FASTA splitter

How to split FASTA files online

The easiest way to split a FASTA file online is to upload or paste a multi-sequence FASTA file, choose how records should be grouped, and run the splitter. ProteinIQ returns downloadable FASTA files and, by default, a summary file that lists the number of records and residues or bases in each output file.

FASTA splitting is useful when downstream tools have per-job sequence limits, when you want one sequence per structure-prediction job, or when a large sequence collection needs to be divided across parallel searches, alignments, or annotation runs.

For a small input like this:

>seq1 sample A
MKTAYIAKQRQISFVKSHFSRQ
>seq2 sample B
GATCGATCGATCGATC
>seq3 sample C
MVLSPADKTNVKAAWGKVGA

Split by sequence count with Sequences per file set to 2 returns one FASTA file containing seq1 and seq2, and another FASTA file containing seq3.

fasta_part_001.fasta:

>seq1 sample A
MKTAYIAKQRQISFVKSHFSRQ
>seq2 sample B
GATCGATCGATCGATC

fasta_part_002.fasta:

>seq3 sample C
MVLSPADKTNVKAAWGKVGA

With the default Sequences per file value of 1, the same input produces three FASTA files. A single-record input produces one FASTA file in every mode; it is never fragmented.

The three numeric split settings must be whole numbers. Sequences per file and Number of output files range from 1 to 10000; Max residues/bases per file ranges from 1 to 10000000. Only the numeric setting for the selected split mode affects grouping. Runs that would produce more than 10,000 FASTA files are rejected; the summary and run log do not count toward this cap.

How FASTA splitting works

A FASTA file stores each sequence as a record with a header line that begins with >, followed by one or more sequence lines. NCBI GenBank FASTA guidance describes the first line as the FASTA definition line and recommends a unique sequence identifier. NCBI BLAST documentation also describes FASTA as a single-line description followed by sequence data lines.

ProteinIQ reads the FASTA records, validates that every header has sequence content, and splits complete records into output files. It does not cut a single FASTA record into smaller sequence fragments. This matters for downstream tools because record identifiers, annotations, and complete biological sequences stay together.

Blank lines are ignored. Comment lines beginning with # or ;, including comments before the first record, are omitted with a warning. Whitespace inside sequence lines, including tabs and non-breaking spaces, is removed and counted in run.log. Wrapped sequence lines are joined into one line per output record, and outputs use LF line endings with a final newline.

Sequence letters retain their original case. Ambiguous letters and the markers -, ., *, and ? are preserved. Length totals count all retained sequence characters, including those markers; they are not molecular-weight calculations or ungapped lengths. The splitter checks file syntax rather than determining whether a sequence is biologically valid for a particular organism or downstream tool.

Each input must contain its own FASTA headers, every header must contain text after >, and every record must have sequence content. Position numbers, commas, FASTQ quality markers, zero-width spaces and other non-sequence characters are rejected with a line number and a suggested correction. The tool does not guess which characters to delete. Duplicate identifiers are preserved and reported when original headers are retained; unique filenames do not make the identifiers inside the files unique.

All splitting runs in your browser. Sequence data is not sent to a server for this client-side utility.

Which FASTA split mode should I use?

The right split mode depends on the downstream constraint you are trying to satisfy.

ProteinIQ preserves sequence order in all modes. Splitting is deterministic, so the same input and settings produce the same groups and filenames.

FASTA output and filename rules

The result panel returns split FASTA files as a file list. You can download files individually or use the download action to collect all output files together.

Output file extensions match supported uploaded FASTA extensions (.fasta, .fa, .fas, .fna, or .faa). Pasted input and .txt uploads use .fasta.

Numbered filenames use the pattern prefix_part_001.fasta. Sequential filenames use prefix_1.fasta, prefix_2.fasta, and so on. Header-based filenames use a sanitized version of the first sequence header in each output file; duplicate names are de-duplicated with numeric suffixes.

Keep Preserve original headers enabled unless a downstream system needs simple generated identifiers. FASTA headers often carry accessions, sample IDs, organism labels, or coordinate information needed to trace results back to the source sequence.

FASTA splitter alternatives

Command-line tools are better for automated pipelines, compressed files, very large datasets, and scripted reproducibility. SeqKit includes split and split2 commands for FASTA and FASTQ files, including splitting by number of parts and records per part. The SeqKit paper describes it as a cross-platform toolkit for FASTA/Q manipulation with high performance on large sequence files.

Use ProteinIQ's FASTA splitter when you need a browser-local, no-code workflow with upload or paste input, record-preserving output, summary files, visible warnings, and convenient downloads. For changing file formats rather than splitting existing FASTA records, use FASTQ to FASTA for sequencing reads, CSV to FASTA for sequence tables, TXT to FASTA for raw sequence text, GenBank to FASTA for annotated GenBank records, or the FASTA converter for alignments and other sequence formats. To list the records of a FASTA file in a spreadsheet instead of splitting it, use FASTA to CSV.

FAQ

Does FASTA Splitter change my sequences?

It preserves supported sequence characters and their case, while removing whitespace and joining wrapped lines. Comment lines are omitted with a warning. Header text is preserved by default, apart from whitespace at the beginning and end of the header line. If Preserve original headers is turned off, the output uses generated headers such as Sequence_1.

Can I split one long sequence into smaller fragments?

No. FASTA Splitter divides a multi-record FASTA file into smaller files, but it does not fragment an individual record. A sequence longer than the Max residues/bases per file setting is kept as one complete record and reported with a warning.

Why did I get fewer files than requested?

Split into number of files creates non-empty files only. If you request 20 files from 8 sequences, the tool creates 8 files and returns a warning.

What FASTA formats are supported?

The splitter accepts FASTA records for protein, DNA, and RNA sequences. Upload .txt, .fasta, .fa, .fas, .fna, or .faa files, or paste FASTA text. Compressed archives, Word and PDF documents, FASTQ, tables and GenBank records need to be converted to FASTA first.

Are my sequences uploaded to a server?

No. This FASTA splitter runs locally in your browser. The sequence text is processed client-side and the output files are generated from that local result.

Sources

• NCBI GenBank FASTA format
• NCBI BLAST FASTA input documentation
• SeqKit usage documentation
• Shen W, Le S, Li Y, Hu F. "SeqKit: A Cross-Platform and Ultrafast Toolkit for FASTA/Q File Manipulation." PLOS ONE 11(10), e0163962 (2016). doi:10.1371/journal.pone.0163962