Redirection

Overview

Teaching: 30 min
Exercises: 20 min

Questions

• How can I search within files?

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

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 being aNy nucleotide at that position in the DNA sequence.

We’ll search for strings inside of our fastq files. Let’s first make sure we are in the correct directory.

$ cd ~/dc_workshop/data/untrimmed_fastq
$ ls  

JC1A_R1.fastq   JC1A_R2.fastq     JP4D_R1.fastq     JP4D_R2.fastq

Suppose we want to see how many reads in our file have really bad segments containing 10 consecutive unknown nucleotides (Ns).

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 your 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.

Let’s search for the string NNNNNNNNNN in the JC1A_R2.fastq file.

$ grep NNNNNNNNNN JC1A_R2.fastq

This command returns a lot of output to the terminal. Every single line in the JC1A_R2.fastq file that contains at least 10 consecutive Ns is printed to the terminal, regardless of how long or short the file is. 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. The -A argument returns 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 JC1A_R2.fastq

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

@MISEQ-LAB244-W7:91:000000000-A5C7L:1:2111:5300:24013 2:N:0:TCGAAG
NNNNNNNNNNNCNANNANNNNNCGCCGGTGTTCTNCTGGGGNACGGANACCGAGTAGATCGGAACAGCGTCGTGGAGNGAAAGAGTGTAGATCCCGGTGGGCGGCGTATCATTAAAAAAAAAACCTGCTGGTCCTTGTCTC
+
AAA11BB3333BGG1GGEC1E?0E0B0BFDGFHD2FBH110A1BEE?A/BAFBDGH///>FEGGG><@/#//?#?/#//????########################################################################################################################################################################

Exercise 1(Begginer): Using grep

1. Search for the sequence GATCGAGAGGGGATAGGCG in the JC1A_R2.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 all 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 GATCGAGAGGGGATAGGCG JC1A_R2.fastq
2. grep -B1 AAGTT *.fastq

Redirecting output

grep allowed us to identify sequences in our FASTQ files that match a particular pattern. All of these sequences were printed to our terminal screen, but in order to work with these sequences and perform other operations 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 are taking what would ordinarily be printed to the terminal screen and redirecting it to another location. In our case, we want to print this information to a file so that we can look at it later and use other commands to analyze 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 JC1A_R2.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

  402   489 50076 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

402 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 2(Intermediate): Using wc

How many sequences in JC1A_R2.fastq contain at least 3 consecutive Ns?

Solution

$ grep NNN JC1A_R2.fastq > bad_reads.txt
$ wc -l bad_reads.txt

596 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 JC1A_R1.fastq > bad_reads.txt
$ wc -l bad_reads.txt

24 bad_reads.txt

The old bad_reads.txt that counts bad quality reads from file JC1A_R2.fastq with 402 lines has been erased. Instead a new bad_reads.txt that contain 24 lines from bad reads from JC1A_R1.fastq has been created. 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 JC1A_R2.fastq > bad_reads.txt
$ wc -l bad_reads.txt

402 bad_reads.txt

$ grep -B1 -A2 NNNNNNNNNN JC1A_R1.fastq >> bad_reads.txt
$ wc -l bad_reads.txt

426 bad_reads.txt

The output of our second call to wc shows that we have not overwritten our original data. The final number of 426 lines results from the adition of 402 reads from JC1A_R2.fastq file + 24 reads from JC1A_R1.fastq file. We can also do this for more files with a single line of code by using a wildcard.

$ rm bad_reads.txt

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

427 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 unintended results.

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 JC1A_R2.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.

Writing for loops

Loops are key to productivity improvements through automation as they allow us to execute commands repeatedly. Similar to wildcards and tab completion, using loops also reduces the amount of typing (and typing mistakes). Loops are helpful when performing operations on groups of sequencing files, such as unzipping or trimming multiple files. We will use loops for these purposes in subsequent analyses, but will cover the basics of them for now.

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 shell programming, this is usually called “expanding” the variable.

$ cd ../untrimmed_fastq/

Let’s write a for loop to show us the first two lines of the fastq files we downloaded earlier. You will notice 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.

$ for filename in *.fastq
> do
> head -n 2 ${filename} >> seq_info.txt
> done

The for loop begins with the formula for <variable> in <group to iterate over>. In this case, the word filename is designated as the variable to be used over each iteration. In our case JC1A_R1.fastq and JC1A_R2.fastq will be substituted for filename because they fit the pattern of ending with .fastq in directory we’ve specified. The next line of the for loop is do. The next line is the code that we want to execute. We are telling the loop to print the first two lines of each variable we iterate over and save the information to a file. Finally, the word done ends the loop.

Note that we are using >> to append the text to our seq_info.txt file. If we used >, the seq_info.txt file would be rewritten every time the loop iterates, so it would only have text from the last variable used. Instead, >> adds to the end of the file.

Using Basename in for loops

Basename is a function in UNIX that is helpful for removing a uniform part of a name from a list of files. In this case, we will use basename to remove the .fastq extension from the files that we’ve been working with.

$ basename JC1A_R2.fastq .fastq

We see that this returns just the SRR accession, and no longer has the .fastq file extension on it.

JC1A_R2

If we try the same thing but use .fasta as the file extension instead, nothing happens. This is because basename only works when it exactly matches a string in the file.

$ basename JC1A_R2.fastq .fasta

JC1A_R2.fastq

Basename is really powerful when used in a for loop. It allows to access just the file prefix, which you can use to name things. Let’s try this.

Inside our for loop, we create a new name variable. We call the basename function inside the parenthesis, then give our variable name from the for loop, in this case ${filename}, and finally state that .fastq should be removed from the file name. It’s important to note that we’re not changing the actual files, we’re creating a new variable called name. The line > echo $name will print to the terminal the variable name each time the for loop runs. Because we are iterating over two files, we expect to see two lines of output.

$ for filename in *.fastq
> do
> name=$(basename ${filename} .fastq)
> echo ${name}
> done

Exercise 3(Advanced): Using basename

Print the file prefix of all of the .txt files in our current directory.

Solution

$ for filename in *.txt
> do
> name=$(basename ${filename} .txt)
> echo ${name}
> done

One way this is really useful is to move files. Let’s rename all of our .txt files using mv so that they have the years on them, which will document when we created them.

$ for filename in *.txt
> do
> name=$(basename ${filename} .txt)
> mv ${filename}  ${name}_2019.txt
> done

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.

• for loops are used for iteration

• basename gets rid of repetitive parts of names