## Pipes, process substitution and why should a biologist ever care

This is the first entry in my series about how to keep DNA sequence processing as simple as possible. Each entry attempts to teach a useful Unix trick or two, focusing on relevance for biologists who might not have much prior Unix experience. Because of the very clever design choices made by the folks behind Unix, every trick can be combined with everything else in ways that lead to a much greater flexibility and expressiveness than any of the tricks alone - I attempt to demonstrate that in the following posts. These tricks work on Bash and Zsh, so be sure that your Unix profile is running one of them. 1

A lot of the computational work that a modern genetically inclined biologist has to do is actually just converting data between incompatible formats and other trivial processing tasks - think converting fastq to fasta, trimming off adapter sequences, removing problematic characters from fasta ids etc… A lot of labs have their stash of Perl and Python scribbles to handle these tasks and certainly these scripts do their thing. However, I personally prefer doing as much of this as possible using just the plain Unix command line and avoid writing scripts if possible. The brevity of expression that Unix command line provides for simple text and sequence processing tasks is about unparalleled. Plus the commands are available on any Unix system, right out of the box.

## What’s the deal with pipes?

Unix command line and standard tools were not designed for DNA sequence processing but nevertheless do that really well. The basic Unix commands form a vocabulary of simple text and sequence processing operations and this vocabulary can be combined into sentences using Unix pipes. For a better introduction for pipes than I can provide here, this video tutorial will get you started. Piping is an easy but powerful concept and definitely worth learning. Here are three common commands in the Unix text and sequence processing vocabulary:

• cat prints contents of a file on screen or sends them down a pipe
• rev reverses any line of text
• tr replaces a set of characters with a different set of characters

Let’s put some of these into practice. A simple example would be taking reverse-complements of DNA sequences in file sequences.txt (which you can download here):

sequences.txt

1AAGTGTCGTA
2GGTCGCAAATG
3GAAGTGCCTAG
4TGAAGATGC
A very simple command pipeline to do the task would be

cat sequences.txt | rev | tr AGCT TCGA

This crunches through the file sequences.txt, first reversing each sequence and then converting each into a respective complement, line by line. The output is - not surprisingly - a reverse-complement of each sequence:

1TACGACACTT
2CATTTGCGACC
3CTAGGCACTTC
4GCATCTTCA

A trick I use all the time is a minor variant of this: during primer design I reverse-complement sequences a lot and want to do that with a minimal typing effort. In that case I won’t even bother to create a file; rather I just send a text string to rev and tr using command echo and the following pipeline. 2

echo TGGTAGTGTAACGCCTACA | rev | tr AGCT TCGA

Pipes can be used to join pretty much any Unix commands together. After all my years with Unix, I’m still discovering new useful ways of piping stuff together.

## When are pipes not enough?

Pipes are great but not always enough. A good example is a more realistic case of reverse-complementing sequences: in real world DNA sequences are typically not provided as simple lists. Rather, a typical format would be fasta where the sequence ids are interspersed by the DNA sequences, as shown in the example below:

example.fasta

1>Sequence_id_1
2AATTGATAGGTCCGTT
3>Sequence_id_2
4TTGTCGTTATATGGTTAC
5>Sequence_id_3
6GTTGAGTCGTGGTCGT

With the kind of command pipeline we used above, the sequence ids would also get reversed and their letter As translated into Ts etc… How could we get around this? I first read about process substitution in this excellent blog entry. Don’t be put off by a scary name - the concept is about as simple as Unix pipes and about as useful. Where piping directs the output of one command to the input of next one, process substitution directs outputs of multiple commands to the input of next one. The figure below attempts to illuminate the concepts and their difference.

Let me demonstrate this using an example. To reverse-complement every sequence in a multiple fasta file and leave the sequence ids untouched, one has to separate the id and sequence parts. For this we also have to introduce two new words into our text processing vocabulary:

• paste joins together two lines of text
• grep finds lines containing (or excluding) a particular text string or character
paste -d "\n" <(grep ">" sequence.fasta) <(grep -v ">" sequence.fasta | rev | tr ATCG TAGC)

That’s it3 - one line of text and quite readable if you get used to the process substitution notation. Let’s open this up a little: after the paste command there are two text strings enclosed inside a <( ... ) -structure. This structure is used to create a stream of text. Since we’re having two of those structures after paste, we’re directing two separate text streams to the paste command. Paste takes these text streams and intersperses their lines. The first text stream is the fasta headers - no processing is taking place for those.

grep ">" sequence.fasta

The second text stream is the sequence parts of the fasta file (filtered by grep using the -v switch) that are, before streaming out to paste, streamed through rev and tr to take a reverse-complement of each sequence.

grep -v ">" sequence.fasta | rev | tr AGCT TCGA

The -d "\n" switch instructs paste to use a newline character as a separator for the text streams. See for yourself what happens if you leave that out! The output of our command pipeline currently gets printed on screen where we can inspect it and see that it’s good. However, we typically want to save the output on disk for subsequent use. There’s a very easy way to redirect the text output from screen to a file - that is the redirection operator >. Just append your command pipeline with character > and the file name, and there you have it. The example below saves the reverse-complemented sequences into a new file called reverse_complemented_sequences.fasta.

paste -d "\n" <(grep ">" sequence.fasta) <(grep -v ">" sequence.fasta | rev | tr ATCG TAGC) > reverse_complemented_sequences.fasta

In this example we only modified the sequence part by taking a reverse-complement. Basically one could do many other modifications to both the sequence and id parts, or convert the file into some entirely different format. There are other standard Unix tools that provide more neat text manipulation tricks and what I find especially useful is a little programming language called Awk. But that’s another story!

1. If you’re not sure which shell you’re running, just run chsh -s /bin/bash on your prompt. That will enable bash for you. You don’t have to worry too much about what this means - Bash is a good general choice for a lot of purposes. [return]
2. Writing this out every single time is actually not lazy enough for me - I will cover lazy stuff like aliases and text editor tricks in future posts. [return]
3. Things get more complicated if the sequence part of the fasta file spans multiple lines, as it often does. The way to deal with that is a bit more involved and will be a topic of a future post.

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