1 Learning objectives

  • Select Docker containers from the docker hub
  • Use multiple Docker container to accomplish related tasks
  • Learn about Docker image and Docker files
  • Bypass “Entrypoint” of Docker images

The main learning objective is to experience different methods of using dockers images and face (and resolve) challenges while doing so.

In class these exercises will be run onto the classroom iMacs.

However, as best as I can I’ll provide Windows hints and instructions when possible, but a basic understanding of line-command under Windows would be more than useful for that (e.g. know what is DOS for example. See APPENDIX C.)

1.1 Requirements

  • Be familiar with Docker or follow workshop 1 “Docker - Beginner Biologist 1” and workshop 2 “Docker - Beginner Biologist 2.”

  • Docker will be used from a line-command terminal: Terminal on a Macintosh in the classroom. A rudimentary knowledge of bash command-line is necessary.

  • If you are a Windows user: PowerShell can be used as a Terminal. However, setting Docker to run on Windows is more involved (not covered in class.)

  • Docker username: downloads will require a (free) username, therefore registration is necessary in order to follow the tutorial. Go to https://hub.docker.com and use the button “Sign up for Docker Hub” to register.

2 Set-up

Tutorials will be held in the Biochemistry classroom 201, and Docker has already be installed.

Instruction for installation can be found on the install link1 of the Docker web site.

Note HTML Version only:

If you are following this document in HTML format the code is shown with a colored background:

White background: standard output of programs.

2.1 Getting started

To get started we need to open a text terminal as detailed below. In class we’ll use a Macintosh.

Do one of the following:.

If you are on a Macintosh:

  1. Find the Terminal icon in the /Applications/Utilities directory. Then double-click on the icon and Terminal will open.
  2. OR use the top-right icon that looks like a magnifying glass (Spotlight Search,) start typing the word Terminal and press return. Terminal will open.

If you are on a PC:

  1. Find Power Shell e.g. using Windows search or Cortana. This will open a suitable text-based terminal.

(Note: Windows cmd does not offer the appropriate commands.)

2.2 Version check

This ensures that Docker is properly installed. The exact running version itself is not very important.

At the $ or > prompt within the window of Terminal, cmd or PowerShell type docker --version to check the version currently installed.

Docker version 19.03.5, build 633a0ea

2.3 Docker login: Required!

Before going further, it is necessary now to login with your Docker Hub ID. You should already have created one before this or the previous workshop. If you need to create an ID now go to https://hub.docker.com to register.

Docker login:.

Login with your Docker ID to push and pull images from Docker Hub. 
If you don't have a Docker ID, head over to https://hub.docker.com 
to create one.
Username: YOUR_DOCKER_ID_HERE
Password: 
Login Succeeded
$

Note: if you do not login first you will receive an error message when tryingt to start docker in the next steps.

3 Choosing a docker image

In the previous workshop we learned how to find, choose, and pull (download) a docker image from the Docker hub.

Today we’ll download multiple images to access programs in each to accomplish preliminary tasks to genomic analysis. These images belong to a larger project called Bioinformatics Docker Images Project2.

This group of docker images provides useful information on how to run the software contained within. However, there are unclear desciptions and errors that we’ll overcome during the workshop.

We will use the following images:

We can get the appropriate pull request from the web pages on the Docker hub.

pull images.

We can list images with:

REPOSITORY            TAG         IMAGE ID          CREATED           SIZE
pegi3s/sratoolkit     latest      12fb29fcba17      2 months ago      306MB
pegi3s/fastqc         latest      5a439982c750      4 months ago      579MB
pegi3s/clustalomega   latest      ed9da1fc309e      4 months ago      290MB

Note on size: Docker images are constructed in layers that can be common over multiple images. Therefore, the actual disk space to store multiple images is less than the sum of the SIZE that is reported in the list.

4 clustalomega

From web clustalomega documentation3: Clustal Omega is a multiple sequence alignment program for aligning three or more sequences together in a computationally efficient and accurate manner. It produces biologically meaningful multiple sequence alignments of divergent sequences.

A full description of the algorithms used by Clustal Omega is available in the Molecular Systems Biology paper Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega (Sievers et al. (2011).) Latest additions to Clustal Omega are described in *Clustal Omega for making accurate alignments of many protein sequences (Sievers and Higgins (2018).)

4.1 multiple sequences fasta file

As a follow-up to our EMBOSS sequence alignment we’ll use this program to make a very small multiple sequence alignment of the short glucagon peptide family that was saved in a shared directory.

Note: If you need to create the shared directory and the sequence files see below in APPENDIX A.

The glucagon family sequence files are in individual fasta format. We first need to create a fasta file that contains them all with a simple cat command:

Combine sequences.

The final, multiple sequence fasta file contains all the data:

>GIP
YAEGTFISDYSIAMDKIRQQDFVNWLLAQ
>GLP-1
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG
>GLP-2
HADGSFSDEMNTILDNLAARDFINWLIQTKITD
>glucagon
HSQGTFTSDYSKYLDSRRAQDFVQWLMNT

4.2 clustalomega image

We are now ready to apply what we learned in the previous workshop: we have downloaded (pulled) the docker image and we know how to share a directory… This should be a breeze right?!

We’ll see!

First we need to explore the container to see where we can “attach” (map) the dockershare directory. In a previous example it was /data but we need to see if that directory actually exists in this image.

It seems that it could exist if we trust the info from the docker hub page for pegi3s/clustalomega:

You should adapt and run the following command:

docker run --rm -v /your/data/dir:/data pegi3s/clustalomega -i /data/sequences.fasta -o /data/sequences_aligned.fasta

In this command, you should replace:

  • /your/data/dir to point to the directory that contains the FASTA file you want to align.
  • sequences.fasta to the actual name of your FASTA file.
  • sequences_aligned.fasta to the actual name of your aligned FASTA file.

So we should be good:

  • /your/data/dir can be $HOME/dockershare
  • We have created the multiple sequence files above and named it sequences.fasta.
  • sequences_aligned.fasta would be the written output.

Since we would share the /data folder henceforth this should work OK.

4.3 clustalomega manual help

We can create a temporary container to request the help provided by the program itself. For this purpose we can add -h or --help. The program will print help informationon the screen.

Note: At this point we do not request a shared directory and the command can be given from within any directory we are at the moment on the local computer.

For more details, the README file is available online4.

Below we’ll follow an example based on the sequences we have and inspired by the example on the docker hub page.

4.4 clustalomega container

We can now run clustalomega from a container. The example given on the web page adds the name of the files on the docker run command itself:

Run command.

After completing the task the container exists and we are back to the host computer prompt.

We e can type the alignment file on the screen:

>GIP
YAEGTFISDYSIAMDKIRQQDFVNWLLAQ----
>GLP-1
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG--
>GLP-2
HADGSFSDEMNTILDNLAARDFINWLIQTKITD
>glucagon
HSQGTFTSDYSKYLDSRRAQDFVQWLMNT----

The - represent the gaps. Therefore it worked.

However, if you re-run the same command (using the same file names) you’ll get this error:

Therefore we learn that adding --force will fix that problem.

Other errors might suggest to look into the help:

The help page is rather long, but we can concentrate on the output file and format information:

We can re-reun the command with a different format which provides a better visual of an alignment. This would be true for the subset clu[stal],msf,phy[lip],selex.

EXERCISE:
Time permitting you can rerun the commands with one or more of these formats.
Do not forget to add --force to overwrite the output file.

For example: (written with line continuation mark \)

CLUSTAL O(1.2.4) multiple sequence alignment


GIP           YAEGTFISDYSIAMDKIRQQDFVNWLLAQ----
GLP-1         HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG--
GLP-2         HADGSFSDEMNTILDNLAARDFINWLIQTKITD
glucagon      HSQGTFTSDYSKYLDSRRAQDFVQWLMNT----
              :::*:* .: .  ::    ::*: **:

EXERCISE 2:
Time permitting create a file (named e.g. sequences2.fasta for longer protein sequence files as detailed in APPENDIX A for “Protein FASTA for clustalomega.”)

Note: There is very little difference between these files, and using the clu format for the output will make the results easier to read.

4.5 Explore the container

Perhaps this should have been the first thing to do?!

Unless special instructions are given when the docker image is created, it shoud be possible to launch the container and explore its content as we have done in previous workshops.

The simplest way is to request a shell on the command e.g. adding /bin/bash or /bin/sh at the end of the docker run command. For example we ran docker run -it alpine /bin/sh in the first workshop.

In the same way the following should work, omitting sharing a directory as we only want to explore:

clustal-omega: unexpected argument "/bin/sh"
For more information try: clustalo --help

The container does not let us in… In this case we were able to run the clustalo software. But it is sometimes useful, or necessary. to dive inside the container… The section below explore these options.

5 Docker files and Entry points

The above problem poses the question of useability of a docker file. In the future we’ll be able to create our own docker files and images but for now we have to rely on existing ones.

As we have done in a previous workshop, the best documentation is to explore the docker container to know what is available. In the above example we assume from the documentation that the /data directory exists… Since the command worked we can suppose that it exists.

As a quick reminder here is the process to create a container from the beginning, even though for now we only downloaded existing images.

  1. Write a docker file containing instructions. This file is plain text.
  2. Create a binary image with this information (with docker build.)
  3. Upload the image on the hub (with docker push.)
  4. Downloadable by other with docker pull.
  5. Create a container from the image with docker run

In order to investigate (and learn useful information at the same time) we have to explore data provided on the Docker hub web page for this image. We will start with the docker file which contains instructions and therefore the blueprint information on the docker image and containers that are derived.

5.1 Entry point

Explore clustalomega docker page.

As of this writing this is what it contains:

This docker file is rather “simple” in the sense that there are only a few lines. This is what is means (\ is the line-continuation code to stipulate that this is a single line to execute.)

  1. FROM ubuntu:18.04 means that this “generic” version of Ubuntu is used as the starting point to which we add the libraries of software that are needed in the next lines.
  2. RUN apt-get update \ update Ubuntu and add a libray
  3. RUN wget download and install clustalomega.
  4. ENTRYPOINT ["clustalo"] when a container is activated, immediately run the clustalo program.

The last point is the critical one: as soon as the container is activated, the software clustalo is started and therefore all data provided on the docker run command line is passed on to the clustalo program. It follows that ou command docker run -it --rm pegi3s/clustalomega /bin/sh failed simply because the argument /bin/sh was given directly to the clustalo program which did not recognize this as a file name containing sequences as the program expects.

Therefore the problem is that we cannot, at this point, “enter and explore” the container itself because of the ENTRYPOINT command that is written “in stone” within the docker file and therefore within the docker image and subsequently the docker container.

5.2 Bypass Entry point

The solution to the problem is to override the ENTRYPOINT instruction by providing an alternate option on the docker run command itself. This is accomplished with the --entrypoint option.

To simply explore the pegi3s/clustalomega container without sharing a directory we can therefore use the following command:

root@faf2a0788794:/#

From this point we can now explore the inside of the docker container:

bin  boot  dev  etc  home  lib  lib64  media  mnt  opt  proc  root  run  sbin  srv  sys  tmp  usr  var

5.2.1 Important note: arguments

(SKIP this section if not enough time.)

If we only want to enter the container with the shell command as above it is clear that in that case the bash shell (/bin/bash) should be installed. That is not always the case. The older, sh alternative (bin/sh) can be substituted but need also be installed.

Other options exist, for example simply asking to list the content of the default landing directory with /bin/ls as the entry point:

However, there are times when an argument needs to be passed along. In that case it may not be obvious to know where to place the arguments within the docker run command5.

For example, why the bash command ls -l works on the terminal, the following command would fail:

docker: Error response from daemon: OCI runtime create failed: container_linux.go:346: starting container process caused "exec: \"/bin/ls -l\": stat /bin/ls -l: no such file or directory": unknown.
ERRO[0001] error waiting for container: context canceled

The reason is that the argument, in this case -l needs to be provided after the container is specified. Therefore the correct command would be:

total 64
drwxr-xr-x   2 root root 4096 May 15 14:07 bin
drwxr-xr-x   2 root root 4096 Apr 24  2018 boot
drwxr-xr-x   5 root root  340 Oct 24 19:34 dev
[...]

For bash commands, the arguments need also to be at the end, but the following command with ls at the end will fail:

/bin/ls: /bin/ls: cannot execute binary file

In this case it is necessary to add -c for the argument to be send to bash properly (see man bash for details.) Therefore the following command will work

EXERCISE: Try the following commands at the end:

  • -c ls -l
  • -c "ls -l"

What is the difference? ________________________________________________

. _______________________________________________________________

6 Two containers for genomics

In this section we’ll use two containers that harbor software that is very useful in the genomics, next generation sequencing (NGS). This will be a short example of using 2 separate docker images to accomplish a task as detailed below.

Task to accomplish:

  • Download “paired-end library” of E.Coli Illumina sequencing from the NCBI archives
  • Check the quality of this NGS sequence reads data

For this we’ll use two diferent containers from the pegi3s series that harbor the following software:

  • sratoolkit: collection of tools and libraries for using data in the INSDC Sequence Read Archives. We will need the fastq-dump utility.
  • fastqc - Sequence read quality assessment.

We already pulled these images earlier. In case you have not done so use the following commands:

6.1 Data

The Sequence Read Archive (SRA, previously known as the Short Read Archive) is a bioinformatics database that provides a public repository for DNA sequencing data, especially the “short reads” generated by high-throughput sequencing (Leinonen, Sugawara, and Shumway (2011)).

The data in the SRA archive is organized by experiment and runs

The data will be from run SRR10314868 of experiment SRX7025998 (see NIH book Understanding SRA Search Results for more details.)

On the page for SRR10314868 the following cascading tabs show the link to download the data

  • Level 1: Browse Tab
  • Level 2: Run Browser Tab
  • Level 3" Data access Tab
SRA

The web address we’ll use later is:
https://sra-download.ncbi.nlm.nih.gov/traces/sra24/SRR/010073/SRR10314868

6.1.1 FastQ

Ultimately we’ll want the data in the fastq format.

The fastq format ia a text-based format for storing both a biological sequence (usually nucleotide sequence) and its corresponding quality scores. Both the sequence letter and quality score are each encoded with a single ASCII character for brevity.6

See APPENDIX B for more details.

7 sratoolkit

The first task is to download Illumina sequence data from the Sequence Read Archives (SRA.)

To download the data we’ll use a utility called wget (web get) that should be present within the container.

While most NGS data is typically available as “fastq” files (sequence and quality) these files, even compressed with the standard utilities (.gz files) are larger thant the format used to archive the data on the SRA.

Therefore, special utility software is necessary to decompressed the files downloaded from the SRA. For this purpose we’ll use the fastq-dump utility.

7.1 Explore sratoolkit container

Let’s first quickly explore the contents of the srapegi3s/sratoolkit` container. We can already add the shared directory as well:

We can now ask if the 2 programs we want to use are available with the which command:

/usr/bin/wget
/opt/sratoolkit.2.9.6-ubuntu64/bin//fastq-dump

We can conclude that indeed these 2 utilities are present and we can now continue.

7.2 Download data

We’ll work within the home directory which now maps alls files that are in the dockershare directory from the computer host.

Donwload data with wget.

We’ll use the wget utility to download the dat from the web addressed determined above within the SRA web site.

[...] 
(sra-download.ncbi.nlm.nih.gov)|130.14.250.27|:443... connected.
HTTP request sent, awaiting response... 200 OK
Length: 80296784 (77M) [application/octet-stream]
Saving to: 'SRR10314868'

SRR10314868      100%[============================>]  76.58M  36.2MB/s    in 2.1s    

2019-10-24 23:03:58 (36.2 MB/s) - 'SRR10314868' saved [80296784/80296784]

This created a file called simply SRR10314868 and we can see how big it is:

-rw-r--r--  1 jsgro  AD\Domain Users    77M Oct 20 19:34 SRR10314868

The file is 77 Megabytes. We can compare this later to the size of the fastq or compressed fastq file that will result from the extraction from this SRA archive.

7.3 Unarchive data

Now we can use fastq-dump to unarchive SRR10314868 in order to extract the sequence information into .fastq formated file(s).

The SRR10314868 archive is that of a “paired-end” run i.e. each fragment was sequence from both forward and reverse directions. Most software will prefer that these be stored in two separate files. From the help we can learn that the modifier --split-file will “Dump each read into separate file. Files will receive suffix corresponding to read number.”7.

We are now looking within the container. Since the shared directory is /home we’ll change to this location before running the command.

Read 850953 spots for SRR10314868
Written 850953 spots for SRR10314868

This may take a one or two minutes. We can then list the files to see how big they are:

-rw-r--r-- 1 root root  77M Oct 21 00:34 SRR10314868
-rw-r--r-- 1 root root 229M Oct 24 23:31 SRR10314868_1.fastq
-rw-r--r-- 1 root root 229M Oct 24 23:31 SRR10314868_2.fastq

We can therefore remark that about 500Mb were compressed into 77Mb within the archive.

Note: to see the content of these text files simply use the more command (q to quit) or alternatively use the head command to see the first 10 lines (default.)

To save space we can optionally delete the SRR10314868 file (now or later) and we can also compress the fastq files. The software that we’ll use in the next step (fastqc) can read compressed files directly.

-rw-r--r-- 1 root root 55M Oct 24 23:31 SRR10314868_1.fastq.gz
-rw-r--r-- 1 root root 58M Oct 24 23:31 SRR10314868_2.fastq.gz

Thefore, while the gzip compression is very good, the SRA compression had achieved a stronger compression level. Both files amount to 113Mbytes while the SRA file was only 77Mbytes.

Conclusion: we now have 2 files from a paired-end experiment that we’ll test for quality with the fastqc program in the next section. The fastqc program will be accessed in a different container.

Since we are now done with this container we can exit. Since we started it with the --rm option it will be automatically be removed from the system:

$

Note: fastq-dump could also be used without running inside the container. In this case it would be necessary to also share a directory, but also specify the output directory because the default directory in the container is / when starting it. The following command would therefore work as well and produce the same .fastq files:

In the next section we’ll analyse the quality of the sequence contained in those sequening files.

8 FastQC

From the FastQC web site8:

“FastQC aims to provide a simple way to do some quality control checks on raw sequence data coming from high throughput sequencing pipelines. It provides a modular set of analyses which you can use to give a quick impression of whether your data has any problems of which you should be aware before doing any further analysis.”

FastQC (Andrews (2010)) can be run in command-line but can also be started with a graphical interface (GUI) in the X11 environment. This presumes that X11 is available and running, and that “sharing” communication be established between the container and the host. This is possible on a Linux or a Macintosh system, perhaps with more difficulty on Windows, but is not trivial for a beginner user. We’ll explore these options in the next workshops.

For today we’ll stick with the command-line option…

8.1 Starting FastQC

We already pulled the docker image earlier. Run this command if you need to download it now:

We could now explore the container briefly to see which directory could be shared. Preferably an empty directory on the container. In the previous section it was /home.

Start the container with the command:

Exception in thread "main" java.awt.HeadlessException: 
No X11 DISPLAY variable was set, but this program performed an operation which requires it.
[truncated output...]
    at uk.ac.babraham.FastQC.FastQCApplication.<init>(FastQCApplication.java:63)
    at uk.ac.babraham.FastQC.FastQCApplication.main(FastQCApplication.java:332)

Should we say “OOPS?”

The error indicated that the container will start immediately with FastQC running in graphical mode for X11 but we are not set for that…

To understand the problem we can go onto the docker page for this image: hub.docker.com/r/pegi3s/fastqc

The paragraph “Using the FastQC image in Linux” has a similar command to start the program that would appear to be what we need, while the next paragraph “Running the FastQC GUI in Linux” details what to do to start the program with X11 graphical display.

In reality this information is misleading as we just found out.

To understand why we can check the tab named Dockerfile as we did earlier with another image. There we’ll see that the last line says:

This means that as soon as the container start the program fastqc will be activated, and since its default is to run as GUI this will cause an error unless X11 is available and running.

As we learned earlier, we’ll have to bypass the entry point, and then we’ll be able to work from within the container.

8.2 Bypass Entry Point

We can circumvent the entrypoint command from the Docker image by specifying an alternate command. If we use /bin/bash as we did before we’ll be able to start the container and obtain an interactive terminal.

Start container.

We bypass entry point and also specify shared directory as before:

root@06d472dffb7c:/#

We can now start working, remembering that we are working within the container. We first change into the shared directory and verify that the compressed archived are present.

SRR10314868_1.fastq.gz  SRR10314868_2.fastq.gz

We can now run the program on one or more files. fastqc can read compressed files and therefore we do not need to uncompress them before use.

To run fastqc on all files the command `fastqc *.gz would do. For today we’ll run it on one of them:

Started analysis of SRR10314868_1.fastq.gz
Approx 5% complete for SRR10314868_1.fastq.gz
Approx 10% complete for SRR10314868_1.fastq.gz
[... truncated]
Approx 95% complete for SRR10314868_1.fastq.gz
Analysis complete for SRR10314868_1.fastq.gz

Two files will be created for each sequence file analyzed.

SRR10314868_1_fastqc.html  SRR10314868_1_fastqc.zip

The information is the same in both and visible through a web browser. Only the HTML format and organization is different.

Open file in web browser.

To see the results simply go to the host computer and open the file SRR10314868_1_fastqc.html with a web browser.

The top of the page will show a summary information table

Measure Value
Filename SRR10314868_1.fastq.gz
File type Conventional base calls
Encoding Sanger / Illumina 1.9
Total Sequences 850953
Sequences flagged as poor quality 0
Sequence length 35-101
%GC 50

The quality score is reported in the form of a “box plot” that here show very high quality:

fastqc base quality box plot

8.3 Exit container

If you are finished, exit the container and return to the host prompt:

$

9 Summary of commands learned or reviewed

Docker Commands Comment
docker --version Short output of version
docker login Required. Register at docker.com
docker pull download a docker image from hub.docker.com
tag some docker images require a specific tag
docker image ls list docker image. Equiv command: docker images
docker run -it --rm -v $HOME/dockershare:/data run shell in container, share dockershare directory
docker container ls -a list all containers, same as command above
docker run -it --rm --entrypoint bypass ENTRYPOINT command
/bin/bash, /bin/ls common “entrypoint” options
Shell Commands Comment
which shell command to find program location
$HOME shell variable designated the default home folder
cd $HOME/dockershare change directory to dockershare located in $HOME
cat > GIP.fa <<- EOF create a file from stdin until EOF
gzip Compress file(s) with GNU zip format
Software within containers Comment
clustalo multiple sequence alignemnt of 3 or more sequences
fastq-dump Extract archived sequence data into fastq files.
fastqc check quality of sequence data.

10 APPENDIX A

10.1 Create shared directory

11 APPENDIX B

11.1 FastQ format

Fastq files (.fq) are usually the “deliverable” data that is given to users, more often in a gnu-zip (gzip) compressed form (.fq.gz) as fastq is a plain text format.

Here are the first 4 lines of one of the files we’ll unarchive today:

@SRR10314868.1.1 1 length=101
TGGGAGCAGATGGTTATGCCTTACACAGACAACCGCTGGAACGAAACCTATTATCGTTCAACGATGATTATTGGTCTGGCACCTGAAGGGAAGGTAAGAGT
+SRR10314868.1.1 1 length=101
GGGGGIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIGIIIIIIIGIIGIIIIIIIGGIGIIIGGGGGGIIGIGGGIGIIIIIIIIIGGIIGIIIIIGIIIG

Fastq files contain 4 lines per recorded sequence:

  • Line 1 begins with a @ character and is followed by a sequence identifier and an optional description (like a FASTA e.g.* a title line).
  • Line 2 is the raw sequence letters (the base calls: A, C, T, G and N).
  • Line 3 begins with a + character as a separator
  • Line 4 encodes the quality values for the sequence in Line 2, and must contain the same number of symbols as letters in the sequence.

The quality values are Phred scores +33 (if it is Illumina data, hence Phred+33) encoded using ASCII characters to represent the numerical quality scores.

There have been a few encoding schemes detailed on the Wiki FastQ format page (scroll down or see below.)

Note: PHRED was the name of a program developped by Philip Palmer Green and acronym for “Phil’s read editor” often used in conjunction with “Phil’s revised assembly program” or PHRAP (Moody (2004).)

See also:

11.1.1 Qualtity scores

The quality scores from the Wiki FastQ format page is reproduced here (HTML version of this document only):

  SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS.....................................................
  ..........................XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX......................
  ...............................IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII......................
  .................................JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ.....................
  LLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLL....................................................
  !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~
  |                         |    |        |                              |                     |
 33                        59   64       73                            104                   126
  0........................26...31.......40                                
                           -5....0........9.............................40 
                                 0........9.............................40 
                                    3.....9..............................41 
  0.2......................26...31........41                              

 S - Sanger        Phred+33,  raw reads typically (0, 40)
 X - Solexa        Solexa+64, raw reads typically (-5, 40)
 I - Illumina 1.3+ Phred+64,  raw reads typically (0, 40)
 J - Illumina 1.5+ Phred+64,  raw reads typically (3, 41)
     with 0=unused, 1=unused, 2=Read Segment Quality Control Indicator (bold) 
     (Note: See discussion above).
 L - Illumina 1.8+ Phred+33,  raw reads typically (0, 41)

12 APPENDIX C

Windows users may run into more difficulties depending on set-up and admin privileges. Docker with fancy variable commands will only run in PowerShell.

Here are useful links:

REFERENCES

Andrews, S. 2010. “FASTQC. A Quality Control Tool for High Throughput Sequence Data.” https://www.bioinformatics.babraham.ac.uk/projects/fastqc/.

Leinonen, R., H. Sugawara, and M. Shumway. 2011. “The sequence read archive.” Nucleic Acids Res. 39 (Database issue): 19–21.

Moody, Glyn. 2004. Digital Code of Life: How Bioinformatics Is Revolutionizing Science, Medici. John Wiley & Sons.

Sievers, F., and D. G. Higgins. 2018. “Clustal Omega for making accurate alignments of many protein sequences.” Protein Sci. 27 (1): 135–45.

Sievers, F., A. Wilm, D. Dineen, T. J. Gibson, K. Karplus, W. Li, R. Lopez, et al. 2011. “Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega.” Mol. Syst. Biol. 7 (October): 539. https://www.ncbi.nlm.nih.gov/pubmed/21988835.

  1. https://docs.docker.com/install/↩

  2. http://pegi3s.github.io/dockerfiles↩

  3. https://www.ebi.ac.uk/seqdb/confluence/display/THD/Clustal+Omega↩

  4. http://www.clustal.org/omega/README↩

  5. https://oprea.rocks/blog/how-to-properly-override-the-entrypoint-using-docker-run/↩

  6. https://en.wikipedia.org/wiki/FASTQ_format↩

  7. https://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?view=toolkit_doc&f=fastq-dump↩

  8. https://www.bioinformatics.babraham.ac.uk/projects/fastqc/↩