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Data Integrity 101 | Verifying Your Files with MD5 Checksums

Why MD5 checksums matter in bioinformatics and how to verify your sequencing data hasn't been corrupted.

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Understanding MD5 Checksums

What is MD5?

MD5 (Message Digest Algorithm 5) is a cryptographic hash function developed by Ronald Rivest in 1991. It takes an input file of any size and produces a fixed 128-bit hash value (typically represented as a 32-character hexadecimal string). Think of it as a unique “fingerprint” for your file.

Why MD5 Matters in Bioinformatics

In bioinformatics, we often work with large sequencing files that are transferred between servers, downloaded from repositories, or stored long-term. Even a single corrupted byte can invalidate your entire analysis. MD5 checksums allow you to verify that your files remain intact and unchanged during these transfers.

How MD5 Verification Works

When a file is created or shared, an MD5 checksum is calculated and stored in an .md5 file. Later, you can recalculate the checksum and compare it to the original—if they match, your file is intact.

Verifying Your Files

Your MD5 file should look something like this:

d41d8cd98f00b204e9800998ecf8427e  file_name.bam

The first part is the MD5 hash, followed by the filename.

Checking Multiple Files

If you have multiple files with their corresponding MD5 files, you can verify them all at once using this simple loop:

for f in *.md5; do
    echo "Checking $f"
    md5sum -c "$f"
done

This will iterate through all .md5 files in your directory and report whether each file passes or fails verification.