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Geo Pertea edited this page Jul 25, 2026 · 2 revisions

The GffRead Utility

The program gffread can be used to validate, filter, convert and perform various other operations on GFF files (use gffread -h to see the various usage options). Because the program shares the same GFF parser code with Cufflinks, Stringtie, and gffcompare, it could be used to verify that a GFF file from a certain annotation source is correctly "understood" by these programs. Thus the gffread utility can be used to simply read the transcripts from the file, and optionally print these transcripts back, in either GFF3 (default) or GTF2 format (with the -T option), while discarding any non-essential attributes, optionally fixing some potential issues with the input file(s). The command line for such a quick cleanup and a quick visual inspection of a given GFF file could be:

gffread -E annotation.gtf -o- | less

This will show the minimalist GFF3 re-formatting of the transcript records found in the input file (annotation.gtf in this example) which could be given in either GFF3 or GTF2 format. The -E option directs gffread to "expose" (display warnings about) any potential issues encountered while parsing the input file.

In order to see the GTF2 version of the same transcripts, the -T option should be added:

gffread -E annotation.gff -T -o- | more

The examples above also show that gffread can be used to convert a file between GTF2 and GFF3 file formats.

Extracting transcript sequences

gffread can also be used to generate a FASTA file with the DNA sequences for all transcripts in a GFF file. For this operation a fasta file with the genomic sequences have to be provided as well. For example, one might want to extract the sequence of all transfrags (defined as transcripts or transcript fragments that result from the assembly process) assembled from a StringTie or Cufflinks assembly session. This can be accomplished with a command line like this:

gffread -w transcripts.fa -g /path/to/genome.fa transcripts.gtf

The file genome.fa in this example would be a multi-fasta file with the genomic sequences of the target genome. This also requires that every contig or chromosome name found in the 1st column of the input GFF file (transcript.gtf in this example) must have a corresponding sequence entry in chromosomes.fa. This should be the case in our example if genome.fa is the file corresponding to the same genome (index) that was used for mapping the reads with Tophat.

Note that the retrieval of the transcript sequences this way is going to be much faster if a fasta index file (genome.fa.fai in this example) is found in the same directory with the genomic fasta file. Such an index file can be created with the samtools utility prior to running gffread, like this:

samtools faidx genome.fa

Then in subsequent runs using the -g option gffread will find this FASTA index and use it to speed up the extraction of transcript sequences.

Obtaining GffRead

Publication

Pertea G and Pertea M. GFF Utilities: GffRead and GffCompare. F1000Research 2020, 9:304 DOI:10.12688/f1000research.23297.1

Contact Information

You can contact us about gffread at: gpertea@jhu.edu

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