FASTA file format

Best practices

The ubiquitous FASTA format is flexible, to a fault. The following best practices will guarantee success in using FASTA files with PacBio software (for example as genome references).

  1. Sequences in FASTA files should be wrapped at a uniform line length, to enable indexing. (A common convention is to wrap lines at 60 characters.) Windows and UNIX line endings are both acceptable.

  2. FASTA files should contain no empty lines (or lines containing only whitespace [1]). FASTA files should contain no unnecessary whitespace (for example, no trailing whitespace on sequence lines).

  3. Sequence contigs in a FASTA file are preceded by identifying header lines. The header, which is the text between the ‘>’ character and the newline, is comprised of:

    • an identifier, which is the first whitespace-delimited token of the header
    • an (optional) comment, which consists of the suffix of the header following the first whitespace. Some APIs may call this string the description or the metadata, but the usage of the comment is completely application-defined.

    Sequence contigs will be identified in the PacBio system and in downstream analysis files using the identifier, so the identifiers contained in a FASTA file must be unique.

  4. Sequences should only contain characters from the following string (IUPAC nucleotide characters, minus ‘-‘ and ‘.’):

  5. Headers should not contain any instances of the ‘>’ character, and identifiers should not begin with an asterisk (‘*’) or contain any of the following characters:

    ',' ':'  '"' (double quote)
  6. PacBio software that imports reference genomes is allowed to outright reject FASTA files that do not meet our requirements; it will never attempt to rewrite/translate a file to fit the requirements.


The following FASTA file would be accepted by the PacBio reference uploader. Downstream files (reports, variant call files) would report the chromosomes as ‘chr1’, ‘chr2’:

>chr1 Jackalope chromosome 1;length=7
>chr2 Jackalope chromosome 2;length=7

The following file would be rejected by the PacBio reference uploader, for violating the identifier uniqueness requirement (in 3, above):

>Jackalope chromosome 1;length=7
>Jackalope chromosome 2;length=7

Implications for bioinformatics tool writers

Since a FASTA presented to a bioinformatics tool may contain characters beyond “GATCN”, tools should use a FASTA reader API capable of normalizing sequence characters. For example, SeqAn uses a Dna5String abstraction to project input sequence characters into “GATCN”; FASTA readers in pbcore will soon offer normalization.

[1]We define a whitespace character as one for which the standard C isspace function returns true (nonzero).