Overview

Teaching: 30 min
Exercises: 25 min

Questions

• How can I get rid of sequence data that doesn’t meet my quality standards?

Objectives

• Clean FASTQ reads using Trimmommatic.

• Select and set multiple options for command-line bioinformatics tools.

• Write for loops with two variables.

Cleaning Reads

In the previous episode, we took a high-level look at the quality of each of our samples using FastQC. We vizualized per-base quality graphs showing the distribution of read quality at each base across all reads in a sample and extracted information about which samples fail which quality checks. We know that all of our samples failed at least one of the quality metrics used by FastQC. This doesn’t mean, though, that our samples should be thrown out! It’s very common to have some reads within a sample, or some positions (near the begining or end of reads) across all reads that are low quality and should be discarded. We will use a program called Trimmomatic to filter poor quality reads and trim poor quality bases from our samples.

Trimmomatic Options

Trimmomatic is a program written in the Java programming language. You don’t need to learn Java to use Trimmomatic (FastQC is also written in Java), but the fact that it’s a Java program helps explain the syntax that is used to run Trimmomatic. The basic command to run Trimmomatic starts like this:

$ java -jar trimmomatic-0.32.jar

java tells our computer that we’re running a Java program. -jar is an option specifying that we’re going to specify the location of the Java program we want to run. The Java program itself will have a .jar file extension.

That’s just the basic command, however. Trimmomatic has a variety of options and parameters. We will need to specify what options we want to use for our analysis. Here are some of the options:

In addition to these options, there are various trimming steps available:

We will use only a few of these options and trimming steps in our analysis. It is important to understand the steps you are using to clean your data. For more information about the Trimmomatic arguments and options, see the Trimmomatic manual.

We said above that a basic command for Trimmomatic looks like this:

$ java -jar trimmomatic-0.32.jar SE

However, a complete command for Trimmomatic will look something like this:

$ java -jar trimmomatic-0.32.jar SE -threads 4 -phred64 SRR_1056.fastq SRR_1056_trimmed.fastq ILLUMINACLIP:SRR_adapters.fa SLIDINGWINDOW:4:20

In this example, we’ve told Trimmomatic:

Running Trimmomatic

Now we will run Trimmomatic on our data. To begin, navigate to your untrimmed_fastq data directory:

$ cd ~/dc_workshop/data/untrimmed_fastq

We are going to run Trimmomatic on one of our single-end samples. We will use a sliding window of size 4 that will remove bases if their phred score is below 20 (like in our example above). We will also discard any reads that do not have at least 20 bases remaining after this trimming step.

$ java -jar ~/Trimmomatic-0.32/trimmomatic-0.32.jar SE SRR098283.fastq SRR098283.fastq_trim.fastq SLIDINGWINDOW:4:20 MINLEN:20

Notice that we needed to give the absolute path to our copy of the Trimmomatic program.

TrimmomaticSE: Started with arguments: SRR098283.fastq SRR098283.fastq_trim.fastq SLIDINGWINDOW:4:20 MINLEN:20
Automatically using 2 threads
Quality encoding detected as phred33
Input Reads: 21564058 Surviving: 17030985 (78.98%) Dropped: 4533073 (21.02%)
TrimmomaticSE: Completed successfully

Exercise

Use the output from your Trimmomatic command to answer the following questions.

1) What percent of reads did we discard from our sample? 2) What percent of reads did we keep?

Solution

1) 21.02% 2) 78.98%

You may have noticed that Trimmomatic automatically detected the quality encoding of our sample. It is always a good idea to double-check this or to enter the quality encoding manually.

We can confirm that we have our output file:

$ ls SRR098283*

SRR098283.fastq  SRR098283.fastq_trim.fastq

The output file is also a FASTQ file. It should be smaller than our input file because we’ve removed reads. We can confirm this:

$ ls SRR098283* -l -h

-rw-r--r-- 1 dcuser dcuser 3.9G Jul 30  2015 SRR098283.fastq
-rw-rw-r-- 1 dcuser dcuser 3.0G Nov  7 23:10 SRR098283.fastq_trim.fastq

We’ve just successfully run Trimmomatic on one of our FASTQ files! However, there is some bad news. Trimmomatic can only operate on one sample at a time and we have more than one sample. The good news is that we can use a for loop to iterate through our sample files quickly!

$ for infile in *.fastq
> do
> outfile="${infile}"_trim.fastq
> java -jar ~/Trimmomatic-0.32/trimmomatic-0.32.jar SE "${infile}" "${outfile}" SLIDINGWINDOW:4:20 MINLEN:20
> done

The new part in our for loop is the line:

> outfile="${infile}"_trim.fastq

infile is the first variable in our loop and takes the value of each of the FASTQ files in our directory. outfile is the second variable in our loop and is defined by adding _trim.fastq to the end of our input file name. Use {} to wrap the variable so that _trim.fastq will not be interpreted as part of the variable name. In addition, quoting the shell variables is a good practice AND necessary if your variables have spaces in them. For more, check Bash Pitfalls. There are no spaces before or after the =.

Go ahead and run the for loop. It should take a few minutes for Trimmomatic to run for each of our six input files. Once it’s done running, take a look at your directory contents.

$ ls

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

If you look very closely, you’ll see that you have three files for the SRR098283 sample. This is because we already had the SRR098283.fastq_trim.fastq file in our directory when we started our for loop (because we had run Trimmomatic on just that one file already). Our for loop included this file in our list of .fastq files and created a new output file named SRR098283.fastq_trim.fastq_trim.fastq, which is the result of running Trimmomatic on our already trimmed file. SRR098283.fastq_trim.fastq and SRR098283.fastq_trim.fastq_trim.fastq should be identical. If you look at your Trimmomatic output in the terminal window, you will see:

TrimmomaticSE: Started with arguments: SRR098283.fastq_trim.fastq SRR098283.fastq_trim.fastq_trim.fastq SLIDINGWINDOW:4:20 MINLEN:20
Automatically using 2 threads
Quality encoding detected as phred33
Input Reads: 17030985 Surviving: 17030985 (100.00%) Dropped: 0 (0.00%)
TrimmomaticSE: Completed successfully

This shows that when we re-trimmed our trimmed file, no new reads were dropped. This is a good thing!

Exercise

Earlier we looked at the first read in our SRR098026.fastq file and saw that it was very poor quality.

$ 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
!!!!!!!!!!!!!!!!#!!!!!!!!!!!!!!!!!!

After filtering out bad reads, what is the first remaining read for this sample? What does its quality look like?

Solution

$ head -n4 SRR098026.fastq_trim.fastq

@SRR098026.342 HWUSI-EAS1599_1:2:1:3:655 length=35
GGATNGGCCTTGTATTTATGATTCTCNGAGTCTGT
+SRR098026.342 HWUSI-EAS1599_1:2:1:3:655 length=35
BB@B!B@AACBBABCCCCBBBBBB@@!B?B<ABB@

Comparing this with our quality scale:

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

We can see that the scores are mostly in the 30+ range. This is pretty good.

We’ve now completed the trimming and filtering steps of our quality control process! Before we move on, let’s move our trimmed FASTQ files to a new subdirectory within our data/ directory. We can also remove our extra, double-trimmed file for the SRR098283 sample.

$ cd ~/dc_workshop/data/untrimmed_fastq
$ mkdir ../trimmed_fastq
$ rm SRR098283.fastq_trim.fastq_trim.fastq
$ mv *_trim* ../trimmed_fastq
$ cd ../trimmed_fastq
$ ls

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

Bonus Exercise (Advanced)

Now that we’ve quality controled our samples, they should perform better on the quality tests run by FastQC. Go ahead and re-run FastQC on your trimmed FASTQ files and visualize the HTML files to see whether your per base sequence quality is higher after trimming.

Solution

In your AWS terminal window do:

$ ~/FastQC/fastqc ~/dc_workshop/data/trimmed_fastq

In a new tab in your terminal do:

$ mkdir ~/Desktop/fastqc_html/trimmed
$ scp dcuser@ec2-34-203-203-131.compute-1.amazonaws.com:~/dc_workshop/data/trimmed_fastq/*.html ~/Desktop/fastqc_html/trimmed
$ open ~/Desktop/fastqc_html/trimmed/*.html

Remember to replace everything between the @ and : in your scp command with your AWS instance number.

Before trimming, one of the sequences gave a warning and another failed the per base sequence quality test. After filtering, all sequences pass that test.

Key Points

• The options you set for the command-line tools you use are important!

• Data cleaning is an essential step in a genomics workflow.