyanobacteria, chloroplast, because of the similarity. But now I'm just confused about how it says p: cyanobacteria (which is bactera) and then c: chloroplast. How can chloroplast be a class and how can it be grouped under bacteria? I know cyanobacteria are considered the ancestors of chloroplasts.

Yeah sorry, I'm just trying to make sense of it, so I can better explain it in my write-up.

Thanks!
Kristin

[kristin oosthuizen]

I saw and older question where a person asked the same question, and Greg Caporaso replied saying it means the sequences are in fact chloroplast rRNA.

I just want to understand how this classification works. So It picks up Kingdom as bacteria, yet class as chloroplast. How is chloroplast a class? I am just interested in the way this classification works. Did the greengenes database originally cluster sequences and identified them as a certain phylum, but then upon curation of this phylum, discovered that some sequences are rather chloroplast, instead of true bacterial sequences, and then within the phylum created another group/class named chloroplast. I know cyanobacteria are able to photosynthesise, but they lack chloroplasts. So how does it group chloroplasts under cyanobacteria? I just want to be able to discuss it, by saying I had a lot of primer cross-reactivity, and this is corroborated by the classification, where my sequences were classified as chloroplast. But I do not understand how it is classified under Kingdom bacteria? Thank you for your assistance, Kristin

[Colin Brislawn]

Hello Kristin,

I think I'm beginning to understand what you are asking... I would split this into two questions:

How can chloroplast be a class and how can it be grouped under bacteria?

This question relates to how the GreenGenes taxonomy database was constructed.  

I am just interested in the way this classification works. 

This question relates to how the script assign_taxonomy.py gives a taxonomy to each OTU centroid sequence.  

They are great questions! I'll start with the first one:

Did the greengenes database originally cluster sequences and identified them as a certain phylum, but then upon curation of this phylum, discovered that some sequences are rather chloroplast, instead of true bacterial sequences, and then within the phylum created another group/class named chloroplast.

The two papers on greengenes are the authoritative resources. Take a look at the newest one:

http://www.nature.com/ismej/journal/v6/n3/full/ismej2011139a.html 

This paragraph is helpful:

Previously, we developed a tool that automatically assigns names to monophyletic groups in large phylogenetic trees (Dalevi et al., 2007), which is useful for naming novel (unclassified) clusters of environmental sequences. Here we describe a method to transfer group names from any existing taxonomy to any tree topology that has overlapping terminal node (tip) names. We used this ‘taxonomy to tree’ approach to annotate the 408 135 sequence tree with the NCBI taxonomy as downloaded in June 2010 (Sayers et al., 2011), supplemented with the Greengenes taxonomy from the previous iteration (Dalevi et al., 2007) and CyanoDB (http://www.cyanodb.cz). Explicit rank information, prefixed to group names, was incorporated into the Greengenes taxonomy to help users orient themselves and to improve the consistency of the classification. 

Greengenes attempted to place reads into a phylogenetic tree, then give names to parts of that tree. It's not perfect, and sometimes strange things happen. For our purposes, I would just ignore the full taxonomy of the chloroplast reads, and just discuss them as coming from chloroplast; we know they did not evolve from 'bacteria' even though that's the best place greengenes could find to put them. 

 

Does that help answer your first question?
Colin 

[kristin oosthuizen]

Hi Colin,

Once again you are super helpful. I really appreciate you pointing me in the right direction. Thank you for all your time and effort in explaining. It makes a lot of sense now.

All the best!
Kristin

[Colin Brislawn]

Hello Kristin,

Glad to help!

Keep in touch,

Colin