Assessing Read Quality

Overview

Teaching: 30 min
Exercises: 20 min

Questions

• How can I describe the quality of my data?

Objectives

• Explain how a FASTQ file encodes per-base quality scores

• Interpret a FastQC plot summarizing per-base quality across all reads

• Use for loops to automate operations on multiple files

Bioinformatics Workflows

When working with high-throughput sequencing data, the raw reads you get off of the sequencer will need to pass through a number of different tools in order to generate your final desired output. The execution of this set of tools in a specified order is commonly referred to as a workflow or a pipeline.

An example of the workflow we will be using for our variant calling analysis is provided below with a brief description of each step.

1. Quality control – Assessing quality using FastQC
2. Quality control – Trimming and/or filtering reads (if necessary)
3. Align reads to reference genome
4. Perform post-alignment clean-up
5. Variant calling

These workflows in bioinformatics adopt a plug-and-play approach in that the output of one tool can be used as input to another tool without any extensive configuration. Having standards for data formats is what makes this feasible. Standards ensure that data is stored in a way that is generally accepted and agreed upon within the community. The tools that are used to analyze data at different stages of the workflow are therefore built under the assumption that the data will be provided in a specific format.

Quality Control

The first step in the variant calling workflow is to take the FASTQ files received from the sequencing facility and assess the quality of the sequence reads.

Details on the FASTQ format

Although it looks complicated (and it is), it’s easy to understand the FASTQ format with a little decoding. Some rules about the format include:

We can view the first complete read in one of the files our dataset by using head to look at the first four lines.

$ head -n4 SRR098026.fastq

@SRR098026.1 HWUSI-EAS1599_1:2:1:0:968 length=35
NNNNNNNNNNNNNNNNCNNNNNNNNNNNNNNNNNN
+SRR098026.1 HWUSI-EAS1599_1:2:1:0:968 length=35
!!!!!!!!!!!!!!!!#!!!!!!!!!!!!!!!!!!

All but one of the nucleotides in this read are unknown (N). This is a pretty bad read!

Line 4 shows the quality for each nucleotide in the read. Quality is interpreted as the probability of an incorrect base call (e.g. 1 in 10) or, equivalently, the base call accuracy (e.g., 90%). To make it possible to line up each individual nucleotide with its quality score, the numerical score is converted into a code where each individual character represents the numerical quality score for an individual nucleotide. For example, in the line above, the quality score line is:

!!!!!!!!!!!!!!!!#!!!!!!!!!!!!!!!!!!

The # character and each of the ! characters represent the encoded quality for an individual nucleotide. The numerical value assigned to each of these characters depends on the sequencing platform that generated the reads. The sequencing machine used to generate our data uses the standard Sanger quality PHRED score encoding, used by Illumina version 1.8 onwards. Each character is assigned a quality score between 0 and 40 as shown in the chart below.

Quality encoding: !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHI
                  |         |         |         |         |
Quality score:    0........10........20........30........40

Each quality score represents the probability that the corresponding nucleotide call is incorrect. This quality score is logarithmic, so a quality score of 10 reflects a base call accuracy of 90%, while a quality score of 20 reflects a base call accuracy of 99%. These probability values are the results from the base calling algorithm and dependent on how much signal was captured for the base incorporation.

Looking back at our read:

@SRR098026.1 HWUSI-EAS1599_1:2:1:0:968 length=35
NNNNNNNNNNNNNNNNCNNNNNNNNNNNNNNNNNN
+SRR098026.1 HWUSI-EAS1599_1:2:1:0:968 length=35
!!!!!!!!!!!!!!!!#!!!!!!!!!!!!!!!!!!

we can now see that the quality of each of the Ns is 0 and the quality of the only nucleotide call (C) is also very poor (# = a quality score of 2). This is indeed a very bad read.

Exercise

What is the last read in the SRR098026.fastq file? How confident are you in this read?

Solution

$ tail -n4 SRR098026.fastq

@SRR098026.249 HWUSI-EAS1599_1:2:1:2:1057 length=35
CNCTNTATGCGTACGGCAGTGANNNNNNNGGAGAT
+SRR098026.249 HWUSI-EAS1599_1:2:1:2:1057 length=35
A!@B!BBB@ABAB#########!!!!!!!######

The second half of this read is poor quality. Many of the positions are unknown (Ns) and the bases that we do have guesses for are of very poor quality (#). However, the beginning of the read is fairly high quality. We will look at variations in position-based quality in just a moment.

Quality Encodings Vary

Although we’ve used a particular quality encoding system to demonstrate interpretation of read quality, different sequencing machines use different encoding systems. This means that, depending on which sequencer you use to generate your data, a # may not be an indicator of a poor quality base call.

This mainly relates to older Solexa/Illumina data, but it’s essential that you know which sequencing platform was used to generate your data, so that you can tell your quality control program which encoding to use. If you choose the wrong encoding, you run the risk of throwing away good reads or (even worse) not throwing away bad reads!

Assessing Quality using FastQC

In real life, you won’t be assessing the quality of your reads by visually inspecting your FASTQ files. Rather, you’ll be using a software program to assess read quality and filter out poor quality reads. We’ll first use a program called FastQC to visualize the quality of our reads. Later in our workflow, we’ll use another program to filter out poor quality reads.

FastQC has a number of features which can give you a quick impression of any problems your data may have, so you can take these issues into consideration before moving forward with your analyses. Rather than looking at quality scores for each individual read, FastQC looks at quality collectively across all reads within a sample. The image below shows a FastQC-generated plot that indicates a very high quality sample:

The x-axis displays the base position in the read, and the y-axis shows quality scores. In this example, the sample contains reads that are 40 bp long. For each position, there is a box-and-whisker plot showing the distribution of quality scores for all reads at that position. The horizontal red line indicates the median quality score and the yellow box shows the 2nd to 3rd quartile range. This means that 50% of reads have a quality score that falls within the range of the yellow box at that position. The whiskers show the range to the 1st and 4th quartile.

For each position in this sample, the quality values do not drop much lower than 32. This is a high quality score. The plot background is also color-coded to identify good (green), acceptable (yellow), and bad (red) quality scores.

Now let’s take a look at a quality plot on the other end of the spectrum.

Here, we see positions within the read in which the boxes span a much wider range. Also, quality scores drop quite low into the “bad” range, particularly on the tail end of the reads. The FastQC tool produces several other diagnostic plots to assess sample quality, in addition to the one plotted above.

Running analyses on Crane

We are going to start doing analyses on our data. So far we’ve been doing all our work on the Crane “login” nodes. It’s fine to use the nodes for simple tasks like examining files with less, editing files with nano, moving files, but for analyses we need to have the work done on the “compute” nodes. For this workshop we will be using the interactive queue. With one command you will start an interactive job, be logged into one of the compute nodes, mount the /common/ directory that we will need later on, and set a timeout of our sesson at 3 hours. The rest of our work will continue on the compute note.

$ srun --pty --time=3:00:00 --license=common $SHELL

[username@cNNNN]$

You will notice that the command prompt will change to say that you are now on a compute node (@cNNN).

$ pwd

/work/group/username

We’ll make sure to cd back to our data/ directory below.

Another important thing to remember is to load any modules we need. We’ll do that before we run each analysis.

Running FastQC

We will be using the FASTQ dataset you transferred yesterday. Please navigate to the files.

$ cd /work/group/username/dc_workshop/data/untrimmed_fastq/

Your directory should look like:

$ ls -F

SRR097977.fastq*  SRR098027.fastq*  SRR098281.fastq*
SRR098026.fastq*  SRR098028.fastq*  SRR098283.fastq*

Exercise

How many FASTQ files are in this dataset? How big are the files? (Hint: Look at the options for the ls command to see how to show file sizes.)

Solution

$ ls -l -h

-rw-r--r-- 1 username group 840M Jul 30  2015 SRR097977.fastq
-rw-r--r-- 1 username group 3.4G Jul 30  2015 SRR098026.fastq
-rw-r--r-- 1 username group 875M Jul 30  2015 SRR098027.fastq
-rw-r--r-- 1 username group 3.4G Jul 30  2015 SRR098028.fastq
-rw-r--r-- 1 username group 4.0G Jul 30  2015 SRR098281.fastq
-rw-r--r-- 1 username group 3.9G Jul 30  2015 SRR098283.fastq

There are six FASTQ files ranging from 840M (840MB) to 4.0G (4.0GB)

To run the FastQC program, we need to load the fastqc module (fastqc). FastQC can accept multiple file names as input, so we can use the *.fastq wildcard to run FastQC on all of the FASTQ files in this directory.

$ module load fastqc
$ fastqc *.fastq

You will see an automatically updating output message telling you the progress of the analysis. It will start like this:

Started analysis of SRR097977.fastq
Approx 5% complete for SRR097977.fastq
Approx 10% complete for SRR097977.fastq
Approx 15% complete for SRR097977.fastq
Approx 20% complete for SRR097977.fastq
Approx 25% complete for SRR097977.fastq
Approx 30% complete for SRR097977.fastq
Approx 35% complete for SRR097977.fastq
Approx 40% complete for SRR097977.fastq
Approx 45% complete for SRR097977.fastq
Approx 50% complete for SRR097977.fastq
...

In total, it should take about five minutes for FastQC to run on all six of our FASTQ files. When the analysis completes, your prompt will return. So your screen will look something like this:

Approx 80% complete for SRR098283.fastq
Approx 85% complete for SRR098283.fastq
Approx 90% complete for SRR098283.fastq
Approx 95% complete for SRR098283.fastq
Analysis complete for SRR098283.fastq

The FastQC program has created several new files within our data/untrimmed_fastq/ directory.

$ ls

SRR097977.fastq        SRR098027.fastq        SRR098281.fastq
SRR097977_fastqc/      SRR098027_fastqc/      SRR098281_fastqc/
SRR097977_fastqc.zip   SRR098027_fastqc.zip   SRR098281_fastqc.zip
SRR098026.fastq        SRR098028.fastq        SRR098283.fastq
SRR098026_fastqc/      SRR098028_fastqc/      SRR098283_fastqc/
SRR098026_fastqc.zip   SRR098028_fastqc.zip   SRR098283_fastqc.zip

For each input FASTQ file, FastQC has created a .zip file and a directory contain an .html report file. The .zip file extension indicates that this is actually a compressed set of multiple output files. We’ll be working with these output files soon. The .html file is a stable webpage displaying the summary report for each of our samples.

We want to keep our data files and our results files separate, so we will move these output files into a new directory within our results/ directory.

$ mkdir /work/group/username/dc_workshop/results/fastqc_untrimmed_reads
$ mv *.zip /work/group/username/dc_workshop/results/fastqc_untrimmed_reads/
$ mv *fastqc/ /work/group/username/dc_workshop/results/fastqc_untrimmed_reads/

Now we can navigate into this results/ directory and do some closer inspection of our output files.

$ cd /work/group/username/dc_workshop/results/fastqc_untrimmed_reads/

Viewing HTML files

If we were working on our local computers, we’d be able to display each of these HTML files as a webpage:

$ open SRR097977_fastqc/fastqc_report.html

However, if you try this on our Crane, you’ll get an error:

Couldn't get a file descriptor referring to the console

This is because Crane doesn’t have any web browsers installed on it, so the remote computer doesn’t know how to open the file. We want to look at the webpage summary reports, so let’s transfer them to our local computers (i.e. your laptop).

To transfer a file from a remote server to our own machines, we will use a variant of the cp command called scp. The “s” stands for “secure” – this is a secure version of copying (cp).

For the cp command, the syntax was:

$ cp my_file new_location

The syntax for scp is the same, but now my_file and new_location are on separate computers, so we need to give an absolute path, including the name of our remote computer. First we will make a new directory on our computer to store the HTML files we’re transferring. Let’s put it on our Desktop for now.

Now we can transfer our HTML files to our local computer using scp.

On Macs or Linux

Open a new tab in your terminal program (you can use the pull down menu at the top of your screen or the Ctrl+t keyboard shortcut) and type:

$ mkdir ~/Desktop/fastqc_html

$ scp username@crane.unl.edu:/work/group/username/dc_workshop/results/fastqc_untrimmed_reads/*.zip ~/Desktop/fastqc_html/

On Windows

Open a Windows command prompt window by typing Windows+r (the Windows key and the ‘r’ key at the same time). You should see a “Run” window open. In the box enter: cmd, a new cmd.exe window should open.

Create a new folder on your desktop:

$ mkdir %HOMEPATH%\Desktop\fastqc_html

(%HOMEPATH% is roughly the same as ~ in Unix based systems.)

Now we can transfer our HTML files to our local computer using pscp. (Note that Linux systems use the forward slash, /, for directories while Windows uses the back slash, \, check that you are using the right ones in the command below.)

> pscp username@crane.unl.edu:/work/group/username/dc_workshop/results/fastqc_untrimmed_reads/*.zip %HOMEPATH%\Desktop\fastqc_html\

Back to everyone

This looks really complicated, so let’s break it down. The first part of the command username@crane.unl.edu is the address for your remote computer. Make sure you replace username with your actual username.

The second part starts with a : and then gives the absolute path of the files you want to transfer from your remote computer. Don’t forget the :. We used a wildcard (*.zip) to indicate that we want all of the zip files.

The third part of the command gives the absolute path of the location you want to put the files. This is on your local computer and is the directory we just created ~/Desktop/fastqc_html/.

You should see a status output like this:

SRR097977_fastqc.zip    100%  130KB 130.1KB/s   00:00
SRR098026_fastqc.zip    100%  183KB 182.8KB/s   00:00
SRR098027_fastqc.zip    100%  129KB 129.0KB/s   00:00
SRR098028_fastqc.zip    100%  192KB 191.7KB/s   00:00
SRR098281_fastqc.zip    100%  195KB 195.0KB/s   00:00
SRR098283_fastqc.zip    100%  192KB 192.0KB/s   00:00

Navigate to this fastqc_html/ directory in your usual file explorer and unzip the files as you usually would. We will learn how to programmatically unzip this files in just a little bit.

Viewing on Mac and Linux systems:

$ cd ~/Desktop/fastqc_html/
$ open */*.html

Viewing on Windows:

> cd %HOMEPATH%\Desktop\fastqc_html
> SRR097977_fastqc.html
> SRR098026_fastqc.html
> SRR098027_fastqc.html
> SRR098028_fastqc.html
> SRR098281_fastqc.html
> SRR098283_fastqc.html

Your computer will open each of the HTML files in your default web browser. Depending on your settings, this might be as six separate tabs in a single window or six separate browser windows.

Exercise

Discuss your results with a neighbor. Which sample(s) looks the best in terms of per base sequence quality? Which sample(s) look the worst?

Solution

SRR097977 and SRR098027 are the best. The other four samples are all pretty bad.

Unzipping Compressed Files

Now that we’ve looked at our HTML reports to get a feel for the data, let’s look more closely at the other output files. Go back to the tab in your terminal program that is connected to Crane and make sure you’re in our results/ subdirectory.

$ cd /work/group/username/dc_workshop/results/fastqc_untrimmed_reads/
$ ls -F

SRR097977_fastqc/     SRR098026_fastqc.zip  SRR098028_fastqc/
SRR098281_fastqc.zip  SRR097977_fastqc.zip  SRR098027_fastqc/
SRR098028_fastqc.zip  SRR098283_fastqc/     SRR098026_fastqc/
SRR098027_fastqc.zip  SRR098281_fastqc/     SRR098283_fastqc.zip

Our .zip files are compressed files. They each contain multiple different types of output files for a single input FASTQ file. To view the contents of a .zip file, we can use the program unzip to list without decompressing these files. Let’s try doing them all at once using a wildcard.

$ unzip -l *.zip

Archive:  SRR097977_fastqc.zip
  Length      Date    Time    Name
---------  ---------- -----   ----
---------                     -------
        0                     0 files

This didn’t work. What happened here is unzip -l expects one .zip argument and we gave it six. We could go through and unzip each file one at a time, but this is very time consuming and error-prone. Someday you may have 500 files to unzip!

A more efficient way is to use a for loop to iterate through all of our .zip files. Let’s see what that looks like and then we’ll discuss what we’re doing with each line of our loop.

$ for filename in *.zip
> do
> unzip -l $filename
> done

When the shell sees the keyword for, it knows to repeat a command (or group of commands) once for each item in a list. Each time the loop runs (called an iteration), an item in the list is assigned in sequence to the variable, and the commands inside the loop are executed, before moving on to the next item in the list.

Inside the loop, we call for the variable’s value by putting $ in front of it. The $ tells the shell interpreter to treat the variable as a variable name and substitute its value in its place, rather than treat it as text or an external command.

In this example, the list is six filenames (one filename for each of our .zip files). Each time the loop iterates, it will assign a file name to the variable filename and run the unzip command. The first time through the loop, $filename is SRR097977_fastqc.zip. The interpreter runs the command unzip on SRR097977_fastqc.zip. For the second iteration, $filename becomes SRR098026_fastqc.zip. This time, the shell runs unzip on SRR098026_fastqc.zip. It then repeats this process for the four other .zip files in our directory.

Follow the Prompt

The shell prompt changes from $ to > and back again as we were typing in our loop. The second prompt, >, is different to remind us that we haven’t finished typing a complete command yet. A semicolon, ;, can be used to separate two commands written on a single line.

Same Symbols, Different Meanings

Here we see > being used a shell prompt, whereas > is also used to redirect output. Similarly, $ is used as a shell prompt, but, as we saw earlier, it is also used to ask the shell to get the value of a variable.

If the shell prints > or $ then it expects you to type something, and the symbol is a prompt.

If you type > or $ yourself, it is an instruction from you that the shell to redirect output or get the value of a variable.

We have called the variable in this loop filename in order to make its purpose clearer to human readers. The shell itself doesn’t care what the variable is called; if we wrote this loop as:

$ for x in *.zip
> do
> unzip -l $x
> done

or:

$ for temperature in *.zip
> do
> unzip -l $temperature
> done

it would work exactly the same way. Don’t do this. Programs are only useful if people can understand them, so meaningless names (like x) or misleading names (like temperature) increase the odds that the program won’t do what its readers think it does.

Multipart commands

The for loop is interpreted as a multipart command. If you press the up arrow on your keyboard to recall the command, it will be shown like so:

$ for filename in *.zip; do unzip -l $filename; done

When you check your history later, it will help your remember what you did!

When we run our for loop, you will see output that starts like this:

Archive:  SRR097977_fastqc.zip
  Length      Date    Time    Name
---------  ---------- -----   ----
        0  12-13-2018 13:28   SRR097977_fastqc/
        0  12-13-2018 13:28   SRR097977_fastqc/Icons/
        0  12-13-2018 13:28   SRR097977_fastqc/Images/
     1561  12-13-2018 13:28   SRR097977_fastqc/Icons/error.png
     1715  12-13-2018 13:28   SRR097977_fastqc/Icons/tick.png
     1450  12-13-2018 13:28   SRR097977_fastqc/Icons/warning.png
     1197  12-13-2018 13:28   SRR097977_fastqc/Icons/fastqc_icon.png
      487  12-13-2018 13:28   SRR097977_fastqc/summary.txt
.
.
.

The unzip program is listing the content of the .zip files and if we ran unzip without the -l flag would decompress these file, creating a new directory (with subdirectories) for each of our samples, to store all of the different output that is produced by FastQC. There are a lot of files here. The one we’re going to focus on is the summary.txt file.

Understanding FastQC Output

If you list the files in our directory now using ls -F you will see:

SRR097977_fastqc/     SRR098026_fastqc.zip  SRR098028_fastqc/     SRR098281_fastqc.zip
SRR097977_fastqc.zip  SRR098027_fastqc/     SRR098028_fastqc.zip  SRR098283_fastqc/
SRR098026_fastqc/     SRR098027_fastqc.zip  SRR098281_fastqc/     SRR098283_fastqc.zip

Let’s see what files are present within one of these output directories.

$ ls -F SRR097977_fastqc/

fastqc_data.txt  fastqc_report.html  Icons/  Images/  summary.txt

Use less to preview the summary.txt file for this sample.

$ less SRR097977_fastqc/summary.txt

PASS  Basic Statistics              SRR097977.fastq
WARN  Per base sequence quality     SRR097977.fastq
PASS  Per sequence quality scores   SRR097977.fastq
PASS  Per base sequence content     SRR097977.fastq
PASS  Per base GC content           SRR097977.fastq
PASS  Per sequence GC content       SRR097977.fastq
PASS  Per base N content            SRR097977.fastq
PASS  Sequence Length Distribution  SRR097977.fastq
WARN  Sequence Duplication Levels   SRR097977.fastq
WARN  Overrepresented sequences     SRR097977.fastq
FAIL  Kmer Content                  SRR097977.fastq

The summary file gives us a list of tests that FastQC ran, and tells us whether this sample passed, failed, or is borderline (WARN).

Documenting Our Work

We can make a record of the results we obtained for all our samples by concatenating all of our summary.txt files into a single file using the cat command. We’ll call this fastqc_summaries.txt and move it to /work/group/username/dc_workshop/docs.

$ cat */summary.txt > /work/group/username/dc_workshop/docs/fastqc_summaries.txt

Exercise

Which samples failed at least one of FastQC’s quality tests? What test(s) did those samples fail?

Solution

We can get the list of all failed tests using grep.

$ cd /work/group/username/dc_workshop/docs
$ grep FAIL fastqc_summaries.txt

FAIL  Per tile sequence quality  SRR097977.fastq
FAIL  Per tile sequence quality  SRR098026.fastq
FAIL  Overrepresented sequences  SRR098026.fastq
FAIL  Kmer Content               SRR098026.fastq
FAIL  Per base sequence quality  SRR098027.fastq
FAIL  Per tile sequence quality  SRR098027.fastq
FAIL  Kmer Content               SRR098027.fastq
FAIL  Per tile sequence quality  SRR098028.fastq
FAIL  Overrepresented sequences  SRR098028.fastq
FAIL  Kmer Content               SRR098028.fastq
FAIL  Overrepresented sequences  SRR098281.fastq
FAIL  Kmer Content               SRR098281.fastq
FAIL  Overrepresented sequences  SRR098283.fastq
FAIL  Kmer Content               SRR098283.fastq

If we want to see all the files that failed at least one test, we can use a combination of grep, cut, sort and uniq.

$ grep FAIL fastqc_summaries.txt | cut -f 3 | sort | uniq

SRR097977.fastq
SRR098026.fastq
SRR098027.fastq
SRR098028.fastq
SRR098281.fastq
SRR098283.fastq

All of our samples failed at least one test. If we want to see a table showing which tests failed, we can use the same command we used above, but this time extract the second field with cut (instead of the third) and add the -c option to uniq to count the number of times each unique value appears.

$ grep FAIL fastqc_summaries.txt | cut -f 2 | sort | uniq -c

6 Kmer Content
4 Overrepresented sequences
1 Per base sequence quality
4 Sequence Duplication Levels

Key Points

• Quality encodings vary across sequencing platforms

• for loops let you perform the same set of operations on multiple files with a single command