tRNAscan-SE(1)tRNAscan-SE(1)NAMEtRNAscan-SE
- a program for improved detection of transfer RNA genes in
genomic sequence
SYNOPSIStRNAscan-SE [options] seqfile(s)DESCRIPTIONtRNAscan-SE searches for transfer RNAs in genomic sequence seqfile(s)
using three separate methods to achieve a combination of speed, sensi‐
tivity, and selectivity not available with each program individually.
tRNAscan-SE was written in the PERL (version 5.0) script language.
Input consists of DNA or RNA sequences in FASTA format. tRNA predic‐
tions are output in standard tabular, ACeDB-compatible, or an extended
format including tRNA secondary structure information. tRNAscan-SE
does no tRNA detection itself, but instead combines the strengths of
three independent tRNA prediction programs by negotiating the flow of
information among them, performing a limited amount of post-processing,
and outputting the result.
tRNAscan-SE combines the specificity of the Cove probabilistic RNA pre‐
diction package (Eddy & Durbin, 1994) with the speed and sensitivity of
tRNAscan 1.3 (Fichant & Burks, 1991) plus an implementation of an algo‐
rithm described by Pavesi and colleagues (1994) which searches for
eukaryotic pol III tRNA promoters (our implementation referred to as
EufindtRNA). tRNAscan and EufindtRNA are used as first-pass prefilters
to identify "candidate" tRNA regions of the sequence. These subse‐
quences are then passed to Cove for further analysis, and output if
Cove confirms the initial tRNA prediction. In this way, tRNAscan-SE
attains the best of both worlds: (1) a false positive rate equally low
to using Cove analysis, (2) the combined sensitivities of tRNAscan and
EufindtRNA (detection of 99% of true tRNAs), and (3) search speed 1,000
to 3,000 times faster than Cove analysis and 30 to 90 times faster than
the original tRNAscan 1.3 (tRNAscan-SE uses both a code-optimized ver‐
sion of tRNAscan 1.3 which gives a 650-fold increase in speed, and a
fast C implementation of the Pavesi et al. algorithm).
tRNAscan-SE was designed to make rapid, sensitive searches of genomic
sequence feasible using the selectivity of the Cove analysis package.
Search sensitivity was optimized with eukaryote cytoplasmic & eubacte‐
rial sequences, but it may be applied more broadly with a slight reduc‐
tion in sensitivity.
In the default tabular output format, each new tRNA in a sequence is
consecutively numbered in the 'tRNA #' column. 'tRNA Bounds' specify
the starting (5') and ending (3') nucleotide bounds for the tRNA.
tRNAs found on the reverse (lower) strand are indicated by having the
Begin (5') bound greater than the End (3') bound.
The 'tRNA Type' is the predicted amino acid charged to the tRNA mole‐
cule based on the predicted anticodon (written 5'->3') displayed in the
next column. tRNAs that fit criteria for potential pseudogenes (poor
primary or secondary structure), will be marked with "Pseudo" in the
'tRNA Type' column (pseudogene checking is further discussed in the
Methods section of the program manual). If there is a predicted intron
in the tRNA, the next two columns indicate the nucleotide bounds. If
there is no predicted intron, both of these columns contain zero.
The final column is the Cove score for the tRNA in bits of information.
Specifically, it is a log-odds score: the log of the ratio of the prob‐
ability of the sequence given the tRNA covariance model used (developed
from hand-alignment of 1415 tRNAs), and the probability of the sequence
given a simple random sequence model. tRNAscan-SE counts any sequence
that attains a score of 20.0 bits or larger as a tRNA (based on empiri‐
cal studies conducted by Eddy & Durbin in ref #2).
OPTIONS-h Prints entire list of program options, each with a brief, one-
line description.
-P This option selects the prokaryotic covariace model for tRNA
analysis, and loosens the search parameters for EufindtRNA to
improve detection of prokaryotic tRNAs. Use of this mode with
prokaryotic sequences will also improve bounds prediction of the
3' end (the terminal CAA triplet).
-A This option selects an archaeal-specific covariance model for
tRNA analysis, as well as slightly loosening the EufindtRNA
search cutoffs.
-O This parameter bypasses the fast first-pass scanners that are
poor at detecting organellar tRNAs and runs Cove analysis only.
Since true organellar tRNAs have been found to have Cove scores
between 15 and 20 bits, the search cutoff is lowered from 20 to
15 bits. Also, pseudogene checking is disabled since it is only
applicable to eukaryotic cytoplasmic tRNA pseudogenes. Since
Cove-only mode is used, searches will be very slow (see -C
option below) relative to the default mode.
-G This option selects the general tRNA covariance model that was
trained on tRNAs from all three phylogenetic domains (archaea,
bacteria, & eukarya). This mode can be used when analyzing a
mixed collection of sequences from more than one phylogenetic
domain, with only slight loss of sensitivity and selectivity.
The original publication describing this program and tRNAscan-SE
version 1.0 used this general tRNA model exclusively. If you
wish to compare scores to those found in the paper or scans
using v1.0, use this option. Use of this option is compatible
with all other search mode options described in this section.
-C Directs tRNAscan-SE to analyze sequences using Cove analysis
only. This option allows a slightly more sensitive search than
the default tRNAscan + EufindtRNA -> Cove mode, but is much
slower (by approx. 250 to 3,000 fold). Output format and other
program defaults are otherwise identical to the normal analysis.
-H This option displays the breakdown of the two components of the
covariance model bit score. Since tRNA pseudogenes often have
one very low component (good secondary structure but poor pri‐
mary sequence similarity to the tRNA model, or vice versa), this
information may be useful in deciding whether a low-scoring tRNA
is likely to be a pseudogene. The heuristic pseudogene detec‐
tion filter uses this information to flag possible pseudogenes
-- use this option to see why a hit is marked as a possible
pseudogene. The user may wish to examine score breakdowns from
known tRNAs in the organism of interest to get a frame of refer‐
ence.
-D Manually disable checking tRNAs for poor primary or secondary
structure scores often indicative of eukaryotic pseudogenes.
This will slightly speed the program & may be necessary for non-
eukaryotic sequences that are flagged as possible pseudogenes
but are known to be functional tRNAs.
-o <file>
Output final results to <file>.
-f <file>
Save final results and Cove tRNA secondary structure predictions
to <file>. This output format makes visual inspection of indi‐
vidual tRNA predictions easier since the tRNA sequence is dis‐
played along with the predicted tRNA base pairings.
-a Output final results in ACeDB format instead of the default tab‐
ular format.
-m <file>
Save statistics summary for run. This option directs tRNAscan-
SE to write a brief summary to <file> which contains the run
options selected as well as statistics on the number of tRNAs
detected at each phase of the search, search speed, and other
bits of information. See Manual documentation for explanation
of each statistic.
-d Display program progress. Messages indicating which phase of
the tRNA search are printed to standard output. If final results
are also being sent to standard output, some of these messages
will be suppressed so as to not interrupt display of the
results.
-l <file>
Save log of program progress in <file>. Identical to -d option,
but sends message to <file> instead of standard output. Note:
the -d option overrides the -l option if both are specified on
the same command line.
-q Quiet mode: the credits & run option selections normally printed
to standard error at the beginning of each run are suppressed.
-b Use brief output format. This eliminates column headers that
appear by default when writing results in tabular output format.
Useful if results are to be parsed or piped to another program.
-N This option causes tRNAscan-SE to output a tRNA's corresponding
codon in place of its anticodon.
-(Option)#
The '#' symbol may be used as shorthand to specify "default"
file names for output files. The default file names are con‐
structed by using the input sequence file name, followed by an
extension specifying the output file type <seqfile.ext> where
'.ext' is:
Extension Option Description
--------------------------
.out -o final results
.stats -m summary statistics file
.log -l run progress file
.ss -f secondary structures save file
.fpass.out -r formatted, tabular output
from first-pass scans
.fpos -F FASTA file of tRNAs identified in
first-pass scans that were found to be
false positives by Cove analysis
Notes:
1) If the input sequence file name has the extensions '.fa' or
'.seq', these extensions will be removed before using the file‐
name as a prefix for default file names. (example -- input file
name Mygene.seq will have the output file name Mygene.out if the
'-o#' option is used).
2) If more than one sequence file is specified on the command
line, the "default" output file prefix will be the name of the
FIRST sequence file on the command line. Use the -p option to
change this default name to something more appropriate when
using more than one sequence file on the command line.
-p <label>
Use <label> prefix as the default output file prefix when using
'#' for file name specification. <label> is used in place of
the input sequence file name.
-y This option displays which of the first-pass scanners detected
the tRNA being output. "Ts", "Eu", or "Bo" will appear in the
last column of Tabular output, indicating that either tRNAscan
1.4, EufindtRNA, or both scanners detected the tRNA, respec‐
tively.
-X <score>
Set Cove cutoff score for reporting tRNAs (default=20). This
option allows the user to specify a different Cove score thresh‐
old for reporting tRNAs. It is not recommended that novice
users change this cutoff, as a lower cutoff score will increase
the number of pseudogenes and other false positives found by
tRNAscan-SE (especially when used with the "Cove only" scan
mode). Conversely, a higher cutoff than 20.0 bits will likely
cause true tRNAs to be missed by tRNAscan (numerous "real" tRNAs
have been found just above the 20.0 cutoff). Knowledgable users
may wish to experiment with this parameter to find very unusual
tRNAs or pseudogenes beyond the normal range of detection with
the preceding caveats in mind.
-L <length>
Set max length of tRNA intron+variable region (default=116bp).
The default maximum tRNA length for tRNAscan-SE is 192 bp, but
this limit can be increased with this option to allow searches
with no practical limit on tRNA length. In the first phase of
tRNAscan-SE, EufindtRNA searches for A and B boxes of <length>
maximum distance apart, and passes only the 5' and 3' tRNA ends
to covariance model analysis for confirmation (removing the bulk
of long intervening sequences). tRNAs containing group I and II
introns have been detected by setting this parameter to over 800
bp. Caution: group I or II introns in tRNAs tend to occur in
positions other than the canonical position of protein-spliced
introns, so tRNAscan-SE mispredicts the intron bounds and anti‐
codon sequence for these cases. tRNA bound predictions, how‐
ever, have been found to be reliable in these same tRNAs.
-I <score>
This score cutoff affects the sensitivity of the first-pass
scanner EufindtRNA. This parameter should not need to be
adjusted from its default values (variable depending on search
mode), but is included for users who are familiar with the
Pavesi et al. (1994) paper and wish to set it manually. See
Lowe & Eddy (1997) for details on parameter values used by
tRNAscan-SE depending on the search mode.
-B <number>
By default, tRNAscan-SE adds 7 nucleotides to both ends of tRNA
predictions when first-pass tRNA predictions are passed to
covariance model (CM) analysis. CM analysis generally trims
these bounds back down, but on occassion, allows prediction of
an otherwise truncated first-pass tRNA prediction.
-g <file>
Use exceptions to "universal" genetic code specified in <file>.
By default, tRNAscan-SE uses a standard universal codon -> amino
acid translation table that is specified at the end of the
tRNAscan-SE.src source file. This option allows the user to
specify exceptions to the default translation table. The user
may use any one of several alternate translation code files
included in this package (see files documentation for specifica‐
tion of file format, or refer to included examples files.
Note: this option does not have any effect when using the -T or
-E options -- you must be running in default or Cove only analy‐
sis mode.
-c <file>
For users who have developed their own tRNA covariance models
using the Cove program "coveb" (see Cove documentation), this
parameter allows substitution for the default tRNA covariance
models. May be useful for extending Cove-only mode detection of
particularly strange tRNA species such as mitochondrial tRNAs.
-Q By default, if an output result file to be written to already
exists, the user is prompted whether the file should be over-
written or appended to. Using this options forces overwriting
of pre-existing files without an interactive prompt. This
option may be handy for batch-processing and running tRNAscan-SE
in the background.
-n <EXPR>
Search only sequences with names matching <EXPR> string. <EXPR>
may contain * or ? wildcard characters, but the user should
remember to enclose these expressions in single quotes to avoid
shell expansion. Only those sequences with names (first non-
white space word after ">" symbol on FASTA name/description
line) matching <EXPR> are analyzed for tRNAs.
-s <EXPR>
Start search at first sequence with name matching <EXPR> string
and continue to end of input sequence file(s). This may be use‐
ful for re-starting crashed/aborted runs at the point where the
previous run stopped. (If same names for output file(s) are
used, program will ask if files should be over-written or
appended to -- choose append and run will successfully be
restarted where it left off).
-T Directs tRNAscan-SE to use only tRNAscan to analyze sequences.
This mode will default to using "strict" parameters with
tRNAscan analysis (similar to tRNAscan version 1.3 operation).
This mode of operation is faster (3-5 times faster than default
mode analysis), but will result in approximately 0.2 to 0.6
false positive tRNAs per Mbp, decreased sensitivity, and less
reliable prediction of anticodons, tRNA isotype, and introns.
-t <mode>
Explicitly set tRNAscan params, where <mode> = R or S
(R=relaxed, S=strict tRNAscan v1.3 params). This option allows
selection of strict or relaxed search parameters for tRNAscan
analysis. By default, "strict" parameters are used. Relaxed
parameters may give very slightly increased search sensitivity,
but increase search time by 20-40 fold.
-E Run EufindtRNA alone to search for tRNAs. Since Cove is not
being used as a secondary filter to remove false positives, this
run mode defaults to "Normal" parameters which more closely
approximates the sensitivity and selectivity of the original
algorithm describe by Pavesi and colleagues (see the next
option, -e for a description of the various run modes).
-e <mode>
Explicitly set EufindtRNA params, where <mode>= R, N, or S
(relaxed, normal, or strict). The "relaxed" mode is used for
EufindtRNA when using tRNAscan-SE in default mode. With relaxed
parameters, tRNAs that lack pol III poly-T terminators are not
penalized, increasing search sensitivity, but decreasing selec‐
tivity. When Cove analysis is being used as a secondary filter
for false positives (as in tRNAscan-SE's default mode), overall
selectivity is not decreased.
Using "normal" parameters with EufindtRNA does incorporate a log
odds score for the distance between the B box and the first
poly-T terminator, but does not disqualify tRNAs that do not
have a terminator signal within 60 nucleotides. This mode is
used by default when Cove analysis is not being used as a sec‐
ondary false positive filter.
Using "strict" parameters with EufindtRNA also incorporates a
log odds score for the distance between the B box and the first
poly-T terminator, but _rejects_ tRNAs that do not have such a
signal within 60 nucleotides of the end of the B box. This mode
most closely approximates the originally published search algo‐
rithm (3); sensitivity is reduced relative to using "relaxed"
and "normal" modes, but selectivity is increased which is impor‐
tant if no secondary filter, such as Cove analysis, is being
used to remove false positives. This mode will miss most
prokaryotic tRNAs since the poly-T terminator signal is a fea‐
ture specific to eukaryotic tRNAs genes (always use "relaxed"
mode for scanning prokaryotic sequences for tRNAs).
-r <file>
Save tabular, formatted output results from tRNAscan and/or
EufindtRNA first pass scans in <file>. The format is similar to
the final tabular output format, except no Cove score is avail‐
able at this point in the search (if EufindtRNA has detected the
tRNA, the negative log likelihood score is given). Also, the
sequence ID number and source sequence length appear in the col‐
umns where intron bounds are shown in final output. This option
may be useful for examining false positive tRNAs predicted by
first-pass scans that have been filtered out by Cove analysis.
-u <file>
This option allows the user to re-generate results from regions
identified to have tRNAs by a previous tRNAscan-SE run. Either
a regular tabular result file, or output saved with the -r
option may be used as the specified <file>. This option is par‐
ticularly useful for generating either secondary structure out‐
put (-f option) or ACeDB output (-a option) without having to
re-scan entire sequences. Alternatively, if the -r option is
used to generate the previous results file, tRNAscan-SE will
pick up at the stage of Cove-confirmation of tRNAs and output
final tRNA predicitons as with a normal run.
Note: the -n and -s options will not work in conjunction with
this option.
-F <file>
Save first-pass candidate tRNAs in <file> that were then found
to be false positives by Cove analysis. This option saves can‐
didate tRNAs found by either tRNAscan and/or EufindtRNA that
were then rejected by Cove analysis as being false positives.
tRNAs are saved in the FASTA sequence format.
-M <file>
This option may be used when scanning a collection of known tRNA
sequences to identify possible false negatives (incorreclty
missed by tRNAscan-SE) or sequences incorrectly annotated as
tRNAs (correctly passed over by tRNAscan-SE). Examination of
primary & secondary structure covariance model scores (-H
option), and visual inspection of secondary structures (use -F
option) may be helpful resolving identification conflicts.
SEE ALSO
User Manual and tutorial: Manual.ps (postscript), MANUAL (text)
BUGS
No major bugs known.
NOTES
This software and documentation is Copyright (C) 1996, Todd M.J. Lowe &
Sean R. Eddy. It is freely distributable under terms of the GNU Gen‐
eral Public License. See COPYING, in the source code distribution, for
more details, or contact me.
Todd Lowe
Dept. of Genetics, Washington Univ. School of Medicine
660 S. Euclid Box 8232
St Louis, MO 63110 USA
Phone: 1-314-362-7667
FAX : 1-314-362-2985
Email: lowe@genetics.wustl.edu
REFERENCES
1. Fichant, G.A. and Burks, C. (1991) "Identifying potential tRNA genes
in genomic DNA sequences", J. Mol. Biol., 220, 659-671.
2. Eddy, S.R. and Durbin, R. (1994) "RNA sequence analysis using
covariance models", Nucl. Acids Res., 22, 2079-2088.
3. Pavesi, A., Conterio, F., Bolchi, A., Dieci, G., Ottonello, S.
(1994) "Identification of new eukaryotic tRNA genes in genomic DNA
databases by a multistep weight matrix analysis of transcriptional con‐
trol regions", Nucl. Acids Res., 22, 1247-1256.
4. Lowe, T.M. & Eddy, S.R. (1997) "tRNAscan-SE: A program for improved
detection of transfer RNA genes in genomic sequence", Nucl. Acids Res.,
25, 955-964.
tRNAscan-SE 1.1 November 1997 tRNAscan-SE(1)
