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File formats used in bioinformatics

A brief introduction to various file formats used in bioinformatics.

Table of contents

Sequence formats

These are file formats for storing nucleotide sequences and/or amino acid (protein) sequences.

FASTA

FASTA is a ubiquitous text-based format for representing nucleotide sequences or amino acid sequences. A FASTA file can contain one sequence or multiple sequences. If a FASTA file contains multiple sequences, it may sometimes be referred to as a “multi-FASTA” file.

Each FASTA entry begins with a > (greater-than) symbol, followed by a comment on the same line describing the sequence that will follow. The actual sequence begins on the line after this comment. Another > (greater-than) symbol denotes the beginning of another FASTA entry (comment describing the sequence + the sequence itself).

Example FASTA file

This example FASTA file contains two linear nucleotide sequences.

>gi|1817694395|ref|NZ_JAAGMU010000151.1| Streptomyces sp. SID7958 contig-52000002, whole genome shotgun sequence
CCGGCTGGCGCGGCTGGCGCTGGCGGTGGGGCTGCGGCTGCTGGAGCTGGGGGTGGCGCTGGAGGCGCAC
GGCCAGAACCTGCTGGTGGTGCTGTCGCCGTCCGGGGAGCCGCGGCGGCTGGTCTACCGCGATCTGGCGG
ACATCCGGGTCTCCCCCGCGCGGCTGGCCCGGCACGGTATCCGGGTTCCGGACCTGCCGGCG

>gi|1643051563|gb|SZWM01000399.1| Citrobacter sp. TBCS-14 contig3128, whole genome shotgun sequence
GCACAGTGAGATCAGCATTCCGTTGGATCTACTGGTCAATCAAAACCTGACGCTGGGTACTGAATGGAAC
CAGCAGCGCATGAAGGACATGCTGTCTAACTCGCAGACCTTTATGGGCGGTAATATTCCAGGCTACAGCA
GCACCGATCGCAGCCCATATTCGAAAGCCGAGATCTTCTCTTTGTTTGCCGAAAACAACATG

FASTA filename extensions

FASTA files usually end with the extension .fasta. This extension is arbitrary, as the content of the file determines its format, not its extension. More descriptive filename extensions can be used instead of .fasta, which are useful as they describe the type of sequence(s) in the file at a glance.

Here are some examples…

  • .fna can be used for FASTA nucleic acids
  • .faa can be used for FASTA amino acids
  • .frn can be used for FASTA non-coding RNA

FASTQ

The FASTQ format is an extension of FASTA that stores both biological sequences (usually nucleotide sequences) and their corresponding quality scores. Both the sequence letter and quality score are encoded with a single character for brevity.

A FASTQ file normally uses four lines per sequence:

  1. A line beginning with @ followed by a sequence identifier and optional description (like the comment line at in a FASTA file)
  2. The raw sequence letters
  3. A line beginning with +, sometimes followed by the same comment as the first line
  4. A line encoding the quality values for the sequence in line 2, with the same numbers of symbols as letters in the sequence

Example FASTQ file 

@SRR8933535.1 1 length=75
NAGGAAACAAAGGCTTACCCGTTATCATTTCCGCAAGAATGCACCCACACGACCATATATCAATGGATGTGGAGT
+SRR8933535.1 1 length=75
#AAAAEEEEEEEEEEEEEEEEEEEEEAEEEEEAEEEEEEEAEAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAE
@SRR8933535.2 2 length=75
NGAGGAGTGGTGGTAGTGTTGCTTGGTGGCAAAGATGTAGTTGGTGGGAAAGCTGAAGTGGTACCGTTGGTTGGA
+SRR8933535.2 2 length=75
#AAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAEEEE

Alignment formats

These are formats for storing alignments of nucleotide or amino acid sequences. The alignment formats discussed here can be manipulated using SAMtools.

SAM

Sequence Alignment/Map (SAM) is a text-based alignment format that supports single- and paired-end reads produced by different sequencing platforms. It can support short and long reads (up to 128Mbp). The format has been extended to include unmapped sequences, and it may contain other data such as base-call & alignment qualities.

The SAM format consists of a header and an alignment section. Headings begin with the @ symbol, which distinguishes them from the alignment section. All lines in a SAM file are tab-delimited. Alignment sections contain 11 mandatory fields, with other fields being optional. Although the mandatory fields must be present, their value can be a * or a 0 depending on the field. Optional fields are presented as key-value pairs in the format TAG:TYPE:VALUE.

The mandatory fields in a SAM file are…

  1. QNAME Query template name. Reads/segments with the same QNAME are from the same template. A read may occupy multiple alignment lines when its alignment is chimeric or when multiple mappings are given.
  2. FLAG Bitwise flag (pairing, strand, mate strand, etc.).
  3. RNAME Reference sequence name.
  4. POS 1-based leftmost mapping position. The first base in a reference sequence has coordinate 1. POS is set to 0 for an unmapped read.
  5. MAPQ Mapping quality. If equal to 255, the mapping quality is not available.
  6. CIGAR Extended Consise Idiosyncratic Gapped Alignment Report string. M for match/mismatch, I for insertion and D for deletion compared with the reference, N for skipped bases on the reference, S for soft clipping, H for hard clipping, and P for padding.
  7. RNEXT Reference name of the mate/next read in the template. For the last read, the next read is the first read in the template.
  8. PNEXT Position of the primary alignment of the mate/next read.
  9. TLEN Observed template length.
  10. SEQ Segment sequence.
  11. QUAL Phred-based base quality (same as the quailty string in FASTQ) plus 33.

SAM files can be manipulated using SAMtools.

BAM

Binary Alignment Map (BAM) is a binary representation of SAM, containing the same information in binary format for improved performance. A position-sorted BAM file can be indexed to allow random access.

CRAM

CRAM is a sequencing read file format that is highly space efficient by using reference-based compression of sequence data and offers both lossless and lossy modes of compression. CRAM files are typically 30 to 60% smaller than their BAM equivalents. CRAM has the following major objectives:

  1. Significantly better lossless compression than BAM
  2. Full compatibility with BAM
  3. Effortless transition to CRAM from using BAM files
  4. Support for controlled loss of BAM data

Stockholm format

The Stockholm format is a system for marking up features in a multiple alignment, used by HMMER, Pfam, and Rfam. The first line of a Stockholm file (.sto, or .stk) states the format and version identifier, currently # STOCKHOLM 1.0. The header is followed by mark-up lines beginning with #. These mark-up lines can annotate features of the alignment file (#=GF, generic per-file annotation), or features of the aligned sequences (#=GS, generic per-sequence annotation). The sequence alignment itself is a series of lines with sequence names (typically in the form name/start-end) followed by a space and the aligned sequence. A line with two forward slashes (//) indicates the end of the alignment.

Example Stockholm file

This is a Stockholm format alignment for Alpha-haemoglobin stabilising protein (AHSP) from Pfam:

# STOCKHOLM 1.0
#=GS G1TJ87_RABIT/6-91   AC G1TJ87.1
#=GS H0XCX0_OTOGA/5-91   AC H0XCX0.1
#=GS F6RTV5_HORSE/5-91   AC F6RTV5.2
#=GS AHSP_BOVIN/5-91     AC Q865F8.1
#=GS AHSP_MOUSE/5-91     AC Q9CY02.1
#=GS G3IJS0_CRIGR/5-91   AC G3IJS0.1
#=GS L9KTP8_TUPCH/5-91   AC L9KTP8.1
#=GS G5BYB6_HETGA/5-104  AC G5BYB6.1
#=GS G3T391_LOXAF/5-91   AC G3T391.1
#=GS F7EDV6_ORNAN/6-92   AC F7EDV6.1
#=GS G3WLQ8_SARHA/5-86   AC G3WLQ8.1
#=GS F7DP52_MONDO/4-90   AC F7DP52.1
#=GS H2NS04_PONAB/5-91   AC H2NS04.1
G1TJ87_RABIT/6-91              .TNKDLISMGLKEF....NVLLNQ.........QVFSDPL.LSQEAMQTVLDDWVNLYVNYYRQQMTGEQQELDKALEELRLELNGLAKPFLNKYSVFLKS
H0XCX0_OTOGA/5-91              QANEDLISAGVKEF....NILLNQ.........QVFNEPF.VSEEAMETVVNDWVNFYMNYYKKQMTGEQGEQEKALQELKQKLNSLANPFLAKYRAFLKS
F6RTV5_HORSE/5-91              QANRDLISTAIKEF....NVLLNQ.........QVFSDPP.VSEEAMVTVVNDWVNFYINYYRRQVVGEQQEKDRALQELRQELNILSAPFLAKYRAFLKS
AHSP_BOVIN/5-91                QTNKDLISKGIKEF....NILLNQ.........QVFSDPA.ISEEAMVTVVNDWVSFYINYYKKQLSGEQDEQDKALQEFRQELNTLSASFLDKYRNFLKS
AHSP_MOUSE/5-91                QSNKDLISTGIKEF....NVLLDQ.........QVFDDPL.ISEEDMVIVVHDWVNLYTNYYKKLVHGEQEEQDRAMTEFQQELSTLGSQFLAKYRTFLKS
G3IJS0_CRIGR/5-91              QTNKELISEGIKQF....NVLLGQ.........QVFDDPL.IPEENMVTVVNDWVNLYINYYKPLVFGKQQEQDKALQELQQELNTLGSQFLTKYRTILKS
L9KTP8_TUPCH/5-91              QVNKDIIATGMKKF....SVLLDQ.........QVFSEPP.ISEEAMVVVVNDWVNFYVNYYGQQVTGEQQEQDRALNELRQELTTMASPFLAKYRAFLKS
G5BYB6_HETGA/5-104             QANKDLIALGMKEFPADYSDMLESHSLSPASHPQVFNYPL.ITEEDMVVVVDDWVNIYINYYRKRLTGEKQDQDRALQELRQELKTLASPFLAKYRACLES
G3T391_LOXAF/5-91              QANKDLISTGMKEF....SILLNQ.........QDMRDNP.IPEEAMVIVVNDWMSFYINYYRQKMTGEQQEQDRALQELQQGLNTLANPFLTKYRDFLKT
F7EDV6_ORNAN/6-92              .SNQDVINSAMAAF....QALLNQ.........QVFSPQIPIPMEAMKIIVRDWIEFYISYFAPKLRGDRQERERAQEDLWETLQAIARPFLDKYRDFLNA
G3WLQ8_SARHA/5-86              QSNQDVISSAMQEF....SKLLDQ.........QEFTKPA.FSETDMVTIVDDWIKFYLSYYSKKMTGNEQEQERAMQKLQEELRTSASPFLDKSQ.....
F7DP52_MONDO/4-90              QSNQDVISSAMQEF....NKLLNQ.........QDFTYAV.ISEKDMVTIVDDWMNYYLSFFSQKMSGDQQEQERAMQKLQEELRSSANPFLDKYRAFLKS
H2NS04_PONAB/5-91              KANKDLISAGLKEF....SVLLNQ.........QVFNDPL.ISEEDMVTVVEDWMNFYINYYRQQVTGEPQERDKALQELRQELNTLANPFLAKYRDFLKS
#=GC seq_cons                  QuNKDLISsGhKEF....slLLNQ.........QVFs-Ph.ISEEsMVTVVsDWVNFYlNYY+pploGEQQEQDRALQELpQELsTLAsPFLsKYRsFLKS
//

VCF

Variant Call Format (VCF) is a format for storing variations between a reference genome and sequences aligned to it, based on SAM/BAM alignments. VCF files begin with a header section: lines in the header section begin with ##. The last line in the header section begins with #; this line gives the headers of the columns used in the VCF file:

  1. CHROM The name of the sequence (typically a chromosome) on which the variation is being called. This sequence is usually known as ‘the reference sequence’, i.e. the sequence against which the given sample varies.
  2. POS The 1-based position of the variation on the given sequence.
  3. ID The identifier of the variation, e.g. a dbSNP rs identifier, or if unknown a “.”. Multiple identifiers should be separated by semi-colons without white-space.
  4. REF The reference base (or bases in the case of an indel) at the given position on the given reference sequence.
  5. ALT The list of alternative alleles at this position.
  6. QUAL A quality score associated with the inference of the given alleles.
  7. FILTER A flag indicating which of a given set of filters the variation has passed.
  8. INFO An extensible list of key-value pairs (fields) describing the variation. Multiple fields are separated by semicolons with optional values in the format: =[,data].
  9. FORMAT An (optional) extensible list of fields for describing the samples.
  10. SAMPLEs For each (optional) sample described in the file, values are given for the fields listed in FORMAT. If multiple samples have been aligned to the reference sequence, each sample will have its own column.

BCF

Binary Call Format (BCF) is a binary representation of VCF, containing the same information in binary format for improved performance.

Generic Feature Formats

The Generic Feature Formats (.gff) are tab-delimited text file formats used for describing genes and other features of DNA, RNA and protein sequences. GFF files are used to annotate genomes, as they describe functional regions of genomes.

There are two widely used versions of the GFF file format:

  1. Gene Transfer Format, a variation of GFF version 2.
  2. Generic Feature Format version 3 (GFF3).

GFF general structure

All GFF formats (GFF2, GFF3 and GTF) are tab delimited with 9 fields per line. They all share the same structure for the first 7 fields, while differing in the content and format of the ninth field. The general structure is as follows:

  1. sequence The name of the sequence where the feature is located.
  2. source Keyword identifying the source of the feature, like a program (e.g. Augustus) or an organization (e.g. SGD).
  3. feature The feature type name, like gene or exon. In a well-structured GFF file, all the children features always follow their parents in a single block (so all exons of a transcript are put after their parent transcript feature line and before any other parent transcript line).
  4. start Genomic start of the feature, with a 1-base offset.
  5. end Genomic end of the feature, with a 1-base offset.
  6. score Numeric value that generally indicates the confidence of the source in the annotated feature. A value of . (a dot) is used to define a null value.
  7. strand Single character that indicates the strand of the feature; it can assume the values of + (positive, or 5'->3'), -, (negative, or 3'->5'), . (undetermined).
  8. phase Phase of coding sequence (CDS) features, indicating where the feature starts in relation to the reading frame. It can be either one of 0, 1, 2 (for CDS features) or . (for everything else).
  9. attributes All the other information pertaining to this feature. The format, structure and content of this field is the one which varies the most between GFF formats.

GTF

GTF files (.gtf) have the following tab-delimited fields (see also GFF general structure):

  1. seqname The name of the sequence. Commonly, this is the chromosome ID or contig ID.
  2. source
  3. feature The following feature types are required: CDS, start_codon, stop_codon. The features 5UTR, 3UTR, inter, inter_CNS, intron_CNS and exon are optional.
  4. start
  5. end
  6. score
  7. strand
  8. frame Similar to phase in the GFF general structure.
  9. attributes All nine features have the same two mandatory attributes at the end of the record: gene_id "value" and transcript_id "value". These are globally unique identifiers for the genomic locus of the transcript and the predicted transcript, respectively. If empty, no gene/transcript is associated with this feature. Attributes are separated by ; (semi-colon).

GTF files also support comments, beginning with # and running until the end of the line. Nothing beyond a hash will be parsed. These may occur anywhere in the file, including at the end of a feature line.

Example GTF file

Here are the first 12 lines of example_genome_annotation.gtf:

#gtf-version 2.2
#!genome-build R64
#!genome-build-accession NCBI_Assembly:GCF_000146045.2
#!annotation-source SGD R64-2-1
NC_001133.9	RefSeq	gene	1807	2169	.	-	.	gene_id "YAL068C"; db_xref "GeneID:851229"; gbkey "Gene"; gene "PAU8"; gene_biotype "protein_coding"; locus_tag "YAL068C"; partial "true"; 
NC_001133.9	RefSeq	exon	1807	2169	.	-	.	gene_id "YAL068C"; transcript_id "NM_001180043.1"; db_xref "GeneID:851229"; gbkey "mRNA"; gene "PAU8"; locus_tag "YAL068C"; partial "true"; product "seripauperin PAU8"; exon_number "1"; 
NC_001133.9	RefSeq	CDS	1810	2169	.	-	0	gene_id "YAL068C"; transcript_id "NM_001180043.1"; db_xref "SGD:S000002142"; db_xref "GeneID:851229"; experiment "EXISTENCE:mutant phenotype:GO:0030437 ascospore formation [PMID:12586695]"; gbkey "CDS"; gene "PAU8"; locus_tag "YAL068C"; note "hypothetical protein; member of the seripauperin multigene family encoded mainly in subtelomeric regions"; product "seripauperin PAU8"; protein_id "NP_009332.1"; exon_number "1"; 
NC_001133.9	RefSeq	start_codon	2167	2169	.	-	0	gene_id "YAL068C"; transcript_id "NM_001180043.1"; db_xref "SGD:S000002142"; db_xref "GeneID:851229"; experiment "EXISTENCE:mutant phenotype:GO:0030437 ascospore formation [PMID:12586695]"; gbkey "CDS"; gene "PAU8"; locus_tag "YAL068C"; note "hypothetical protein; member of the seripauperin multigene family encoded mainly in subtelomeric regions"; product "seripauperin PAU8"; protein_id "NP_009332.1"; exon_number "1"; 
NC_001133.9	RefSeq	stop_codon	1807	1809	.	-	0	gene_id "YAL068C"; transcript_id "NM_001180043.1"; db_xref "SGD:S000002142"; db_xref "GeneID:851229"; experiment "EXISTENCE:mutant phenotype:GO:0030437 ascospore formation [PMID:12586695]"; gbkey "CDS"; gene "PAU8"; locus_tag "YAL068C"; note "hypothetical protein; member of the seripauperin multigene family encoded mainly in subtelomeric regions"; product "seripauperin PAU8"; protein_id "NP_009332.1"; exon_number "1"; 
NC_001133.9	RefSeq	gene	2480	2707	.	+	.	gene_id "YAL067W-A"; db_xref "GeneID:1466426"; gbkey "Gene"; gene_biotype "protein_coding"; locus_tag "YAL067W-A"; partial "true"; 
NC_001133.9	RefSeq	exon	2480	2707	.	+	.	gene_id "YAL067W-A"; transcript_id "NM_001184582.1"; db_xref "GeneID:1466426"; gbkey "mRNA"; locus_tag "YAL067W-A"; partial "true"; product "uncharacterized protein"; exon_number "1"; 
NC_001133.9	RefSeq	CDS	2480	2704	.	+	0	gene_id "YAL067W-A"; transcript_id "NM_001184582.1"; db_xref "SGD:S000028593"; db_xref "GeneID:1466426"; gbkey "CDS"; locus_tag "YAL067W-A"; note "hypothetical protein; identified by gene-trapping, microarray-based expression analysis, and genome-wide homology searching"; product "uncharacterized protein"; protein_id "NP_878038.1"; exon_number "1";

GFF3

GFF3 files (.gff3 or .gff) have the same tab-delimited fields as GTF, with the following differences:

  1. Column/field 3 is referred to as type instead of feature.
  2. In column/field 9, feature attributes are in the format tag=value, as opposed to tag "value". Multiple tag=value pairs are still separated by semicolons.
  3. All attributes that begin with an uppercase letter are reserved for later use. Attributes that begin with a lowercase letter can be used freely by applications.
  4. The attribute Parent indicates the parent of a feature. A parent ID can be used to group exons into transcripts, transcripts into genes, an so forth. A feature may have multiple parents. Parent can only be used to indicate a “part of” relationship (i.e. that a feature is a smaller part of a larger feature).

Example GFF3 file

From the specification page for GFF3:

##gff-version 3.2.1
##sequence-region ctg123 1 1497228
ctg123 . gene            1000  9000  .  +  .  ID=gene00001;Name=EDEN
ctg123 . TF_binding_site 1000  1012  .  +  .  ID=tfbs00001;Parent=gene00001
ctg123 . mRNA            1050  9000  .  +  .  ID=mRNA00001;Parent=gene00001;Name=EDEN.1
ctg123 . mRNA            1050  9000  .  +  .  ID=mRNA00002;Parent=gene00001;Name=EDEN.2
ctg123 . mRNA            1300  9000  .  +  .  ID=mRNA00003;Parent=gene00001;Name=EDEN.3
ctg123 . exon            1300  1500  .  +  .  ID=exon00001;Parent=mRNA00003
ctg123 . exon            1050  1500  .  +  .  ID=exon00002;Parent=mRNA00001,mRNA00002
ctg123 . exon            3000  3902  .  +  .  ID=exon00003;Parent=mRNA00001,mRNA00003
ctg123 . exon            5000  5500  .  +  .  ID=exon00004;Parent=mRNA00001,mRNA00002,mRNA00003
ctg123 . exon            7000  9000  .  +  .  ID=exon00005;Parent=mRNA00001,mRNA00002,mRNA00003
ctg123 . CDS             1201  1500  .  +  0  ID=cds00001;Parent=mRNA00001;Name=edenprotein.1

References