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The rarefaction.shared command will generate inter-sample rarefaction curves using a re-sampling without replacement approach. The traditional way that ecologists use rarefaction is not to randomize the sampling order within a sample, rather between samples. For instance, if we wanted to know the number of OTUs in the human colon, we might sample from various sites within the colon, and sequence a bunch of 16S rRNA genes. By determining the number of OTUs in each sample and comparing the composition of those samples it is possible to determine how well you have sampled the biodiversity within the individual. mothur has the ability to generate data for inter-sample rarefaction curves for the number of observed species. For this tutorial you should download and decompress Patient70Data.zip
mothur > make.shared(list=patient70.fn.list, group=patient70.sites.groups)
By default, the rarefaction.shared() command will perform 1,000 randomizations of the order in which the samples are considered. So if you run rarefaction.shared() multiple times, you will get slightly different results. To get the default behavior enter:
mothur > rarefaction.shared(shared=patient70.fn.shared)
This will result in output to the screen looking like:
unique 1 0.00 2 0.01 3 0.02 4 0.03 5 0.04 6 0.05 7 0.06 8 0.07 9 0.08 10 0.09 11 0.10 12
The left column indicates the label for each line in the data set and the right column indicates the row number in the data set. Execution of rarefaction.shared() will generate the file patient70.fn.shared.rarefaction, which looks like:
samples unique lci hci 0.00 lci hci ... 1 443.7500 164.7345 722.7655 182.8480 86.3489 279.3471 ... 2 850.0090 490.6902 1209.3278 308.3940 187.8304 428.9576 ... 3 1248.1000 855.0973 1641.1027 413.4350 291.1591 535.7109 ... 4 1634.4600 1251.6258 2017.2942 502.5470 392.0305 613.0635 ... 5 2002.1400 1655.6566 2348.6234 582.0000 485.6954 678.3046 ... 6 2375.1600 2107.6512 2642.6688 650.0620 578.2567 721.8673 ... 7 2742.0000 2742.0000 2742.0000 713.0000 713.0000 713.0000 ...
The actual file has many more columns, but this should give you the idea. The first column is the number of samples that have been considered. Since their were 7 group names in the group file, rarefaction.shared() rarefied the data over the 7 groups. The rest of the columns are in triplets. The first column of the triplet is the average number of OTUs observed after sampling 1, 2, 3, etc. groups. The second and third columns of each triplet are the bounds on the 95% confidence interval of average. If you look closely, you will find that the average for 1 sample is the average number of OTUs observed across all of the samples. Also, if you look at the data in the row for 7 samples you will see that the bounds on the 95% confidence interval are the same as the average. Finally, the richness observed for sample 7 is the total number of OTUs you would observe if you processed patient70.fn.list through summary.single().
There may only be a couple of lines in your OTU data that you are interested in generating the rarefaction curve for. There are two options. You could: (i) manually delete the lines you aren't interested in from your list file; (ii) or use the label option. To use the label option with the rarefaction.shared() command you need to know the labels you are interested in. If you want the rarefaction curve data for the lines labeled unique, 0.03, 0.05 and 0.10 you would enter:
mothur > rarefaction.shared(shared=patient70.fn.shared, label=unique-0.03)
In the file patient70.fn.shared.rarefaction you would see something like:
numsampled unique lci hci 0.03 lci hci 1 437.9010 156.7862 719.0158 58.6599 32.9568 84.3630 2 844.5540 484.9140 1204.1940 76.2709 52.1988 100.3430 3 1234.6800 850.1886 1619.1714 86.6658 64.5309 108.8007 4 1617.0100 1239.6273 1994.3927 94.2071 74.2022 114.2120 5 2003.0800 1661.2946 2344.8654 100.1300 82.7736 117.4864 6 2376.2700 2105.6267 2646.9133 105.6120 92.6939 118.5301 7 2742.0000 2742.0000 2742.0000 111.0000 111.0000 111.0000
To improve the accuracy of the calculations you can change the number of randomizations that are performed using the iters option; the default value is 1,000. Running 10,000 randomization should take 10-times as long as the default:
mothur > rarefaction.shared(shared=patient70.fn.shared, iters=10000)
The processors option allows you to reduce the processing time by using multiple processors. By default mothur will use all the processors found. You can set the processors with the following option:
mothur > rarefaction.shared(shared=patient70.fn.shared, processors=2)
Obviously, the goal of rarefaction is to randomize across the samples; however, if you just want a collector's curve across the samples you can use the jumble option:
mothur > rarefaction.shared(shared=patient70.fn.shared, jumble=0)
The design parameter allows you to assign your groups to sets. If provided mothur will run rarefaction.shared on a per set basis.
mothur > rarefaction.shared(shared=final.an.0.03.subsample.0.03.pick.shared, design=mouse.sex_time.design)
The sets parameter allows you to specify which of the sets in your design file you would like to analyze. The set names are separated by dashes. The default is all sets in the design file.
mothur > rarefaction.shared(shared=final.an.0.03.subsample.0.03.pick.shared, design=mouse.sex_time.design, sets=F003Late)
If you are running rarefaction.shared with a design file and would like your results collated in multiple files, set groupmode=f. (Default=True).
mothur > rarefaction.shared(shared=final.an.0.03.subsample.0.03.pick.shared, design=mouse.sex_time.design, groupmode=f)
If you had started this tutorial with the following comamnds:
mothur > get.group(shared=patient70.fn.shared)
You would have seen that there were 7 groups here: 70A-70F and 70S. The sequences from 70S were collected from Patient 70's stool sample those from samples 70A-70F were from their mucosa. These 7 groups would yield 21 lies if you ran the rarefaction.shared() command; however, if you were only interested in the comparisons between each mucosa site you could use the groups option:
mothur > rarefaction.shared(shared=patient70.fn.shared, groups=70A-70B-70C-70D-70E-70F)
Alternatively, if you want all of the pairwise comparisons you can either not include the group option or set it equal to "all".
mothur > rarefaction.shared(shared=patient70.fn.shared, calc=sharedobserved, groups=all)
The subsample parameter allows you to enter the size pergroup of the sample or you can set subsample=T and mothur will use the size of your smallest group.
The subsampleiters parameter allows you to choose the number of times you would like to run the subsample. Default=1000.
Visualization with R
To visual your data follow Pat's tutorial here.