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As we have done with: fastqc, cutadapt, and bowtie2, we want to install samtools and bcftools into a new environment (we'll call this one GVA-samtoolsSNV). Once again, having access to conda-forge will be required to install the most recent version.

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title	See if you can use what you know from installing bowtie to install the new environment without the additional help contained in this drop down. The code will still be hidden if you need a hint.

https://anaconda.org/bioconda/samtools and https://anaconda.org/bioconda/bcftools both show the bioconda channel as being the best source for installing the programs.
The instructions above list having the second channel "conda-forge" as being required to install the most recent versions
In the read mapping tutorial we saw that we could install programs to a new environment when we created said environment without needing the install keyword

Code Block

language	bash
title	Complete answer for installing samtools and bcf tools
collapse	true

conda create -n GVA-samtoolsSNV -c bioconda samtools bcftools -c conda-forge

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title	Expand here for detailed descriptions of the troubleshooting that took place for this last year.

The assumption last year was that the correct command would be: conda install -c bioconda samtools as it is what was listed at https://anaconda.org/bioconda/samtools. Instead the correct command ended up being: conda install -c bioconda samtools bcftools openssl=1.0

There are 2 different things going on in this command.

Forcing the installation of a specific version of openssl. In this case, a lower version than would normally be installed if samtools were installed by itself. According to https://github.com/bioconda/bioconda-recipes/issues/12100 my understanding is that when the conda package was put together there is an error wherein samtools specifically says to get version 1.1 of openssl, but the samtools program specifically requires version 1.0 to be present.
We are installing both samtools and bcftools at the same time. This can clean up some installation problems when there are conflicts between individual packages and you want to use them in a single environment. An alternative would be to have a samtools environment and a bcftools environment, but that creates unnecessary steps of having to change environments in the middle of your analysis.

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title	Click here to expand and see what the outcome of the assumed installation command is, what the problem is, and steps you could take to fix it.

Warning

title	This box contains example commands and outputs showing you something that does NOT work for educational and diagnostic purposes. If you use the code listed in this box, be sure you use ALL the code or you may run into downstream problems with this tutorial.

Code Block

language	bash

conda install -c bioconda samtools
samtools --version

The above command will appear to install correctly as other programs have, but the second command which you would expect to show you the version of samtools instead returns the following error:

No Format
samtools: error while loading shared libraries: libcrypto.so.1.0.0: cannot open shared object file: No such file or directory

Googling the entire error the top results clearly mention conda and several pages list problems associated "fixes" with different conda installation commands. One of the suggested fixes was to add access to the conda-forge channel as we have done this year.

Other "fixes" involved commands that may work, but at the expense of altering existing packages in our environment ~~(conda install -c bioconda samtools=1.9 --force-reinstall)~~ while this command may work based on community feedback, the number of programs that would be downgraded is concerning to me. If I were going to go this route, I would instead copy my existing conda environment to a new "test" environment (conda create --name GVA2021-samtools-test --clone GVA2021), run the struck-through command listed above, and make sure it works as expected before weighing what exact programs were being downgraded and what the immediate effects would be on other analysis. Most likely I would ultimately rename the environment to something more permanent, and keep samtools as a separate step (conda create --name GVA2021-samtools --clone GVA2021-samtools-test; conda env remove --name GVA2021-samtools-test)

Rather than going through all that. My solution is simply to install an older version of samtools deliberately from the start as several webpages (including: https://github.com/bioconda/bioconda-recipes/issues/13958 suggest that the issue is specific to the 1.12 version). In order to do this, I first had to remove the existing (incorrect) samtools version.

Code Block

language	bash

conda remove samtools
conda install -c bioconda samtools==1.11
samtools --version

which now gives a reasonable output of:

No Format
samtools 1.11 Using htslib 1.11 Copyright (C) 2020 Genome Research Ltd.

Unfortunately, this would then create a downstream problems with installing bcftools and a different set of conflicts. Therefore we will again remove our (now functioning) samtools package, and install samtools, bcftools, and openssl version 1.0 in a single command.

Code Block

language	bash

conda remove samtools
conda install -c bioconda samtools bcftools openssl=1.0

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