5. Juzenas, S. et al.
(2017) A comprehensive, cell specific
the evidence of these papers, another
curtain appears behind it. To pull this
second curtain back, we must develop
strategies to isolate cells directly from
tissues. What this successful technology
will be is not yet clear, but without this
information, our understanding of the
roles miRNAs play in cellular differentia-
tion and homeostasis remains incom-
plete. Accurate miRNA expression
estimates are even more important as
we learn about the importance of the
relative abundance of miRNAs to their
targets and sponges [13].
All together, these insights and resources
greatly advance miRNA research.
Acknowledgments
M.K.H. and M.N.M. are supported by grant
1R01HL137811 from the National Institutes of Health.
M.K.H. is also supported by an American Heart Asso-
ciation Grant-in-Aid (17GRNT33670405). M.N.M. is
also supported by the University of Rochester CTSA
award number UL1 TR002001 from the National Cen-
ter for Advancing Translational Sciences of the National
Institutes of Health. B.F. is supported by the South-
Eastern Norway Regional Health Authority (Grant No.
2014041). K.J.P. is supported by NASA-Ames.
1
Department of Pathology, Johns Hopkins University
School of Medicine, Baltimore, MD 21205, USA
2
Department of Tumor Biology, Institute for Cancer
Research, The Norwegian Radium Hospital, Oslo
University Hospital, N-0424 Oslo, Norway
3
Department of Biological Sciences, Dartmouth College,
Hanover, NH 03755, USA
4 three definitions are in use. We
Department of Biostatistics and Computational Biology,
University of Rochester Medical Center, Rochester, NY
14642, USA
@
Twitter: @Marc_Halushka
*Correspondence: mhalush1@jhmi.edu (M.K. Halushka).
URL: http://labs.pathology.jhu.edu/halushka/.
https://doi.org/10.1016/j.tig.2017.12.015
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Forum
The Definition of Open
Reading Frame
Revisited
Patricia Sieber,1
Matthias Platzer,2 and
Stefan Schuster1,*
The term open reading frame
(ORF) is of central importance to
gene finding. Surprisingly, at least
discuss several molecular biologi-
cal and bioinformatics aspects,
and we recommend using the
definition in which an ORF is
bounded by stop codons.
Open reading frame (ORF) is a basic term
in molecular genetics and bioinformatics.
The detection of ORFs is an important
step in finding protein-coding genes in
genomic sequences, including analyses
based on highly fragmented draft (meta)
genome assemblies [1–3]. ORFs can be
detected by simple in silico analysis,
while proving that a sequence is really
protein-coding requires more effort. Sur-
prisingly, in many textbooks not much
attention is spent on defining the term
ORF, apparently taking its meaning for
granted. Moreover, the given definitions
are often not perfectly clear-cut. For
example, the standard textbook Genes
VII by Lewin [4] states on p. 26: ‘A reading
frame that consists exclusively of triplets
representing amino acids is called an
open reading frame or ORF. A sequence
that is translated into protein has a read-
ing frame that starts with a special initia-
tion codon (AUG) and that extends
through a series of triplets representing
amino acids until it ends at one of the
three types of termination codon’. The
first sentence defines an ORF as bounded
by stop codons (stop/stop definition)
whereas the second sentence may be
(mis)understood as beginning with a start
codon (start/stop definition). Currently at
least three definitions are in use, which
differ in the location of the ORF bound-
aries [5] (Box 1).
Before going into detail, it is worth recall-
ing the different meanings of the term
‘definition’ itself. A ‘lexical definition’
reports the most common usage of a
term [6,7]. It is the definition likely to be
found in a dictionary and can change over
time. An ‘operational definition’ focuses
on a specific objective or application and
may differ from the lexical definition [6]. In
our case, the main objective is gene find-
ing using bioinformatics software. The
question arises of how the term ORF devi-
ates from that of a coding DNA sequence
(CDS). A CDS means a nucleotide
sequence that is eventually translated into
a protein [8]. This implies that the CDS of a
particular protein is bounded by transla-
tion start and stop codons. In some cases
the term ORF is considered equivalent to
that of CDS [9]. Other authors describe an
ORF as a potential protein-coding
sequence which can be determined by
sequence features alone [8]. Note that
there is a difference between the
Trends in Genetics, March 2018, Vol. 34, No. 3 167
concepts of reading frame and ORF. A
reading frame is one of six possibilities
for translating a given double-stranded
genomic sequence into amino acids. For
a particular reading frame, an ORF is a
region that is not interrupted by a stop
codon and is bounded in accordance with
a particular definition (Box 1) [5]. Thus, an
ORF is a sequence region that is ‘open’ for
translation. It is an indicator for a potential
protein-coding gene [3].
We revisit and discuss here the different
ORF definitions to finally recommend one
definition universally applicable in finding
protein-coding genes by bioinformatics
tools. All three ORF definitions currently
in use (Box 1 and Figure 1) consider stop
codons. In the most widely used genetic
code, three of 64 triplets encode stop
codons (TAG, TGA, and TAA). In DNA
sequences not subject to selection and
with a G+C content of 50%, the average
distance between stop codons is 64/
3 ffi 21 codons. It is even shorter when
the G + C content is lower [10]. By con-
trast, the median length of protein-coding
sequences is considerably higher than 21
codons [5,10].
Definition 1 is the current lexical definition
because most textbooks use it. This may
have historical reasons because the first
completely sequenced genomes (except
viruses) were prokaryotic, and gene
structure in prokaryotes is less complex
than in eukaryotes because of the
absence of splicing. Definition 1 focuses
on identifying potential CDSs in prokary-
otic genomes. In eukaryotes, it is applica-
ble only to mature mRNAs, the few genes
containing only a single translated exon,
and to genes with introns of a length
divisible by three and not containing stop
codons in the respective reading frame.
However, in both prokaryotes and eukar-
yotes there is the problem that internal
methionine codons can be mistaken for
start codons [11]. Context information
(such as potential promoter sequences
168 Trends in Genetics, March 2018, Vol. 34, No. 3
or homology search) can be included to ORF according to Definition 2 is not much
find the correct start of the ORF [11]. In longer than when beginning with a start
the case of multiple ATG triplets, several codon [9]. Furthermore, it is easier to
tools based on Definition 1 only consider apply than Ddefinition 1 because stop
the first as the start codon. codons simply need to be found. Among
others, Definition 2 was applied in the
In eukaryotic genomes, it is much more algorithm of OrfM [3], which shows to
complicated to predict CDSs because be significantly faster than methods that
most introns contain stop codons and/ search for start and stop codons. By con-
or cause shifts between reading frames trast, Definition 3 is limited to eukaryotic
(when comparing mature transcripts with internal and (potentially) completely pro-
the DNA sequence). Furthermore, it is tein-coding exons because they are iden-
difficult to identify the correct splice sites. tified by specific algorithms of eukaryotic
Splicing can be considered easily when gene annotation before determining
applying Definition 2, which can deal with translation start and stop positions.
stop codons located within introns. An Although finding splice sites is more com-
ORF according to this definition does plicated than finding start and stop
not necessarily contain an entire CDS, codons, Definition 3 is useful for these
but a potential exon or group of exons. algorithms, but is only rarely mentioned
In addition, metagenomic or other frag- in the literature. All three definitions are
ments such as transcript contigs based on operational rules and are well-
obtained by RNA-seq with missing start suited for being implemented. All three
or stop codons can be analyzed. In this are employed in ORF prediction software,
case, the search for ATG triplets is often as shown in Box S1 in the supplemental
meaningless. The definition should then information online.
be relaxed by considering a maximal
stretch of a nucleotide sequence not In this context, it is also worthwhile com-
interrupted by internal stop codons in paring the concepts of ORF and exon.
the considered reading frame. It can be First, they obviously differ because stop
applied to complete genomic sequences codons and splice sites are clearly not
as well, and could be proposed as gen- identical. Second, there may not be a
eral definition. Another point is that the 50 - stop codon in the neighboring introns,
untranslated region (50 -UTR) frequently and an ORF may therefore include more
contains stop codons such that the than one exon (legend to Figure 1).
Box 1. Three ORF Definitions Currently in Use
In all definitions, an ORF is regarded as a stretch of nucleotide sequence that is not interrupted by stop
codons in a given reading framea [5], while they differ as follows:
Definition 1: an ORF is a sequence that has a length divisible by three and begins with a translation start
codon (ATG) and ends at a stop codon [2,8–10].
Definition 2: an ORF is a sequence that has a length divisible by three and is bounded by stop codons
[3,5,12].
Definition 3: an ORF is a sequence delimited by an acceptor and a donor splice site [1]. Thus, it refers to a
potentially translated eukaryotic internal exon. 50 - and 30 -terminal exons of a putative gene are determined at
the end of the gene prediction process and are not considered for the actual ORF detection.
a
This overarching ‘boundless’ definition is inherent in all three definitions and is necessary when analyz-
ing very short sequence stretches, as in the case of metagenome assemblies.
Prokaryotes
ATG Stop
DefiniƟon 1
5'UTR + CDS
DNA strand

Stop ATG Stop

DefiniƟon 2
5'UTR + CDS
DNA strand

Eukaryotes

Stop ATG Stop Stop Stop

DefiniƟon 1
Exon Exon
DNA strand

Stop ATG Stop Stop Stop

DefiniƟon 2
Exon Exon
DNA strand

Stop ATG Stop Stop Stop

DefiniƟon 3
Exon Exon
DNA strand

Figure 1. Applying the Three Definitions Leads to Different Open Reading Frames (ORFs) (Indicated by Orange Lines) Concerning Their Boundaries.
The corresponding ORFs vary between prokaryotes and eukaryotes. An ORF is delimited by a start codon and a stop codon (Definition 1; in the case of prokaryotes
practically redundant with CDS), two stop codons (Definition 2), or donor and acceptor splice sites (Definition 3; only for eukaryotes). In all cases the ORFs are not
interrupted by internal stop codons in the considered reading frame. According to Definition 2, the ORFs of a eukaryotic gene need not lie in the same reading frame. An
ORF according to Definitions 1 or 2 may involve more than one exon if there are no stop codons in the intronic region in between and if they lie in the same reading frame.

Definition 2 distinguishes clearly between sequences. Overall, we are coming to Acknowledgments

ORF, CDS, and exon. It can easily be
processed by a computer and is the most
general definition. Furthermore, this defi-
nition can be applied even in the case of
prokaryotes and metagenomic
the conclusion that Definition 2 is to be We thank Günter Theißen and Martin Hölzer for stim-
preferred, and we suggest making it the ulating discussions. Financial support by the Univer-
lexical definition in the future. Definition 2 sity of Jena and the Deutsche
Forschungsgemeinschaft (Transregio 124 FungiNet,
is preferable from an operational, prag-
project B1) is gratefully acknowledged.
matic point of view: from stop to stop.

Trends in Genetics, March 2018, Vol. 34, No. 3 169

Supplemental Information
Supplemental information associated with this article
can be found online at https://doi.org/10.1016/j.tig.
2017.12.009.
1
Department of Bioinformatics, Friedrich Schiller
University Jena, Ernst-Abbe-Platz 2, 07743 Jena,
Germany
2
Leibniz Institute on Aging – Fritz Lipmann Institute (FLI),
Beutenbergstraße 11, 07745 Jena, Germany
*Correspondence: stefan.schu@uni-jena.de (S. Schuster).
https://doi.org/10.1016/j.tig.2017.12.009
170 Trends in Genetics, March 2018, Vol. 34, No. 3
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