Overview

We analyzed a single lambda phage evolution experiment as both an introduction to variant visualization in the first part of Wednesday's class, but breseq is designed to be able to with "small" genomes, not just "tiny" genomes. It also has more advanced tools for visualizing mutations across multiple genomes and more complete identification of all mutations.

Learning Objectives

1. Run breseq on 7 different E. coli clones evolved for 40,000 generations
2. Use the packaged gdtools commands to generate a comparison table of all mutations from all 7 samples
3. Learn shortcuts for compressing, transferring, and extracting multiple folders/files with single commands
4. Visualize the data on your local computer

Get some data

The data files for this example are in the following path: $BI/ngs_course/ecoli_clones/data/ go ahead and copy them to a new folder in your $SCRATCH directory called GVA_breseq_multi-sample:

mkdir $SCRATCH/GVA_breseq_multi-sample
cd $SCRATCH/GVA_breseq_multi-sample
cp $BI/ngs_course/ecoli_clones/data/* .

if everything worked correctly, you should see the following files. We've provided a bit more context to what those files actually are:

Setting up the run

As we begin working with multiple samples it becomes more important or at least helpful to organize our output in a more meaningful way. Typically this means storing input files in 1 location, output folders in another, and information about the analysis in a 3rd location.

mkdir run_output logs

Note in the above command we are making deliberate use of having a space on the command line to create 2 different directories as was mentioned in the introduction tutorial.

Normally, we would use all of our data and use both R1 and R2 data, but to speed things up for the class, we will only use R1 data. Put the following commands into a new file called "commands" using nano. As the run will take ~40 minutes to complete, don't worry about not understanding what each part of the commands mean, after the run is started, there will be a section explaining what was done.

breseq -j 6 -r NC_012967.1.gbk -o run_output/00K SRR030252_1.fastq &> logs/00K.log.txt &
breseq -j 6 -r NC_012967.1.gbk -o run_output/02K SRR030253_1.fastq &> logs/02K.log.txt &
breseq -j 6 -r NC_012967.1.gbk -o run_output/05K SRR030254_1.fastq &> logs/05K.log.txt &
breseq -j 6 -r NC_012967.1.gbk -o run_output/10K SRR030255_1.fastq &> logs/10K.log.txt &
breseq -j 6 -r NC_012967.1.gbk -o run_output/15K SRR030256_1.fastq &> logs/15K.log.txt &
breseq -j 6 -r NC_012967.1.gbk -o run_output/20K SRR030257_1.fastq &> logs/20K.log.txt &
breseq -j 6 -r NC_012967.1.gbk -o run_output/40K SRR030258_1.fastq &> logs/40K.log.txt &

Be sure to write (ctrl + o) before you exit (ctrl +x) nano.

breseq -j 6 -r NC_012967.1.gbk -o run_output/00K SRR030252_1.fastq SRR030252_2.fastq &> logs/00K.log.txt &
breseq -j 6 -r NC_012967.1.gbk -o run_output/02K SRR030253_1.fastq SRR030253_2.fastq &> logs/02K.log.txt &
breseq -j 6 -r NC_012967.1.gbk -o run_output/05K SRR030254_1.fastq SRR030254_2.fastq &> logs/05K.log.txt &
breseq -j 6 -r NC_012967.1.gbk -o run_output/10K SRR030255_1.fastq SRR030255_2.fastq &> logs/10K.log.txt &
breseq -j 6 -r NC_012967.1.gbk -o run_output/15K SRR030256_1.fastq SRR030256_2.fastq &> logs/15K.log.txt &
breseq -j 6 -r NC_012967.1.gbk -o run_output/20K SRR030257_1.fastq SRR030257_2.fastq &> logs/20K.log.txt &
breseq -j 6 -r NC_012967.1.gbk -o run_output/40K SRR030258_1.fastq SRR030258_2.fastq &> logs/40K.log.txt &

Executing the run

chmod +x commands
./commands

^The chmod command makes the commands file executable.

^ './commands'  # executes the commands file. the './' portion tells the command line to execute a file from the current directory as it is not in your $PATH variable.

Since we started running all of the breseq runs in the background, and are redirecting all of the output to log files (underlined portions will be explained below), there won't actually be anything printed to the screen, and we will get our prompt back. DON'T worry! things are likely working fine despite you not seeing anything happening. 

ps  
tail logs/*.log.txt  

^ the ps command will return information on what processes are currently running. You should see 7 breseq commands initially though as they run they will spawn their own processes with names like perl and bowtie.

^ the tail command will show the last 10 lines the run information about all 7 samples. Errors should be obvious.

You will want to periodically use these commands to check that the files continue to run. Eventually all 7 log files will show "+++   SUCCESSFULLY COMPLETED" as the last line. 

tail -n 1 logs/*|grep -c "+++   SUCCESSFULLY COMPLETED"

Understanding the breseq command that is being used.

While it is running, we'll look back at what you did and explain some of the new things you are encountering/using. you should move onto reading through the next section learning how you will create comparison tables to easily visualize the data across the different samples. If you have all that figured out you may want to start one of the other tutorials while the above commands finish. Just remember to check back periodically as the 'completion' notifications of background jobs can be easy to miss.

breseq -j 6 -r NC_012967.1.gbk -o run_output/<XX>K <ID>_1.fastq <ID>_2.fastq &> logs/<XX>K.log.txt &

Using gdtools to compare samples.

Assuming everything is running as it should (you should ask if you are not sure), we need to start exploring the gdtools commands. gdtools is a series of commands designed to function on "genome diff" (.gd) files (which are the main data type produced by breseq which is used by downstream analysis) that come packaged with breseq. As we have seen with numerous other programs throughout the course typing gdtools by itself will return a list of options. In the case of gdtools, it returns the list of subcommands that are available. Each has its own use, but for the purpose of this tutorial we are interested in comparing the output of multiple samples to one another. 

ANNOTATE (or COMPARE)  annotate the effects of mutations and compare multiple samples

Since gdtools compare seems to be the command and subcommand that we wanted to run to generate the comparison. Try typing just that to get a list of expected arguments.

gdtools ANNOTATE/COMPARE [-o annotated.html] -r reference.gbk input.1.gd [input.2.gd ... ]

In our command we sent the output of each sample to its own folder using the -o run_output/<XX>K portion of the command line. Among the multiple directories that breseq created within each output directory, you will find one named output which contains a file named output.gd. These are the files that we need to feed into the gdtools compare command (along with the NC_012967.1.gbk reference file) to generate the comparison table we want. In this course we have mentioned that using consistent nomenclature is important which it is, but now we come to a point where the consistency has created a problem. Because all of the files are named "output.gd" we can neither move all of the commands into a single folder (they would overwrite one another as they have the same name) or the resulting table would display sample IDs as "output" "output" "output" ... not the most helpful information to say the least.

To circumvent that we will need to rename each file to something more meaningful (say the name of the directory that the output/output.gd file was found in (aka the sample name)). 

cd $SCRATCH/GVA_breseq_multi-sample
tail -n 1 logs/*|grep -c "+++   SUCCESSFULLY COMPLETED"

Grouping .gd files together

cd $SCRATCH/GVA_breseq_multi-sample/run_output
for d in *;do cp -i $d/output/output.gd $d.gd;done;mkdir gd_files;mv -i *.gd gd_files
mv gd_files ..

This will copy the .gd file from each run into a new directory and then move that directory up 1 level so that it will be easier to use when making the compare table

Generating a compare table

cd $SCRATCH/GVA_breseq_multi-sample/
gdtools compare -r NC_012967.1.gbk -o multi-sample-compare-table.html gd_files/*.gd

where:

• -r ... is the reference
• -o ... is the output table we want
• gd_files/*.gd lists all the samples we want to have on our table

As the .html ending suggests, it will be an html output similar to the other breseq output you have seen and thus needs a web browser to be able to view it. Therefore you should use the scp command to transfer the multi-sample-compare-table.html file back to your local computer. Remember assistance with the scp command can be found here.

Evaluating compare table

Once you open the file on your local computer consider the following broad explanations:

1. Each row represents a unique mutation
2. The first 2 columns state what the position in the genome is, and what mutation ocurred
3. Columns 3-9 represent each individual sample
4. the remainder of the columns provide information from the annotation describing what genes are hit, and what the effect of the mutation is

As columns 3-9 show an increase in evolutionary time (from 2,000 to 20,000 generations), you will notice multiple examples where the first few columns are empty but the last 2,3,4 columns show 100% showing that the mutation was not detected until a certain time point then arose before the next sampling, and remained in the population to the end of our time course.

Can you find any examples of mutations that arose, but were out competed by the end of the evolution experiment? (do you understand what those should look like)

One downside of compare tables is that while it shows what mutations breseq called, you lose the ability to click on individual mutations to understand how/why that mutation was called. Therefore while compare tables are great, they are nearly always used in conjuncture with evaluating the individual breseq output as well. The next section will deal with a helper script to more quickly export multiple samples.

Exporting multiple breseq results using a single .tar.gz file

I have a small shell script for dealing with breseq data that copies, renames, compresses, groups each samples output directory into a single new directory, and compresses that directory so you can easily transfer it. Where can you get ahold of such a time saving script you ask? In the BioITeam directories of course!

cd $SCRATCH/GVA_breseq_multi-sample/run_output
ls
ls ..
export-breseq.sh
ls
ls ..

The above code block:

1. moves to the directory that contains the samples we just ran through breseq
2. shows you what is in that directory
3. shows you what is in the parent directory
4. executes the bash script described above
5. shows you the new output is in the parent directory not the current directory

Using the SCP tutorial if necessary transfer the compressed archive 05_Output_Export.tar.gz back to your computer.

Inflating compressed archives of breseq results.

# scp command ... 05_Output_Export.tar.gz .
tar -xvzf 05_Output_Export.tar.gz
cd 05_Output_Export
ls

Now you will see 7 .tar.gz files in the directory, and you can extract each of them 1 at a time by using the tar -xvzf <FILE> command. This seems acceptable for 7 samples but not when dealing with 100s of samples. The 1 liner below will extract .tar.gz files sequentially:

for f in *.tar.gz;do tar xvzf $f;done

This command will extract all files ending in .tar.gz regardless of their origin. If you find yourself doing this routinely you may want to turn this command into a small bash script as i have done. This is as simple as the following 2 commands and moving the detar.sh file to a location in your $PATH. Remember you can check what directories are in your $PATH by typing 'echo $PATH'

echo "for f in *.tar.gz;do tar xvzf $f;done" > detar.sh
chmod +x detar.sh

This command will work on either your personal computer or TACC the same way, but in my experience, the export-breseq.sh is used on TACC while detar.sh is used on my local computer.

Comparing breseq output to compare table

Now you can click through each individual sample's output to see the different mutations as you could in the intro to breseq tutorial.

Other useful tutorials

Data analysis with breseq (like all other programs) is only as good as the data that goes into it. The MultiQC and Trimmomatic tutorials work well upstream of breseq, while the identification of novel DNA elements may be useful for things such as trying to identify unknown plasmids and makes use of the genome assembly tutorial.

Alternatively, again consider going back to the intro breseq tutorial to rerun the sample that you took through read QC, mapping, SNV calling, and visualization.

Back to the GVA2020 page