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Table of Contents

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Expand
titleSee 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
languagebash
titleComplete answer for installing samtools and bcf tools
collapsetrue
conda create -n GVA-SNV -c conda-forge -c bioconda samtools bcftoolssamtools bcftools

for reasons discussed https://github.com/bioconda/bioconda-recipes/issues/34190 and https://edcarp.github.io/introduction-to-conda-for-data-scientists/03-using-packages-and-channels/index.html listing conda-forge before bioconda is critical.


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


Info

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.

  1. 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.
  2. 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.
Expand
titleClick 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
titleThis 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
languagebash
conda install -c bioconda samtools
samtools --version

The above command appeared to install correctly as other conda installations did, but the second command which you would expect to show 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 mentioned conda and several pages listed problems associated "fixes" with different conda installation commands:

  1. One of the suggested fixes was to add access to the conda-forge channel (as we have done this year).
  2. Additionally, last year the entire course was taught with the hope of sequentially adding new programs to a single growing environment. As mentioned yesterday, such an approach is not always optimal/easy, and hence why this year we are creating a number of additional environments. Working from the assumption that you wanted to keep a single environment, one fix that appeared to be working well based on community feedback was conda install -c bioconda samtools=1.9 --force-reinstall, but at the expense of altering existing packages in the environment. When running the command, the number of programs that would be downgraded was nearly 2 full screens long.
  3. Rather than jumping to the "force-reinstall" solution it was suggested to copy the existing conda environment to a new "test" environment (conda create --name GVA2021-samtools-test --clone GVA2021). Once in the new environment, using the "force-reinstall" command above would have given access to samtools, but would then also require testing other programs in the environment (such as bowtie2, cutadapt, fastqc). Assuming all programs still (seemed) to work you could then rename the environment (conda create --name GVA2021-V2 --clone GVA2021-samtools-test; conda env remove --name GVA2021-samtools-test). Obviously the more programs that are added, the more likely running into these kinds of conflicts (where different versions of the same dependency are required), and the more programs you would have to check to see if the "test" envvironment broke any previously installed programs. 
  4. As there was information that suggested the issues were specific to version 1.12 (including: https://github.com/bioconda/bioconda-recipes/issues/13958), another solution was simply to install an older version of samtools deliberately from the start. In order to do this, I first had to remove the existing (incorrect) samtools version (conda remove samtools) and then specify the older version we wanted to use (conda install -c bioconda samtools==1.11). This then allowed the samtools --version command to give expected output of version 1.11 Unfortunately, during testing it quickly became obvious that the next program to install (bcftools) was going to create an entirely new set of installation problems meaning that samtools again hat to be uninstalled and samtools bcftools and the downgraded openssl version installed together. conda remove samtools ; conda install -c bioconda samtools bcftools openssl=1.0. 





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No Format
bcftools 1.17
Using htslib 1.17
Copyright (C) 2023 Genome Research Ltd.
License GPLv3+: GNU GPL version 3 or later <http://gnu.org/licenses/gpl.html>
This is free software: you are free to change and redistribute it.
There is NO WARRANTY, to the extent permitted by law.


Warning

If you get something else, such as an error message with bcftools --version:

"bcftools: error while loading shared libraries: libgsl.so.25: cannot open shared object file: No such file or directory" 

do not proceed, check your conda install command and if it matches the one listed above, get my attention.

Calling variants in reads mapped by bowtie2

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