Redirection

Overview

Teaching: 15 min
Exercises: 15 min

Questions

• How can I search within files?

• How can I combine existing commands to do new things?

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

Objectives

• Employ the grep command to search for information within files.

• Print the results of a command to a file.

• Construct command pipelines with two or more stages.

Searching files

We discussed in a previous episode how to search within a file using less. We can also search within files without even opening them, using grep. grep is a command-line utility for searching plain-text files for lines matching a specific set of characters (sometimes called a string) or a particular pattern (which can be specified using something called regular expressions). We’re not going to work with regular expressions in this lesson, and are instead going to specify the strings we are searching for. Let’s give it a try!

Nucleotide abbreviations

The four nucleotides that appear in DNA are abbreviated A, C, T and G. Unknown nucleotides are represented with the letter N. An N appearing in a sequencing file represents a position where the sequencing machine was not able to confidently determine the nucleotide in that position. You can think of an N as a NULL value within a DNA sequence.

Suppose we want to see how many reads in our file have really bad segments containing 10 consecutive unknown nucleoties (Ns). Let’s search for the string NNNNNNNNNN in the SRR098026 file.

Determining quality

In this lesson, we’re going to be manually searching for strings of Ns within our sequence results to illustrate some principles of file searching. It can be really useful to do this type of searching to get a feel for the quality of your sequencing results, however, in research you will most likely use a bioinformatics tool that has a built-in program for filtering out low-quality reads. You’ll learn how to use one such tool in a later lesson.

$ grep NNNNNNNNNN SRR098026.fastq

This command returns a lot of output to the terminal. Each line in the SRR098026 file which contains at least 10 consecutive Ns is printed to the terminal. We may be interested not only in the actual sequence which contains this string, but in the name (or identifier) of that sequence. We discussed in a previous lesson that the identifier line immediately precedes the nucleotide sequence for each read in a FASTQ file. We may also want to inspect the quality scores associated with each of these reads. To get all of this information, we will return the line immediately before each match and the two lines immediately after each match.

We can use the -B argument for grep to return a specific number of lines before each match and the -A argument to return a specific number of lines after each matching line. Here we want the line before and the two lines after each matching line so we add -B1 -A2 to our grep command.

$ grep -B1 -A2 NNNNNNNNNN SRR098026.fastq

One of the sets of lines returned by this command is:

@SRR098026.177 HWUSI-EAS1599_1:2:1:1:2025 length=35
CNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
+SRR098026.177 HWUSI-EAS1599_1:2:1:1:2025 length=35
#!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

Exercise

1) Search for the sequence GNATNACCACTTCC in the SRR098026.fastq file. Have your search return all matching lines and the name (or identifier) for each sequence that contains a match.

2) Search for the sequence AAGTT in both FASTQ files. Have your search return all matching lines and the name (or identifier) for each sequence that contains a match.

Solution

1) grep -B1 GNATNACCACTTCC SRR098026.fastq
2) grep -B1 AAGTT *.fastq

Redirecting output

grep allowed us to identify sequences in our FASTQ files that match a particular pattern. But all of these sequences were printed to our terminal screen. In order to work with these sequences and perform other opperations on them, we will need to capture that output in some way.

We can do this with something called “redirection”. The idea is that we’re redirecting what was output to the terminal to another location. In our case, we want to print this information to a file, so that we can look at it later and do other analyses with this data.

The command for redirecting output to a file is >.

Let’s try out this command and copy all the records (including all four lines of each record) in our FASTQ files that contain ‘NNNNNNNNNN’ to another file called ‘bad_reads.txt’.

$ grep -B1 -A2 NNNNNNNNNN SRR098026.fastq > bad_reads.txt

File extensions

You might be confused about why we’re naming our output file with a .txt extension. After all, it will be holding FASTQ formatted data that we’re extracting from our FASTQ files. Won’t it also be a FASTQ file? The answer is, yes - it will be a FASTQ file and it would make sense to name it with a .fastq extension. However, using a .fastq extension will lead us to problems when we move to using wildcards later in this episode. We’ll point out where this becomes important. For now, it’s good that you’re thinking about file extensions!

The prompt should sit there a little bit, and then it should look like nothing happened. But type ls. You should see a new file called bad_reads.txt.

We can check the number of lines in our new file using a command called wc. wc stands for word count. This command counts the number of words, lines, and characters in a file.

$ wc bad_reads.txt

  537  1073 23217 bad_reads.txt

This will tell us the number of lines, words and characters in the file. If we want only the number of lines, we can use the -l flag for lines.

$ wc -l bad_reads.txt

537 bad_reads.txt

Because we asked grep for all four lines of each FASTQ record, we need to divide the output by four to get the number of sequences that match our search pattern.

Exercise

How many sequences in SRR098026.fastq contain at least 3 consecutive Ns?

Solution

$ grep NNN SRR098026.fastq > bad_reads.txt
$ wc -l bad_reads.txt

249 bad_reads.txt

We might want to search multiple FASTQ files for sequences that match our search pattern. However, we need to be careful, because each time we use the > command to redirect output to a file, the new output will replace the output that was already present in the file. This is called “overwriting” and, just like you don’t want to overwrite your video recording of your kid’s first birthday party, you also want to avoid overwriting your data files.

$ grep -B1 -A2 NNNNNNNNNN SRR098026.fastq > bad_reads.txt
$ wc -l bad_reads.txt

537 bad_reads.txt

$ grep -B1 -A2 NNNNNNNNNN SRR097977.fastq > bad_reads.txt
$ wc -l bad_reads.txt

0 bad_reads.txt

Here, the output of our second call to wc shows that we no longer have any lines in our bad_reads.txt file. This is because the second file we searched (SRR097977.fastq) does not contain any lines that match our search sequence. So our file was overwritten and is now empty.

We can avoid overwriting our files by using the command >>. >> is known as the “append redirect” and will append new output to the end of a file, rather than overwriting it.

$ grep -B1 -A2 NNNNNNNNNN SRR098026.fastq > bad_reads.txt
$ wc -l bad_reads.txt

537 bad_reads.txt

$ grep -B1 -A2 NNNNNNNNNN SRR097977.fastq >> bad_reads.txt
$ wc -l bad_reads.txt

537 bad_reads.txt

The output of our second call to wc shows that we have not overwritten our original data.

We can also do this with a single line of code by using a wildcard.

$ grep -B1 -A2 NNNNNNNNNN *.fastq > bad_reads.txt
$ wc -l bad_reads.txt

537 bad_reads.txt

File extensions - part 2

This is where we would have trouble if we were naming our output file with a .fastq extension. If we already had a file called bad_reads.fastq (from our previous grep practice) and then ran the command above using a .fastq extension instead of a .txt extension, grep would give us a warning.

grep -B1 -A2 NNNNNNNNNN *.fastq > bad_reads.fastq

grep: input file ‘bad_reads.fastq’ is also the output

grep is letting you know that the output file bad_reads.fastq is also included in your grep call because it matches the *.fastq pattern. Be careful with this as it can lead to some surprising output.

We’ve now created separate files to store the results of our search for reads matching particular criteria. Since we might have multiple different criteria we want to search for, creating a new output file each time has the potential to clutter up our workspace. We also so far haven’t been interested in the actual contents of those files, only in the number of reads that we’ve found. We created the files to store the reads and then counted the lines in the file to see how many reads matched our criteria. There’s a way to do this, however, that doesn’t require us to create these intermediate files - the pipe command (|).

This is probably not a key on your keyboard you use very much, so let’s all take a minute to find that key. What | does is take the output that is scrolling by on the terminal and uses that output as input to another command. When our output was scrolling by, we might have wished we could slow it down and look at it, like we can with less. Well it turns out that we can! We can redirect our output from our grep call through the less command.

$ grep -B1 -A2 NNNNNNNNNN SRR098026.fastq | less

We can now see the output from our grep call within the less interface. We can use the up and down arrows to scroll through the output and use q to exit less.

Redirecting output is often not intuitive, and can take some time to get used to. Once you’re comfortable with redirection, however, you’ll be able to combine any number of commands to do all sorts of exciting things with your data!

None of the command line programs we’ve been learning do anything all that impressive on their own, but when you start chaining them together, you can do some really powerful things very efficiently. Let’s take a few minutes to practice.

Exercise

Now that we know about the pipe (|), write a single command to find the number of reads in the SRR098026.fastq file that contain at least one region of 8 unknown nucleotides in a row. Do this without creating a new file.

Solution

$ grep "NNNNNNNN" SRR098026.fastq | wc -l

 223

Key Points

• grep is a powerful search tool with many options for customization.

• >, >>, and | are different ways of redirecting output.

• command > file redirects a command’s output to a file.

• command >> file redirects a command’s output to a file without overwriting the existing contents of the file.

• command_1 | command_2 redirects the output of the first command as input to the second command.