How to convert FASTA to FASTQ format

Overview

CZ ID pipelines only accept sequencing files in FASTQ format. If you have sequence data in FASTA format, you have to convert it to FASTQ format prior to upload. Note that FASTQ files include sequence data alongside quality scores. Quality score information is lost in FASTA format, which only includes sequence information. Therefore, you cannot create a legitimate FASTQ file from a FASTA file. If you are confident about read quality (e.g., if you already removed poor quality reads using another program), you can generate a FASTQ file from a FASTA file using dummy quality scores.

Here we list steps to convert sequencing files from FASTA to FASTQ format using Seqtk. If you have questions or need help, contact our team by sending an email to [e-mail]. 

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Convert FASTA to FASTQ Using Seqtk

Seqtk is a tool for processing sequences in FASTQ or FASTA format in Mac and other Linux operating systems (if you have a PC, see Note for Windows OS).

To convert sequence file format:

1. Install Seqtk

Option 1: If you have Homebrew installed on your computer, install Seqtk with the following command:

brew install seqtk

Option 2: Install Seqtk from Git repository using the following commands:

git clone https://github.com/lh3/seqtk.git;
cd seqtk; make

2. Set the directory to the folder where your FASTA sequence files are located.

3. To convert files to FASTQ format, run the following command where "I" is used as the dummy quality score:

seqtk seq -F 'I' input_filename.fa > output_filename.fq

Note: By using "I" as the quality score, you are assigning a quality score (Q-score) of 40 to each nucleotide in sequence reads. See Quality Score Encoding for details about symbols used for different Q-score values. You can assign any Q-score you want, but keep in mind that CZ ID pipelines will remove remove reads with quality scores < 20.

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Note for Windows OS

Seqtk is designed to run on Linux systems. You can install a Windows Subsystem for Linux (WSL) in Windows 10 (version 20 and higher) or Windows 11. The WSL enables you to run a Linux file system, along with Linux command-line tools and GUI applications, directly on Windows. Click here for instructions on how to install the WSL.