Step-by-Step Guide: Convert Multiline FASTA to Single-Line FASTA

Introduction

FASTQ and FASTA are standard formats in bioinformatics. Sometimes, it’s necessary to convert multiline FASTA sequences to a single-line format to meet specific software requirements or simplify manual inspection. Below, you’ll find a beginner-friendly manual with step-by-step instructions and scripts using UNIX commands and Perl for this conversion.

Prerequisites

1. Basic understanding of the FASTA format:
   • A header line starting with > followed by sequence description.
   • Sequence lines spanning multiple lines.
2. Access to a UNIX/Linux shell or Windows Subsystem for Linux (WSL). Alternatively, Perl must be installed on your system.
3. A sample FASTA file, e.g., input.fasta.

Step-by-Step Instructions

Option 1: Using awk (Linux/UNIX Shell)

1. Open a terminal and navigate to the directory containing the FASTA file.
2. Run the following awk command:
   bash

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   awk '/^>/ {if (NR > 1) printf("\n"); printf("%s\n", $0); next;} {printf("%s", $0);} END {printf("\n");}' input.fasta > output.fasta

   • Explanation:
     • ^>: Identifies lines starting with > (headers).
     • if (NR > 1) printf("\n");: Adds a newline before each new header (except the first).
     • printf("%s", $0);: Concatenates sequence lines without a newline.
   • Output: A single-line FASTA file named output.fasta.
3. Verify the output:
   bash

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   cat output.fasta


Option 2: Using perl (One-Liner Script)

1. Open a terminal and navigate to the directory containing the FASTA file.
2. Run the following Perl command:
   bash

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   perl -pe '$. > 1 and /^>/ ? print "\n" : chomp' input.fasta > output.fasta

   • Explanation:
     • $. > 1: Ensures the newline is added only after the first header.
     • /^>/: Detects header lines.
     • chomp: Removes newline characters from sequence lines.
3. Verify the output:
   bash

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   cat output.fasta


Option 3: Using a Dedicated Perl Script

1. Create a new Perl script file, e.g., linearize_fasta.pl.
2. Add the following script content:
   perl

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   #!/usr/bin/perl
   use strict;
   use warnings;

   while (<>) {
if (/^>/) {
print "\n" if $. > 1;
print;
} else {
chomp;
print;
}
}
print "\n";


3. Save the script and make it executable:
   bash

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   chmod +x linearize_fasta.pl

4. Run the script on your input file:
   bash

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   ./linearize_fasta.pl input.fasta > output.fasta


Option 4: Using sed

For quick inline edits using sed:

• Explanation:
  • Combines all lines until a header (>) is encountered.

Optional: Quality Check

After converting the FASTA file:

1. Check that each header is followed by a single line of sequence:
   bash

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   grep -v "^>" output.fasta | awk '{if(length($0)>0) print length($0);}' | sort -nu

   • Ensures uniform line lengths.
2. Count headers:
   bash

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   grep -c "^>" output.fasta


Windows Users

1. Use WSL to access a UNIX shell and follow the above steps.
2. Alternatively, install Perl for Windows via Strawberry Perl here.

Conclusion

Converting a multiline FASTA file into a single-line FASTA is simple and can be accomplished using awk, perl, or sed scripts. These approaches are efficient, flexible, and suitable for various operating systems.