A real world example may help to illuminate.
tacg has several output formats ranging from a summary of hits to a complete linear map including full double stranded sequence, all hits, and co-translation in all frames. Almost all output is preceded by a notification that a subselection has been made (if it has been), the name of the input filename (if entered via commandline via STDIN, this is noted as fake filename; if entered via the web interface, it will be noted correctly), and the sequence composition, including degeneracies.
Here are the details, in roughly the order of popularity:
Summary of Hits:
This flag simply finds all patterns in the rebase file and summarizes the numeric results in a table. It is influenced by the RE selection criteria, so that as you make the criteria more selective, you will get fewer total patterns reported.
If you don't specify specific patterns via the -p or -x options, tacg will go searching for the pattern database called rebase.data in the current directory, then in your HOME directory, then in the TACGLIB directory, before giving up. If you give it a restricted number of patterns, then -s will only show them. Also, if you filter the number of patterns with selection filters such as -o, -n, -cost, etc, the results will only show the ones that pass ALL the filters.
== Enzymes that DO NOT MAP to this sequence:
AscI SgfI SrfI
== Total Number of Hits per Enzyme:
FseI 1 PacI 9 SfiI 2
HaeII 9 PmeI 1 SwaI 8
NotI 1 SbfI 2
Table of Sites
Produces a table of Site information, ordered by the appearance of the patterns in the rebase file or by the order that they have been entered from the command line if using the -p flag. If the -c flag is used, the Site entries will be ordered by number of hits, and then alphabetically.
== Match Sites by Enzyme or Pattern BbvCI CC'TCA_GC (0 Err) - 57 Match(s) found (7.98 sites exp) 4067 4202 5878 7174 8430 10266 10460 12134 12439 18307 18540 21426 28450 36914 41496 42408 53190 55192 55653 61004 61798 63690 66002 75553 79089 80860 80995 84331 90403 92047 93813 94656 96199 96519 99306 100053 101127 104854 107617 107959 109923 112474 113130 113264 116563 116681 118254 118388 118640 118775 120402 124173 126590 128419 129257 131402 140255 FseI GG_CCGG'CC (0 Err) - 1 Match(s) found (0.75 sites exp) 18786 HaeII AWWRTAANNWWGNNNC (0 Err) - 9 Match(s) found (5.04 sites exp) 22421 27566 33732 48560 58182 125115 135815 136926 137462 NotI GC'GGCC_GC (0 Err) - 1 Match(s) found (0.75 sites exp) 17798
(remainder omitted)
Table of Fragments
Produces tables of fragments much like the sites example above, with the fragments ordered by occurrence (-F1), size (-F2), or both (-F3). This flag is also affected by the -c flag, as above.
The output ordered by occurrence is:
== Fragment Sizes by Enzyme or Pattern (UNSORTED) HaeII AWWRTAANNWWGNNNC (0 Err) - 10 Fragment(s) found (5.04 sites exp) 22421 5145 6166 14828 9622 66933 10700 1111 536 6356
The output ordered by size is:
HaeII AWWRTAANNWWGNNNC (0 Err) - 10 Fragment(s) found (5.04 sites exp) 536 1111 5145 6166 9622 10700 14828 22421 66933 6356
Linear Map
Minimally, produces a complete linear map of the sequence with all bases included. The patterns included are those that pass the selection filters by min, max, cost, overhang, size of recognition sequence, dam, and dcm methylation. The map can be modified in many ways, both to include extra information by requesting co-translations and less information to create a more compact map (see box below).
The following is the basic output format: generated by
== Linear Map of Sequence:
BseMII~
DpnI
Sau3AI
BstYI DdeI
NlaIII BglII BsmAI~ BbsI MboII
\ \ \ \ \ \ \ \
1 ttggcatgagcaactgaaataatagatctgccatttcctgagacagagaagactatgaag 60
aaccgtactcgttgactttattatctagacggtaaaggactctgtctcttctgatacttc
^ * ^ * ^ * ^ * ^ * ^ *
Following is a stanza from a subsequence. The top strand is numbered startign at 1, The bottom strand is numbered starting at the original numbering (the output was generated with the command:
% tacg -L -n4 -b 4444 < seqfile
== Linear Map of Sequence:
Tsp509I
ScrFI Bsp1286I
BstNI BseSI
PspGI BfaI MnlI~ TspRI
BssKI XbaI BseMII~ DdeI Bst4CI
\ \ \ \\ \ \ \ \ \
1 tttgtgccaggaattgttctagacatcagatatactgaggtaaacaaaacagtgcccctg 60
4444 aaacacggtccttaacaagatctgtagtctatatgactccatttgttttgtcacggggac 4503
^ * ^ * ^ * ^ * ^ * ^ *
Options used only in the Linear Map
Linear Co-Translation
This option provides co-translation beneath the DNA sequence in 1,3, or 6 frames in 1 or 3 letter codes. It provides co-translation without any implication about ORFs, simply translating the nucleic acid triplets into amino acids. It uses the requested Codon Translation table for the translation. See also the -O flag and the —orfmap flag for additional Translation features.
% tacg -L -n4 -T3,1
== Linear Map of Sequence:
BseMII~
DpnI
Sau3AI
BstYI DdeI
NlaIII BglII BsmAI~ BbsI MboII
\ \ \ \ \ \ \ \
1 ttggcatgagcaactgaaataatagatctgccatttcctgagacagagaagactatgaag 60
aaccgtactcgttgactttattatctagacggtaaaggactctgtctcttctgatacttc
^ * ^ * ^ * ^ * ^ * ^ *
1 L A * A T E I I D L P F P E T E K T M K
2 W H E Q L K * * I C H F L R Q R R L * R
3 G M S N * N N R S A I S * D R E D Y E G
Alternative sequence numbering
Allows for starting the numbering for the linear map with something other than 1, for example to start numbering a genomic fragment containing a transcriptional unit to be numbered relative to the start site, you might use —numstart=-241 as below. Note that if you use the —numstart option, the lower strand will be numbered with the original numbering so that you can correlate the two (unless you also specify —strands=1 —notics, which will leave only the top strand alternatively numbered.
== Linear Map of Sequence:
BseMII~
DpnI
Sau3AI
BstYI DdeI
NlaIII BglII BsmAI~ BbsI MboII
\ \ \ \ \ \ \ \
-241 ttggcatgagcaactgaaataatagatctgccatttcctgagacagagaagactatgaag -182
1 aaccgtactcgttgactttattatctagacggtaaaggactctgtctcttctgatacttc 60
^ * ^ * ^ * ^ * ^ * ^ *
Sequence Tic Markers
Reduces the output by one line per linear map stanza by removing the 5 and 10 base markers normally below the sequence; when combined with the —strands=1 (below) reduces it by 2, important for some.
Complimentary Strand
Reduces the output by one line per linear map stanza by removing the complementary strand; can be combined or used independently with the —notics option above.
BseMII~
DpnI
Sau3AI
BstYI DdeI
NlaIII BglII BsmAI~ BbsI MboII
\ \ \ \ \ \ \ \
-241 ttggcatgagcaactgaaataatagatctgccatttcctgagacagagaagactatgaag -182
Ladder Map
Requests a ladder map, much like GCG's MAPPLOT output. When marking REs, the plot marks 5' overhangs with a \, a 3' overhang with / and blunt ends with a |. Patterns entered from the commandline are marked with a |. The Ladder map can be widened with the -w flag to increase resolution and the patterns can be Strider-sorted with the -c flag to collect patterns that hit the same number of times.
The Ladder Map ends with a Summary Map which plots infrequent patterns on a single line, using the same width and markers as the Ladder Map. The cutoff for the number of sites plotted is sequence-length dependent - the longer the sequence, the greater the cutoff.
shown without -c sorting option
== Ladder Map of Enzyme Sites:
# hits
1000 2000 3000 4000 5000 6000 7000 8000 9000 10000
: : : : : : : : : :
BbvCI -----------------------\\---------\-------\-------\--------- 5
PacI ---/----------/--------------------------------------------- 2
SapI -\------------------------------\--------------------------- 2
SwaI ----|------------------------------------------------------- 1
shown with the -c sorting option
== Ladder Map of Enzyme Sites:
# hits
1000 2000 3000 4000 5000 6000 7000 8000 9000 10000
: : : : : : : : : :
SwaI ----|------------------------------------------------------- 1
PacI ---/----------/--------------------------------------------- 2
SapI -\------------------------------\--------------------------- 2
BbvCI -----------------------\\---------\-------\-------\--------- 5
Pseudo Gel Map
Produces a crude simulation of an electrophoretic gel separation of the digest performed. It uses a simple log() approximation of the distance travelled. The LoCutOff sets the lower bound of the fragment size you want; the HiCutOff if entered, sets the upper bound - convenient if you want to compare different digestions sets or zoom into a particular region to get more resolution. The output (see Example output) uses patterns labels on the vertical axis and presents the fragment mobilities as moving from the right to the left. Single bands are indicated as a |. If there are more than one band that maps to a character position, that position is marked with the number of bands that the character position contains. More than 9 bands are marked with a *. As noted above, if the region in which you're interested has multiple bands, you can 'zoom' in by specifying a smaller bracketing LoCutOff & HiCutOff, and also by expanding the width of the output. As in other restriction analyses, the output reflects the filtering by min/max # of hits , magnitude of site, overhang, cost, etc. the output can be further Strider-sorted with the -c flag.
== Pseudo-Gel Map of Digestions:
# frags
100 1000 10000
. . . . . . . . .. . . . . . . . ..
BbvCI | 2 || | 6
PacI | | | 3
SapI | | | 3
SwaI | | 2
Cloning Assistance
The —clone flag allows you to search for sequence segments amenable for cloning. You can specify segments where a cut MUST occur (#x#) and segments where they must NOT occur (#_#), where the # is a number in the range of the sequence length. You can chain up to 15 of these criteria together. The regions that satisfy the criteria either completely or partially will be printed in order of best-fit. You can additionally filter the REs used to attempt the cloning fit with the usual filtering agents: overhang, min, max, cost, etc.
This is a hard output to describe, but below is the output for the following command:
% tacg --clone '2340_4553,4554x5556,5557_23453,23461x35388' -e 36000 -w 75 < sequence
== Here's the Clone output for the 4 Criteria:
Rule 1: 2340_4553
Rule 2: 4554x5556 [summary of the rules]
Rule 3: 5557_23453
Rule 4: 23461x35388
REs that met ALL criteria:
SapI [only one enzyme met all the criteria]
== Ladder Map of Enzyme Sites:
# hits
3000 6000 9000 12000 15000 18000 21000 24000 27000 30000 33000 36000
: : : : : : : : : : : :
SapI \---------\-------------------------------------------\----------------- 3
^
[looks like it missed the range but it's right on the edge]
Results (listed by Rules) that met SOME criteria & did not violate a hits-forbidden range.
--- For Rule 1 (2340_4553), the following REs matched:
MluI NruI PvuI SalI SapI SexAI SnaBI SwaI
== Ladder Map of Enzyme Sites:
# hits
3000 6000 9000 12000 15000 18000 21000 24000 27000 30000 33000 36000
: : : : : : : : : : : :
MluI ---------------------------------------------------------2\\------------ 4
NruI -----------------------------------------------------------4||---------- 6
PvuI ---------------------------------------------------------2-/------------ 3
SalI ---\-----------------------------------------------------\-\------------ 3
: : : : : : : : : : : :
SapI \---------\-------------------------------------------\----------------- 3
SexAI ---------------------------------------------------\----------------\--- 2
SnaBI ---|-----------------------------------------------------3|2-3---------- 10
SwaI -|--------------------------------------------------------2---------|--- 4
--- For Rule 2 (4554x5556), the following REs matched:
SapI
== Ladder Map of Enzyme Sites:
# hits
3000 6000 9000 12000 15000 18000 21000 24000 27000 30000 33000 36000
: : : : : : : : : : : :
SapI \---------\-------------------------------------------\----------------- 3
--- For Rule 3 (5557_23453), the following REs matched:
MluI NruI PvuI SalI SapI SexAI SnaBI SwaI
== Ladder Map of Enzyme Sites:
# hits
3000 6000 9000 12000 15000 18000 21000 24000 27000 30000 33000 36000
: : : : : : : : : : : :
MluI ---------------------------------------------------------2\\------------ 4
NruI -----------------------------------------------------------4||---------- 6
PvuI ---------------------------------------------------------2-/------------ 3
SalI ---\-----------------------------------------------------\-\------------ 3
: : : : : : : : : : : :
SapI \---------\-------------------------------------------\----------------- 3
SexAI ---------------------------------------------------\----------------\--- 2
SnaBI ---|-----------------------------------------------------3|2-3---------- 10
SwaI -|--------------------------------------------------------2---------|--- 4
--- For Rule 4 (23461x35388), the following REs matched:
MluI NruI PvuI SalI SapI SexAI SnaBI SwaI
== Ladder Map of Enzyme Sites:
# hits
3000 6000 9000 12000 15000 18000 21000 24000 27000 30000 33000 36000
: : : : : : : : : : : :
MluI ---------------------------------------------------------2\\------------ 4
NruI -----------------------------------------------------------4||---------- 6
PvuI ---------------------------------------------------------2-/------------ 3
SalI ---\-----------------------------------------------------\-\------------ 3
: : : : : : : : : : : :
SapI \---------\-------------------------------------------\----------------- 3
SexAI ---------------------------------------------------\----------------\--- 2
SnaBI ---|-----------------------------------------------------3|2-3---------- 10
SwaI -|--------------------------------------------------------2---------|--- 4
--- Results (listed by RE) that met SOME criteria & did not violate a hits-forbidden range
RE Name Rule 1 Rule 2 Rule 3 Rule 4
MluI X X X
NruI X X X
PvuI X X X
SalI X X X
SapI X X X X
SexAI X X X
SnaBI X X X
SwaI X X X
Open Reading Frame Tables
This flag will search any combination of the 6 frames for ORFs and report the ones greater than the MinSize in a table that includes:
-
Frame of the Current ORF
-
Sequence # of the Current ORF in that frame
-
Offset from the start in both bases and AAs
-
Size of the ORF in AAs and KDa
-
estimated pI
-
ORF in 1 letter code for external analysis
The specification for the frames is simple but odd. All the frame numbers you want analysed are caoncatenated together. For example, if you wanted to analyze only frames 1 & 2 for ORFs larger than 150 AAs, the option string would be -O 12,150. For frames 1,3,4,6, the option string would be -O 1346,150.
== Open Reading Frame Analysis: == ORF Analysis for Frame 1: 2 ORF(s) > 150 AAs F# ORF# Begin(bp/AAs) End(bp/AAs) #AAs MWt(KDa) pI > 1 1 12031 / 4010 12615 / 4205 195 21854.84 10.566 MFSLFYTMCSRMVTEKSQREQREKERELYREPASAEGWFCFRNWNVSRLDFYFQFTFQIQLPKLFDLSPCLLTRK RTLGLLSWELGAHVERLAQIHILAKFSHTKGPAREKRKDESGSRRDFKGRAAARGXAQAPPSADIRHGSSARAAF PRASPTRGAGRGTRRAGGKEGEGLDTAAVLPAGFSRPVPMPSSGA > 1 2 108490 / 36163 109191 / 36397 234 24692.54 11.919 MLKMQTWGGQEYHTQGCHFTLGGLHRGRAEAPLTSQMGQQPGRGAPHFPDGAAAAQKRPSLPRRGGSRAEAPLTX QTGRXPGRGAPHFPDGAAAGQRRPSLPRRGGSRAEAPLTSQTGRQPGRGAPHFPDGAAAGQRRPSLPRRGGSRAE APLTSQTGRQPGRGAPHFPDGAAAGQRCPSPPRQGGSRAEALLSYQSVGWPGRRVPHFPVSWAARQRHSSLPRWG SQAEGLLTC == ORF Analysis for Frame 3: 1 ORF(s) > 150 AAs F# ORF# Begin(bp/AAs) End(bp/AAs) #AAs MWt(KDa) pI > 3 1 31008 / 10335 31523 / 10507 172 20300.56 10.219 MFXVXXXXXXXXSXXXXXXXXXXXXXXXXXXGXVXXXXXXXFXXAXRXXXXLXXFXXXXXXXPKXGXXXXXXXXX XXXXXXXXXXXXXXXXXXXXXXXXVFLLFKXFFFFSXSSKXSXXXXXXXXXLNXYIXXGQVFSXWXPGTPLQIFA TTHQHGXLKNNYGHIIFVYWLK == ORF Analysis for Frame 4: 2 ORF(s) > 150 AAs F# ORF# Begin(bp/AAs) End(bp/AAs) #AAs MWt(KDa) pI > 4 1 10599 / 3533 10149 / 3382 151 17502.81 9.657 MKAAGLGDGWGSRVPRSLWHGPRLPRVQTPPEMXXLXXLPPARSNPQPLPIVWKGLYSNFNWHLESTFYFLFSFF FFFEKESHFVAQAGVQWHDLGSLQPPPSRFKRFSCLSLPSSWDYRHLPPCPANFCNFSRDGVSPYWPGWSQISNL R > 4 2 8253 / 2751 7785 / 2594 157 17320.85 7.666 MPHNSPCLIEQPKSSFNISYLWLPSPYVSCYLASSCSLLSSTTGSPAVPSECKAHARTCTHTHTHTHTCTHTHTH TIHLSPCCLCPEAPGQVDVHRAHSLTSFRFCSDSPSLELVILKNSHPPKPSAFTPIPVTLHFFYGLHLLLLSLLG LFFILTY
The output format is described here but note that especially with degenerate sequences, the filtering is quite lax; degenerate nucleic acid sequence will simply cause unknown amino acids to be translated, and the resulting ORFs can be quite large, albeit weird (see above for Frame 3)
If you want extended information on each ORF, append an x to the frame specification (-o 1346x,150) and 3 more lines will be generated for each ORF entry listing the number and percentage of each Amino Acid.
== ORF Analysis for Frame 1: 2 ORF(s) > 150 AAs F# ORF# Begin(bp/AAs) End(bp/AAs) #AAs MWt(KDa) pI > 1 1 12031 / 4010 12615 / 4205 195 21854.84 10.566 #L A C D E F G H I K L M N P Q R S T V W X Y ## 22 3 6 13 13 17 4 4 10 16 4 2 12 7 24 17 9 5 3 1 3 #% 11.28 1.54 3.08 6.67 6.67 8.72 2.05 2.05 5.13 8.21 2.05 1.03 6.15 3.59 12.31 8.72 4.62 2.56 1.54 0.51 1.54 MFSLFYTMCSRMVTEKSQREQREKERELYREPASAEGWFCFRNWNVSRLDFYFQFTFQIQLPKLFDLSPCLLTRK RTLGLLSWELGAHVERLAQIHILAKFSHTKGPAREKRKDESGSRRDFKGRAAARGXAQAPPSADIRHGSSARAAF PRASPTRGAGRGTRRAGGKEGEGLDTAAVLPAGFSRPVPMPSSGA > 1 2 108490 / 36163 109191 / 36397 234 24692.54 11.919 #L A C D E F G H I K L M N P Q R S T V W X Y ## 30 3 4 7 6 37 9 0 2 15 3 0 29 19 31 17 11 3 4 2 2 #% 12.82 1.28 1.71 2.99 2.56 15.81 3.85 0.00 0.85 6.41 1.28 0.00 12.39 8.12 13.25 7.26 4.70 1.28 1.71 0.85 0.85 MLKMQTWGGQEYHTQGCHFTLGGLHRGRAEAPLTSQMGQQPGRGAPHFPDGAAAAQKRPSLPRRGGSRAEAPLTX QTGRXPGRGAPHFPDGAAAGQRRPSLPRRGGSRAEAPLTSQTGRQPGRGAPHFPDGAAAGQRRPSLPRRGGSRAE APLTSQTGRQPGRGAPHFPDGAAAGQRCPSPPRQGGSRAEALLSYQSVGWPGRRVPHFPVSWAARQRHSSLPRWG SQAEGLLTC
Note that the actual translation of the DNA depends on the Codon table selected (see -C flag). Despite using the appropriate codon table, the translation may not be accurate because some organisms use non-MET initiators and tacg has not been coded to act on that information; it only starts ORFs on METs.(read more about this in the codon.data file in the Data directory)
NB: tacg is not bright enough to know that translations that cross the begin/end transition should be continued if the sequence is circular, so it will miss the ORFs that do this. If you are concerned about this, split your sequence in the middle and stick the begin/ends together and re-run the analysis. This also applies to the ORF maps, immediately below.
ORF maps
(—orfmap) When used with the -o flag above, this option generates a pseudographical ORF map showing the location and orientation of each ORF found, as well as a map of all starts and stops in the requested frames. Note that the ORF Map Summary will only show those ORFs that meet the length criteria.
== ORF Map Summary
2000 4000 6000 8000 10000 12000 14000 16000 18000 20000
: : : : : : : : : :
1 -->
6 <---
: : : : : : : : : :
2000 4000 6000 8000 10000 12000 14000 16000 18000 20000
== MET / STOP Map Summary
2000 4000 6000 8000 10000 12000 14000 16000 18000 20000
: : : : : : : : : :
1 |||>||||||>|>||| |>||>|||||||||||||>||| > |> |||||>||||>>||| >|| ||||
3 ||||>||>|>|||>|| ||||||>|||||>|>||||>>>||>|||||||>|>|||>| || >|||>||
4 <|||||||||||||||<|<<|||||<<||||<<<||<<||<||<|||||||<|||||||||||<|||<||
6 |<||<<|||<|||||||||||<|||||||||<|||<<|<|||<|<|| |<|||<|| ||< <||||||
: : : : : : : : : :
2000 4000 6000 8000 10000 12000 14000 16000 18000 20000
Plasmid Maps
These two options will generate a circular plasmid map (again, a la DNA Strider) in Postscript format (—ps) or PDF format (—pdf). The PDF format is the one that almost everyone will want; the postscript version is generated 1st and then converted to PDF so it's identical. The conversion does require a working ghostscript installed as (or linked to) /usr/bin/gs, altho the creation of the map does not.
The map is appended to the file tacg_Map.ps in the current directory, and further runs will be appended to the same file, so it may be disconcerting to view the file at first; the later plots are at the end. If this behavior is not acceptable, prepend the tacg command with an rm tacg_Map.ps;
The sequence going into the plasmid map will be converted to a circular map regardless of whether the sequence is circular or not.
When the plasmid map option is called, the subroutine also calls the logdegen() function, which notes the start and end of every run of degeneracies. This allows the map to have the degeneracies marked along the rim as a grey band. This is particularly useful because it allows you to compose plasmid maps of known and unknown sequence and therefore get at least known sites marked correctly. Using the Gel Map option you can verify if the known sites generate valid fragment sizes.
Figure: The pdf plasmid map output looks like this.
Codon Usage Table Selection
The following translation tables are supported by tacg. The actual table is a slight modification of NCBI's comprehensive Codon table collection. In that file (and in codon.data file, you can read the complete description of each.
| # | Organism | # | Organism | # | Organism |
|---|---|---|---|---|---|
| 0 | Standard* | 6 | Echino_Mito | 12 | Blepharisma_Nucl |
| 1 | Vert_Mito | 7 | Euplotid_Nucl | 13 | Chlorophycean_Mito |
| 2 | Yeast_Mito | 8 | Bacterial | 14 | Trematode_Mito |
| 3 | Mold_Mito | 9 | Alt_Yeast_Nucl | 15 | Scenedes_Mito |
| 4 | Invert_Mito | 10 | Ascidian_Mito | 16 | Thrausto_Mito |
| 5 | Ciliate_Nucl | 11 | Alt_Flatworm_mito |
Note that tacg does not provide as accurate translation as other tools. Specifically, it does not take into account the alternative initiator codons that some other species use, nor does it translate through the start/stop sequence in a file if you use the -f 0 flag to indicate a circular topology. These are known bugs and are already on the TODO list.
