Mistake when running the protocol about astrobiomike.github.io

[XELong]

Thanks a lot!

[AstrobioMike]

Hi there, @XELong :)

Sorry you've had some trouble. I certainly can't always update things whenever a new version of something comes out, but since it's been so long in this case, I think it's a good idea to try. Actually when I last put this together I still had to use some earlier versions of some things that weren't yet available in conda, like R v4, but at least that one's not a problem anymore. And I think it's important to have that self-contained environment for folks who can't or don't want to setup on their computer, so I am somewhat constrained to what I know how to build in a binder, and wouldn't want to update the page just based on all current versions if they can't be in the environment.

So as soon as I can, I will try to update things 👍 but it also sounds like that particular problem you listed isn't a version problem. It's telling us that the rank "Phylum" isn't in the object. Maybe this is just a mistake in the code I have there (if you're using the tutorial data).

Can you tell me what you get back if you run rank_names(ASV_physeq)? I suspect "Phylum" won't be in there, maybe it's lowercase?

[XELong]

Thanks for your answer! I have update the mamba environment based on your tutorial. Here is the codes:
mamba create -y -n XXXX(envs) -c conda-forge -c bioconda -c r -c defaults
cutadapt=4.1 r-base=4.1 rstudio-desktop r-tidyverse=1.3.2
r-vegan=2.6_4 r-dendextend=1.16.0 r-viridis=0.6.2
bioconductor-phyloseq=1.38.0 bioconductor-deseq2=1.34.0 bioconductor-dada2=1.22.0
bioconductor-biocgenerics=0.40 bioconductor-decipher=2.22.0 bioconductor-decontam=1.14.0
bioconductor-biomformat=1.22.0 r-biocmanager=1.30.18
r-matrix=1.5_1 libopenblas=0.3.21
However, it also has a small problem. The problem occured in this step " filtered_out <- filterAndTrim(forward_reads, filtered_forward_reads, reverse_reads, filtered_reverse_reads, maxEE=c(2,2), rm.phix=TRUE, minLen=175, truncLen=c(250,200))". Error reports that " Error in filterAndTrim(forward_reads, filtered_forward_reads, reverse_reads, :
Some input files do not exist." I don't know how to fix. Fortunately, I ran this step in the latest version of dada2 and R base. It works.

[AstrobioMike]

So that isn't a version problem either. It just means the variable holding the input reads, either the forward_reads variable or reverse_reads one, isn't holding the correct path to the reads, OR our R environment isn't in the location we think it is. That it worked when you were working with a different version was just a coincidence, and whatever the initial problem was must have been resolved while re-doing something. When hitting the problem, you could have checked by seeing what was stored in those variables, ("forward_reads" and "reverse_reads"), and checking if you are where you think you are in the R environment, with getwd(), and looking for the expected files with list.files() to ensure they are where we think they are and that it matches what's in the variables that are supposed to be pointing to them.

It sounds like a lot of problems you're hitting are just normal growing pains of getting used to things in R that we all have to fight through :)

[XELong]

Thanks! One more questions, ls R1.fq | cut -f1 -d "" > samples, what means for this code. I am not familiar with the Unix. When I used my own data to run the process. There is a problem like this "ls: cannot access '_R1.fq': No such file or directory". I have revised the file format just like the example in the tutorial. For example, "XYK0310020_sub_R2.fq.gz, XYK0526080_sub_R2.fq.gz, XYK0812040_sub_R2.fq.gz, XYK10280100_sub_R2.fq.gz, XYK0312040_sub_R1.fq.gz". Could you tell me why ? Thank you very much!

[AstrobioMike]

The details make all the difference here, and it looks like what you're typing in as having done (ls R1.fq | cut -f1 -d "" > samples), isn't matching what the error says, as it says it was looking for "_R1.fq" – with an underscore in front of R1.fq. Because this entirely depends on your location and the exact file names, it's not easy to troubleshoot this way. But going on what you posted for the file names, something like this might work if in the same folder/directory as the files:

ls *_R1.fq.gz | cut -f 1 -d "_" > samples

You can always check first like this and seeing what prints out:

ls *_R1.fq.gz | cut -f 1 -d "_"

More importantly though, if wanting to do this type of work, getting this skill set is way more valuable than getting help on one specific instance like this. So I definitely recommend going through the 5-part unix crash course :) It will get you introduced and exposed to these things 👍

https://astrobiomike.github.io/unix/unix-intro