Assessing Read Quality


Teaching: 30 min
Exercises: 20 min
  • How can I describe the quality of my data?

  • 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.

Bioinformatic 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 easily 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.

Starting with Data

Often times, the first step in a bioinformatic workflow is getting the data you want to work with onto a computer where you can work with it. If you have outsourced sequencing of your data, the sequencing center will usually provide you with a link that you can use to download your data. Today we will be working with publicly available sequencing data.

We are studying a population of Escherichia coli (designated Ara-3), which were propagated for more than 50,000 generations in a glucose-limited minimal medium. We will be working with three samples from this experiment, one from 5,000 generations, one from 15,000 generations, and one from 50,000 generations. The population changed substantially during the course of the experiment, and we will be exploring how with our variant calling workflow.

The data are paired-end, so we will download two files for each sample. We will use the European Nucleotide Archive to get our data. The ENA “provides a comprehensive record of the world’s nucleotide sequencing information, covering raw sequencing data, sequence assembly information and functional annotation.” The ENA also provides sequencing data in the fastq format, an important format for sequencing reads that we will be learning about today.

To download the data, run the commands below. It will take about 10 minutes to download the files.

mkdir -p ~/dc_workshop/data/untrimmed_fastq/
cd ~/dc_workshop/data/untrimmed_fastq

curl -O
curl -O
curl -O
curl -O
curl -O
curl -O 

Faster option

If your workshop is short on time or the venue’s internet connection is weak or unstable, learners can avoid needing to download the data and instead use the data files provided in the .backup/ directory.

$ cp ~/.backup/untrimmed_fastq/*fastq.gz .

This command creates a copy of each of the files in the .backup/untrimmed_fastq/ directory that end in fastq.gz and places the copies in the current working directory (signified by .).

The data comes in a compressed format, which is why there is a .gz at the end of the file names. This makes it faster to transfer, and allows it to take up less space on our computer. Let’s unzip one of the files so that we can look at the fastq format.

$ gunzip SRR2584863_1.fastq.gz 

Quality Control

We will now assess the quality of the sequence reads contained in our fastq files.


Details on the FASTQ format

Although it looks complicated (and it is), we can understand the fastq format with a little decoding. Some rules about the format include…

Line Description
1 Always begins with ‘@’ and then information about the read
2 The actual DNA sequence
3 Always begins with a ‘+’ and sometimes the same info in line 1
4 Has a string of characters which represent the quality scores; must have same number of characters as line 2

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 -n 4 SRR2584863_1.fastq 
@SRR2584863.1 HWI-ST957:244:H73TDADXX:1:1101:4712:2181/1

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 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, using 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:;<=>?@ABCDEFGHIJ
                   |         |         |         |         |
Quality score:    01........11........21........31........41                                

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

Looking back at our read:

@SRR2584863.1 HWI-ST957:244:H73TDADXX:1:1101:4712:2181/1

we can now see that there is a range of quality scores, but that the end of the sequence is very poor (# = a quality score of 2).


What is the last read in the SRR2584863_1.fastq file? How confident are you in this read?


$ tail -n 4 SRR2584863_1.fastq 
@SRR2584863.1553259 HWI-ST957:245:H73R4ADXX:2:2216:21048:100894/1

This read has more consistent quality at its end than the first read that we looked at, but still has a range of quality scores, most of them high. We will look at variations in position-based quality in just a moment.

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 one 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. This is much shorter than the reads we are working with in our workflow. 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 1st 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 absolute range, which covers the lowest (0th quartile) to highest (4th quartile) values.

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 FastQC

We will now assess the quality of the reads that we downloaded. First, make sure you’re still in the untrimmed_fastq directory

$ cd ~/dc_workshop/data/untrimmed_fastq/ 


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


$ ls -l -h
-rw-rw-r-- 1 dcuser dcuser 545M Jul  6 20:27 SRR2584863_1.fastq
-rw-rw-r-- 1 dcuser dcuser 183M Jul  6 20:29 SRR2584863_2.fastq.gz
-rw-rw-r-- 1 dcuser dcuser 309M Jul  6 20:34 SRR2584866_1.fastq.gz
-rw-rw-r-- 1 dcuser dcuser 296M Jul  6 20:37 SRR2584866_2.fastq.gz
-rw-rw-r-- 1 dcuser dcuser 124M Jul  6 20:22 SRR2589044_1.fastq.gz
-rw-rw-r-- 1 dcuser dcuser 128M Jul  6 20:24 SRR2589044_2.fastq.gz

There are six FASTQ files ranging from 124M (124MB) to 545M.

FastQC can accept multiple file names as input, and on both zipped and unzipped files, so we can use the *.fastq* wildcard to run FastQC on all of the FASTQ files in this directory.

$ 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 SRR2584863_1.fastq
Approx 5% complete for SRR2584863_1.fastq
Approx 10% complete for SRR2584863_1.fastq
Approx 15% complete for SRR2584863_1.fastq
Approx 20% complete for SRR2584863_1.fastq
Approx 25% complete for SRR2584863_1.fastq
Approx 30% complete for SRR2584863_1.fastq
Approx 35% complete for SRR2584863_1.fastq
Approx 40% complete for SRR2584863_1.fastq
Approx 45% complete for SRR2584863_1.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 SRR2589044_2.fastq.gz
Approx 85% complete for SRR2589044_2.fastq.gz
Approx 90% complete for SRR2589044_2.fastq.gz
Approx 95% complete for SRR2589044_2.fastq.gz
Analysis complete for SRR2589044_2.fastq.gz

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

$ ls 
SRR2584863_1.fastq        SRR2584866_1_fastqc.html  SRR2589044_1_fastqc.html
SRR2584863_1_fastqc.html   SRR2584866_1.fastq.gz     SRR2589044_1.fastq.gz
SRR2584863_2_fastqc.html  SRR2584866_2_fastqc.html  SRR2589044_2_fastqc.html
SRR2584863_2.fastq.gz     SRR2584866_2.fastq.gz     SRR2589044_2.fastq.gz

For each input FASTQ file, FastQC has created a .zip file and a .html 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 -p ~/dc_workshop/results/fastqc_untrimmed_reads 
$ mv *.zip ~/dc_workshop/results/fastqc_untrimmed_reads/ 
$ mv *.html ~/dc_workshop/results/fastqc_untrimmed_reads/ 

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

$ cd ~/dc_workshop/results/fastqc_untrimmed_reads/ 

Viewing the FastQC results

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

$ open SRR2584863_1_fastqc.html 

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

Couldn't get a file descriptor referring to the console

This is because the AWS instance we’re using 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 scp, which we learned yesterday in the Shell Genomics lesson.

First we will make a new directory on our computer to store the HTML files we’re transfering. Let’s put it on our desktop for now. Open a new tab in your terminal program (you can use the pull down menu at the top of your screen or the Cmd+t keyboard shortcut) and type:

$ mkdir -p ~/Desktop/fastqc_html 

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

$ scp*.html ~/Desktop/fastqc_html

As a reminder, the first part of the command is the address for your remote computer. Make sure you replace everything after dcuser@ with your instance number (the one you used to log in).

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 (*.html) to indicate that we want all of the HTML 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:

SRR2584863_1_fastqc.html                      100%  249KB 152.3KB/s   00:01    
SRR2584863_2_fastqc.html                      100%  254KB 219.8KB/s   00:01    
SRR2584866_1_fastqc.html                      100%  254KB 271.8KB/s   00:00    
SRR2584866_2_fastqc.html                      100%  251KB 252.8KB/s   00:00    
SRR2589044_1_fastqc.html                      100%  249KB 370.1KB/s   00:00    
SRR2589044_2_fastqc.html                      100%  251KB 592.2KB/s   00:00  

Now we can go to our new directory and open the HTML files.

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


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?


All of the reads contain usable data, but the quality decreases toward the end of the reads.

Decoding the other FastQC outputs

We’ve now looked at quite a few “Per base sequence quality” FastQC graphs, but there are nine other graphs that we haven’t talked about! Below we have provided a brief overview of interpretations for each of these plots. For more information, please see the FastQC documentation here

Working with the FastQC text output

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 your AWS instance (the tab label will start with dcuser@ip) and make sure you’re in our results subdirectory.

$ cd ~/dc_workshop/results/fastqc_untrimmed_reads/ 
$ ls 
SRR2584863_1_fastqc.html  SRR2584866_1_fastqc.html  SRR2589044_1_fastqc.html
SRR2584863_2_fastqc.html  SRR2584866_2_fastqc.html  SRR2589044_2_fastqc.html

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 decompress these files. Let’s try doing them all at once using a wildcard.

$ unzip *.zip 
caution: filename not matched:
caution: filename not matched:
caution: filename not matched:
caution: filename not matched:
caution: filename not matched:

This didn’t work. We unzipped the first file and then got a warning message for each of the other .zip files. This is because unzip expects to get only one zip file as input. 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 like we learned in the Shell Genomics lesson 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 $filename
> done

In this example, the input 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 The interpreter runs the command unzip on For the second iteration, $filename becomes This time, the shell runs unzip on It then repeats this process for the four other .zip files in our directory.

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

   creating: SRR2589044_2_fastqc/
   creating: SRR2589044_2_fastqc/Icons/
   creating: SRR2589044_2_fastqc/Images/
  inflating: SRR2589044_2_fastqc/Icons/fastqc_icon.png  
  inflating: SRR2589044_2_fastqc/Icons/warning.png  
  inflating: SRR2589044_2_fastqc/Icons/error.png  
  inflating: SRR2589044_2_fastqc/Icons/tick.png  
  inflating: SRR2589044_2_fastqc/summary.txt  
  inflating: SRR2589044_2_fastqc/Images/per_base_quality.png  
  inflating: SRR2589044_2_fastqc/Images/per_tile_quality.png  
  inflating: SRR2589044_2_fastqc/Images/per_sequence_quality.png  
  inflating: SRR2589044_2_fastqc/Images/per_base_sequence_content.png  
  inflating: SRR2589044_2_fastqc/Images/per_sequence_gc_content.png  
  inflating: SRR2589044_2_fastqc/Images/per_base_n_content.png  
  inflating: SRR2589044_2_fastqc/Images/sequence_length_distribution.png  
  inflating: SRR2589044_2_fastqc/Images/duplication_levels.png  
  inflating: SRR2589044_2_fastqc/Images/adapter_content.png  
  inflating: SRR2589044_2_fastqc/fastqc_report.html  
  inflating: SRR2589044_2_fastqc/fastqc_data.txt  
  inflating: SRR2589044_2_fastqc/  

The unzip program is decompressing the .zip files and 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.

If you list the files in our directory now you will see:

SRR2584863_1_fastqc       SRR2584866_1_fastqc       SRR2589044_1_fastqc
SRR2584863_1_fastqc.html  SRR2584866_1_fastqc.html  SRR2589044_1_fastqc.html
SRR2584863_2_fastqc       SRR2584866_2_fastqc       SRR2589044_2_fastqc
SRR2584863_2_fastqc.html  SRR2584866_2_fastqc.html  SRR2589044_2_fastqc.html

The .html files and the uncompressed .zip files are still present, but now we also have a new directory for each of our samples. We can see for sure that it’s a directory if we use the -F flag for ls.

$ ls -F 
SRR2584863_1_fastqc/      SRR2584866_1_fastqc/      SRR2589044_1_fastqc/
SRR2584863_1_fastqc.html  SRR2584866_1_fastqc.html  SRR2589044_1_fastqc.html
SRR2584863_2_fastqc/      SRR2584866_2_fastqc/      SRR2589044_2_fastqc/
SRR2584863_2_fastqc.html  SRR2584866_2_fastqc.html  SRR2589044_2_fastqc.html

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

$ ls -F SRR2584863_1_fastqc/ 
fastqc_data.txt  fastqc_report.html	Icons/	Images/  summary.txt

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

$ less SRR2584863_1_fastqc/summary.txt 
PASS    Basic Statistics        SRR2584863_1.fastq
PASS    Per base sequence quality       SRR2584863_1.fastq
PASS    Per tile sequence quality       SRR2584863_1.fastq
PASS    Per sequence quality scores     SRR2584863_1.fastq
WARN    Per base sequence content       SRR2584863_1.fastq
WARN    Per sequence GC content SRR2584863_1.fastq
PASS    Per base N content      SRR2584863_1.fastq
PASS    Sequence Length Distribution    SRR2584863_1.fastq
PASS    Sequence Duplication Levels     SRR2584863_1.fastq
PASS    Overrepresented sequences       SRR2584863_1.fastq
WARN    Adapter Content SRR2584863_1.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). Remember, to quit from less you must type q.

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 ~/dc_workshop/docs.

$ cat */summary.txt > ~/dc_workshop/docs/fastqc_summaries.txt 


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


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

$ cd ~/dc_workshop/docs
$ grep FAIL fastqc_summaries.txt
FAIL    Per base sequence quality       SRR2584863_2.fastq.gz
FAIL    Per tile sequence quality       SRR2584863_2.fastq.gz
FAIL    Per base sequence content       SRR2584863_2.fastq.gz
FAIL    Per base sequence quality       SRR2584866_1.fastq.gz
FAIL    Per base sequence content       SRR2584866_1.fastq.gz
FAIL    Adapter Content SRR2584866_1.fastq.gz
FAIL    Adapter Content SRR2584866_2.fastq.gz
FAIL    Adapter Content SRR2589044_1.fastq.gz
FAIL    Per base sequence quality       SRR2589044_2.fastq.gz
FAIL    Per tile sequence quality       SRR2589044_2.fastq.gz
FAIL    Per base sequence content       SRR2589044_2.fastq.gz
FAIL    Adapter Content SRR2589044_2.fastq.gz

Other notes – Optional

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!

Same Symbols, Different Meanings

Here we see > being used as 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 should redirect output or get the value of a variable.

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.