Hi All,
I am tying to analyse 16 of my 16S samples using mothur. I sequenced them using Miseq 2x300 and I used illumina primers to target V3 and V4 regions. I followed the procedure as described in MiSeq_SOP and up to a stage where I am doing OTU based analysis and generate a .rarefaction file. I am having trouble plotting a rarefaction curve using the R script mentioned in “Sogin Data Analysis” but not successful. Also, are there any script or program to visualize the outputs of mothur? I would be grateful if you can help me on this.
I did it very old school and opened the rarefaction file in excel and then plotted no of new otus vs no of seqs.
I can open almost every output from mothur in one of excel, a text editor or internet explorer (for svg files)
Ditto. Excel and TextWrangler are your friends.
I did some nice plots by opening the rarefaction files in Excel, copy/paste into my graphics program, and going from there.
Thank you very much for your response. As I am a beginner in mothur and haven’t plotted these type of data previously, I would like more specific suggestion in plotting the output files of mothur. For example first few lines of my taxonomy file looks like this when I open with gedit:
OTU Size Taxonomy
Otu00001 73549 Bacteria(100);Firmicutes(100);Bacilli(100);Lactobacillales(100);Streptococcaceae(100);Streptococcus(100);
Otu00002 15193 Bacteria(100);Proteobacteria(100);Alphaproteobacteria(100);Caulobacterales(100);Caulobacteraceae(100);Brevundimonas(100);
Otu00003 14192 Bacteria(100);Proteobacteria(100);Gammaproteobacteria(100);Enterobacteriales(100);Enterobacteriaceae(100);Klebsiella(84);
Otu00004 13367 Bacteria(100);Bacteroidetes(100);Flavobacteriia(100);Flavobacteriales(100);Flavobacteriaceae(100);Chryseobacterium(100);
Otu00005 6191 Bacteria(100);Proteobacteria(100);Gammaproteobacteria(100);Xanthomonadales(100);Xanthomonadaceae(100);Stenotrophomonas(100);
Otu00006 5051 Bacteria(100);Proteobacteria(100);Betaproteobacteria(100);Neisseriales(100);Neisseriaceae(100);Neisseria(100);
Similarly a portion of my rarefaction file looks like:
numsampled 0.03-Joey-1 lci-Joey-1 hci-Joey-1 0.03-Joey-10 lci-Joey-10 hci-Joey-10 0.03-Joey-11 lci-Joey-11 hci-Joey-11 0.03-Joey-12 lci-Joey-12 hci-Joey-12 0.03-Joey-13 lci-Joey-13 hci-Joey-13 0.03-Joey-14 lci-Joey-14 hci-Joey-14 0.03-Joey-15 lci-Joey-15 hci-Joey-15 0.03-Joey-16 lci-Joey-16 hci-Joey-16 0.03-Joey-2 lci-Joey-2 hci-Joey-2 0.03-Joey-3 lci-Joey-3 hci-Joey-3 0.03-Joey-4 lci-Joey-4 hci-Joey-4 0.03-Joey-5 lci-Joey-5 hci-Joey-5 0.03-Joey-6 lci-Joey-6 hci-Joey-6 0.03-Joey-7 lci-Joey-7 hci-Joey-7 0.03-Joey-8 lci-Joey-8 hci-Joey-8 0.03-Joey-9 lci-Joey-9 hci-Joey-9
1 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000
100 24.2160 18.0000 32.0000 9.0480 6.0000 14.0000 11.8560 7.0000 17.0000 7.9040 4.0000 12.0000 18.6960 13.0000 24.0000 18.8160 13.0000 25.0000 14.5440 10.0000 21.0000 27.5120 21.0000 36.0000 5.8640 3.0000 10.0000 15.2000 10.0000 21.0000 12.1600 8.0000 17.0000 18.4720 13.0000 24.0000 19.9120 15.0000 26.0000 4.3360 2.0000 7.0000 10.0880 6.0000 15.0000 11.4080 7.0000 16.0000
PCoA file:
group axis1 axis2 axis3 axis4 axis5 axis6 axis7 axis8 axis9 axis10 axis11 axis12 axis13 axis14 axis15 axis16
Joey-1 0.261483 0.027870 0.006770 -0.002237 -0.046385 -0.069473 -0.001724 0.010141 -0.031051 0.026252 0.007876 0.009403 0.006114 -0.045523 -0.000000 0.000000
Joey-10 0.253738 0.017898 0.000785 -0.005209 0.071452 0.056495 0.081197 0.001852 -0.028346 0.031921 0.110913 -0.019841 0.032796 0.007811 0.000000 0.000000
Joey-11 0.248479 0.016303 -0.000362 -0.005915 0.094788 0.086524 0.106990 -0.002011 -0.019074 0.033341 -0.045081 -0.063541 -0.019963 -0.064291 0.000000 0.000000
Joey-12 -0.610356 0.423260 0.000672 -0.003587 0.021041 0.092296 -0.049521 0.265468 -0.003800 -0.000051 0.000183 0.000179 0.000031 -0.000474 0.000000 0.000000
Joey-13 0.261302 0.020440 0.002461 -0.003817 0.027203 0.022463 0.042601 0.004240 0.007703 0.014482 -0.013745 -0.048753 -0.063882 0.078904 0.000000 0.000000
Joey-14 0.250429 0.020121 0.004877 -0.003752 0.018271 0.002055 0.061164 0.013844 -0.014044 -0.177285 0.003038 -0.000911 0.002768 -0.006294 0.000000 0.0000
Thanks. Would appreciate your help.
You just have to open those files with excel, then they are normal tables which you can graph with standard charts in excel. I removed the lci and hci columns in the rarefaction data and plot with ‘scatter with smooth lines’. You can only plot 2 axes for PcoA with excel, unless you use R or other graphing software. Though someone did create 3D scatterplot with excel macros: http://www.doka.ch/Excel3Dscatterplot.htm, which only requires you to copy and paste your data. Or look at this tutorial, http://www.r-bloggers.com/getting-fancy-with-3-d-scatterplots/, if you fancy doing it with R.
Thank you very much Soon for your kind help. Learned a lot of things. I am still stuck in graphically representing the composition of different bacteria in each of my samples. Which output file from mothur to be used for this purpose and can that be done using excel as well? Thanks.
If you are alright with simple piechart or barchart, you can do it with excel. If you are looking at presence or absence of otu, use .cons.tax.summary file. Relative abundance of otu - look at shared file.