LTRharvest user`s manual
Contents
1. be conducted motifmisn This option requires the option motif Specify the allowed number of mismatches by the argument n If this option is not set then a search for the exact motif will be conducted The non negative integer n has to be chosen from the range 0 3 vicl Specify the number of nucleotide positions to the left and to the right the vicinity that will be searched for TSDs and or one motif around the 5 and 3 predicted boundary of a LTR retrotransposon This option has only an effect if option mintsd and or option motif is switched on If this option is not selected by the user the default value of is 60 overlaps no best all Specify the output with regard to nested and or overlapping LTR retrotransposon predictions If the argument no is selected then neither nested nor overlapping predictions will be reported in the output If the argument best is selected then in the case of two or more nested or overlapping predictions solely the LTR retrotransposon prediction with the highest similarity between its LTRs will be reported If the argument all is selected then all LTR retrotransposon predictions will be reported whether there are nested and or overlapping predictions or not If this option is not selected by the user the option with argument best is set by default 2 5 Alignment options An X drop extension process permits the search for degenerated LTRs The alignment options pro vide the opportunity2. set to 15000 by default similar similaritythreshold Specify the minimum similarity value between the two LTRs The argument similaritythreshold has to be chosen from the range 0 100 and means a percentage If this option is not selected by the user the default value is set to 85 mintsd TSDminlen If this option is selected a search for target site duplications TSDs will be conducted with a minimum TSD length of 7SDminlen If this option is not selected by the user a search for TSDs with minimum TSD length 4 will be conducted If this option is set but no maximum TSD length is specified by the option maxtsd then the maximum TSD length is set to 20 by default maxtsd TSDmaxlen This option requires the option mintsd If this option is selected a search for target site duplications TSDs will be conducted with a maximum TSD length of TSDmaxlen motif expr Specify 2 nucleotides for the starting motif and 2 nucleotides for the ending motif at the begin ning and the ending of each LTR respectively Only palindromic motif sequences where the motif sequence is equal to its complementary sequence read backwards are allowed e g tgca Type the nucleotides without any space separating them If this option is not selected by the user then candidate pairs will not be checked if they contain a motif If this options is set but no allowed number of mismatches is specified by the option motifmis then a search for the exact motif will
3. verbose mode longoutput additional motif TSD output requires mintsd or motif help show all options The option index is mandatory range z y Specify the range in the input sequence s to be searched for LTR retrotransposon candidates That is if x 1000 and y 10000 then candidates are only reported if they start after position 1000 and end before position 10000 in their respective sequence coordinates If x y 0 default then the whole sequences are searched 2 3 Output options Results are reported in tabular fashion on stdout and can easily be written to a file using the notation gt resultfile as in the following gt ltrharvest index indexname options gt resultfile In addition the following options write extra information to files out outputfile Specify the name of a file where the predictions will be written to Each prediction will be represented by an individual FASTA entry outinner outputfile Specify the name of a file where the inner regions the prediction sequences without the flanking LTR sequences will be written to Each prediction will be represented by an individual FASTA entry gff3 outputfile Specify the name of a file where the predictions will be written to Each prediction will be represented by an individual GFF3 2 entry 2 4 Filter options The filter options provide the opportunity to exclude predictions with unwanted sequence length or distance features If a particula
4. 2 19970727 fsa TIME overall 1 37 Now we can use the index for LTRharvest The first example call will just use the default parameters for the filter and alignment options without searching for TSD or an LTR start end motif gt ltrharvest index chr02 19970727 fsa args index chr02 19970727 fsa predictions are reported in the following way s ret e ret l ret s 1LTR e 1LLTR 1 1LTR s rLTR e rLTR 1 rLTR sim LTRs seq nr where s starting position e ending position 1 length ret LTR retrotransposon 1LTR left LTR cLTR right LTR sim similarity seq nr sequence number 259532 265448 5917 259532 259863 332 427672 430021 2350 427672 428170 499 29632 35597 5966 29632 29969 338 35259 220989 226919 5931 220989 221339 351 265117 429522 265448 430021 339 226574 226919 332 500 98 82 346 99 40 91 60 0 97 15 0 0 0 Each comment line starts with the comment symbol Each non comment line denotes a LTR retro transposon prediction with starting and ending positions of the whole LTR retrotransposon the left LTR instance and the right LTR instance respectively Furthermore for each of these elements the corresponding element length is reported as well as a percentage similarity of the two LTRs The last integer of each line denotes the number of the input sequence the LTR retrotransposon prediction occurs in The input sequence numbers are counted fro
5. 5 338 gaaat 5 tg ca 100 00 15 56452 62375 5924 56452 56788 337 gttat 5 tg ca 62039 62375 337 gttat 5 tg ca 100 00 15 3 3 Sequence clustering of LTRharvest output optional In addition to the prediction process done by LTRharvest a cluster analysis on the predicted sequences is recommended Here we choose the single linkage cluster analysis program from the Vmatch software tool 7 which is not part of the GenomeTools binary gt in order to show how this task can be accomplished An index needs to be constructed from the predicted sequences by the program mkvt ree which is part of Vmatch mkvtree db pred chrAll fsa dna pl allout v reading file pred chrAll fsa overall space peak main 2 61 MB 10 01 bytes symbol secondary 0 53 MB Now we can use vmatch which is part of Vmatch to perform a clustering of the predicted se quences A reasonable parameter set is given in Tab 2 page 11 See the Vmatch manual 7 for an explanation of the options The following command computes clusters of all database sequences in pred chrA11 fsa Each cluster has a unique cluster number followed by the sequence number contained in the cluster vmatch dbcluster 95 7 Cluster pred chrAll p d seedlength 50 1 1101 exdrop 9 pred chrAll fsa args dbcluster 95 7 Cluster pred chrAll p d seedlength 50 1 1101 exdrop 9 pred chrAll fsa 3 clusters 45 elements out of 46 97 83 are in clusters 1 elements out of 46 2 17 are sin
6. 70 499 429522 9632 35597 5966 29632 29969 338 35259 20989 226919 5931 220989 221339 351 226574 39597 6339 265448 430021 226919 332 99 40 0 500 91 60 0 98 82 346 97 15 0 Searching additionally for TSD and a LTR start end motif we use the options mintsd maxtsd gt ltrharvest index chr02 19970727 fsa mintsd 5 maxtsd 20 motif tgca motif and motifmis motifmis 0 args index chr02 19970727 fsa mintsd 5 maxtsd 20 motif tgca motifmis 0 predictions are reported in the following way S 2 2 2 s ret e ret l ret s 1LTR e 1LTR 1 1LTR s rLTR e rLTR eq nr where s starting position e ending position 1 length ret LTR retrotransposon 1LTR left LTR rLTR right LTR sim similarity seq nr sequence number 59532 265448 5917 259532 259863 332 265117 9632 35590 5959 29632 29953 332 35259 20996 226911 5916 220996 221329 334 226575 35590 332 265448 226911 1 rLTR sim LTRs 332 99 40 0 99 70 337 97 33 0 Finally if we are interested in the sequence and the length of the TSD as well as the sequence of the motif we select the option longoutput This also is an example for specifying all filter and alignment options by the user gt ltrharvest index chr02 19970727 fsa seed 30 xdrop 5 mat 2 mis 2 ins del 3 minlenltr 100 maxlenltr 1000 mindistltr 1000 maxdistltr 15000 similar 90 0 o
7. LTRharvest a manual David Ellinghaus Stefan Kurtz Ute Willhoeft Research Group for Genomeinformatics Center for Bioinformatics University of Hamburg Bundesstrasse 43 20146 Hamburg Germany willhoeft zbh uni hamburg de In any documentation or publication about research using LTRhar vest please cite the following paper D Ellinghaus S Kurtz and U Willhoeft L7Rharvest an efficient and flexible software for de novo detection of LTR retrotransposons BMC Bioinformatics 2008 9 18 http www biomedcentral com 1471 2105 9 18 August 6 2012 1 Introduction This document describes LTRharvest 1 a software tool for de novo predictions of LTR retrotrans posons in genomic sequences 4 LTRharvest computes boundary positions of potential LTR retro transposons on a persistent index structure of the genomic target sequence the enhanced suffix array 3 Usually the genomic target sequence is a complete chromosomal DNA sequence in FASTA for mat Nevertheless every DNA sequence in multiple FASTA format can be passed to the software For the prediction LTRharvest implements several filters These are consecutively applied on the sequence data to reject candidates which are not conform with sequence length or distance features of LTR retrotransposons Since these features are mostly species specific every filter can be switched on or switched off and is free for parameterisation of a certain LTR retrotransposon model LTRhar
8. est needs the corresponding tables Furthermore we specify option dna as we are processing DNA sequences gt suffixerator db chrAll1 19971001 fsa gz indexname chrAl1 19971001 fsa tis suf lcp des sds dna dna yes indexname chrAl11 19971001 fsa TIME overall 108 35 Now we can use the index with LTRharvest In addition to the filter and alignment options we choose option out for printing the predicted LTR retrotransposon sequences to a file gt ltrharvest index chrAll 19971001 fsa seed 100 minlenltr 100 maxlenltr 1000 mindistltr 1000 maxdistltr 15000 xdrop 5 mat 2 mis 2 ins 3 del 3 similar 90 0 overlaps best mintsd 5 maxtsd 20 motif tgca motifmis 0 vic 60 longoutput out pred chrAll fsa args index chrAll 19971001 fsa seed 100 minlenltr 100 maxlenltr 1000 mindistltr 1000 maxdistltr 15000 xdrop 5 mat 2 mis 2 ins 3 del 3 similar 90 0 overlaps best mintsd 5 maxtsd 20 motif tgca motifmis 0 vic 60 longoutput out pred chrAll fsa predictions are reported in the following way s ret e ret l ret s 1LLTR e 1LTR 1 1LTR TSD 1 TSD m 1LTR s rLTR e rLTR 1 rLTR TSD 1 TSD m rLTR sim LTRs seq nr where 160239 166163 5925 160239 160575 337 ggttc 5 tg ca 165827 166163 337 ggttc 5 tg ca 100 00 Q 29632 35590 5959 29632 29963 332 ataat 5 tg ca 35259 35590 332 ataat 5 tg ca 99 70 1 844407 850335 5929 844407 844744 338 gaaat 5 tg ca 849998 85033
9. glets luster of size 2 luster of size 3 cluster of size 40 O 32 41 42 36 39 13 40 43 37 30 44 38 18 21 17 34 35 31 12 20 33 23 26 27 6 28 3 29 251249192722 4 1050 t ga LS ed EE 2 14 45 aaoo 1 1 1 References 1 http www zbh uni hamburg de ltrharvest 2 GFF3 tab delimited file format for genome annotation http www sequenceontology org gff3 shtml 10 Table 2 A default parameter set for Vmatch s single linkage clustering program Parameter name Value Comment dbcluster 95 Emin x 100 1 Match covers at least 95 of the smaller sequence and formula value of the larger sequence d Compute direct matches p Compute palindromic matches seedlength 50 Minimal length of the exact repeats l Dmin Lmin Minimal length of matches exdrop 9 Xdrop score when extending a seed in both directions 3 M I Abouelhoda S Kurtz and E Ohlebusch Replacing suffix trees with enhanced suffix arrays Journal of Discrete Algorithms 2 53 86 2004 4 D Ellinghaus S Kurtz and U Willhoeft LTRharvest an efficient and flexible software for de novo detection of LTR retrotransposons BMC Bioinformatics 9 18 2008 5 G Gremme The GENOMETOOLS genome analysis system http genometools org 6 J M Kim S Vanguri J D Boeke A Gabriel and D F Voytas Transposable elements and genome organisation a comprehensive survey of retrotransposons revealed by the co
10. m 0 Invoking LTRharvest with the optional argument v gives more information about enabled and dis abled options as well as additional information about the enhanced suffix array index and time space consumption Moreover specifying options out and outinner the run results in two multiple FASTA files gt ltrharvest index chr02 19970727 fsa v out pred chr02 fsa outinner pred inner chr02 fsa pred inner chr02 fsa user defined options and values verbosemode On indexname chr02 19970727 fsa outputfile pred chr02 fsa xdropbelowscore 5 Similaritythreshold 85 00 minseedlength 30 matchscore 2 mismatchscore 2 insertionscore 3 deletionscore 3 minLTRlength 100 maxLTRlength 1000 minLTRdistance 1000 maxLTRdistance 15000 overlaps best minTSDlength 0 maxTSDlength 20 palindromic motif motifmismatchesallowed 4 vicinity 60 nt s ret e ret l ret s 1LTR e 1LTR 1 1LTR seq nr where s starting position e ending position 1 length outputfile inner region pred inner chr02 fsa predictions are reported in the following way e rLTR args index chr02 19970727 fsa v out pred chr02 fsa outinner 1 rLTR sim LTRs Se Se SR HE 2 4 2 2 ret LTR retrotransposon 1LTR left LTR rLTR right LTR sim similarity seq nr sequence number 59532 265448 5917 259532 259863 332 265117 27672 430021 2350 427672 4281
11. mplete Sac charomyces cerevisiae genome sequence Genome Research 8 5 464 478 1998 7 S Kurtz The VMATCH large scale sequence analysis software http www vmatch de 11
12. nhanced suffix array index range specify the range in the input sequences to be searched for LTR retrotransposon candidates Output options out specify outputfilename for predictions in multiple FASTA format gff3 specify outputfilename for predictions in GFF3 format outinner specify outputfilename for multiple FASTA file of the inner regions of predictions Filter options seed specify minimum seed length for exact maximal repeats minlenltr specify minimum length for each LTR maxlenltr mi ndistltr maxdistltr specify maximum length for each LTR specify minimum distance of LTR startpositions specify maximum distance of LTR startpositions similar specify similaritythreshold in range 1 100 mintsd specify minimum length for each TSD maxtsd specify maximum length for each TSD requires mint sd motif specify palindromic motif consisting of 2 nucleotides startmotif 2 nucleotides endmotif motifmis specify maximum number of mismatches in motif requires mot i f vic specify the number of nucleotides to the left and to the right that will be searched for TSDs and or motifs around 5 and 3 boundary overlaps specify no best all Alignment options xdrop specify xdrop value for seed extension mat specify matchscore for seed extension mis specify mismatchscore for seed extension ins specify insertionscore for seed extension del specify deletionscore for seed extension Miscellaneous options v
13. ongoutput This option additionally prints information about the detected TSD and or the motif to stdout if a search for TSD and or for the motif has been selected by the user This option requires the option mintsd and or motif help LTRharvest will show a summary of all options on stdout and terminate with exit code 0 3 Examples In this section example applications of LTRharvest are presented In Subsection 3 1 examples for using different options of LTRharvest are given Subsection 3 2 then gives an example for the pre diction of LTR retrotransposons on the entire S cerevisiae genome In Subsection 3 3 an example for a clustering process of the LTRharvest output is shown Please note that this step is not part of LTRharvest and is carried out by Vmatch 7 3 1 Using different options of LTRharvest As target sequence file we use some FASTA file chr02 19970727 fsa gz containing the S cere visiae genome sequence chromosome 2 Note that the file is a compressed file in gzip format because of the ending gz This format can be handled by the program Suffixerator First we create the enhanced suffix array We invoke gt suffixerator with options tis suf 1cp des ssp and sds since LTRharvest needs the corresponding tables Furthermore we spec ify dna as we process DNA sequences gt suffixerator db chr02 19970727 fsa gz indexname chr02 19970727 fsa tis suf lcp des ssp sds da a dna yes indexname chr0
14. r option is not set by the user a default value for this options will be set except for the options mintsd maxtsd and motif Thus if none of the filter options is set by the user a prediction of LTR retrotransposons will be conducted without searching for target site duplications TSD or a particular LTR start end motif seed Lex Specify the minimum length for the exact maximal repeats Only those repeats with the spec ified minimum length are analyzed in the process of finding candidate pairs Exact maximal repeats of length below that threshold are not taken further into account Les has to be a positive integer If this option is not selected by the user then Les is set to 30 by default minlenltr Lmin Specify the minimum length of each LTR Lmin has to be specified as a positive integer If this option is not selected by the user then Lmin is set to 100 by default 3 maxlenltr Lmar Specify the maximum length of each LTR Lmas has to be specified as a positive integer If this option is not selected by the user then Lmax is set to 1000 by default mindistltr Dmin Specify the minimum distance of LTR starting positions Dmin has to be specified as a positive integer If this option is not selected by the user then Dmin is set to 1000 by default maxdistltr Dmaz Specify the maximum distance of LTR starting positions Dmax has to be specified as a positive integer If this option is not selected by the user then Dmax is
15. to control this extension of the maximal repeat seeds If a particular alignment option is not set by the user a default value for this options will be set xdrop X Specify the xdrop value X for extending a seed repeat in both directions allowing for matches mismatches insertions and deletions The argument X must be a positive integer or 0 The X drop extension process stops as soon as the extension involving matches mismatches inser tions and deletions has a score smaller than T X where T denotes the largest score seen so far If this option is not selected by the user then X is set to 5 by default mat score Specify the positive match score for the X drop extension process If the option is not selected by the user the default value is 2 mis score Specify the negative mismatch score for the X drop extension process If this option is not selected by the user the default value is 2 ins score Specify the negative insertion score for the X drop extension process If this option is not selected by the user the default value is 3 del score Specify the negative deletion score for the X drop extension process If this option is not selected by the user the default value is 3 2 6 Miscellaneous options This option enables the verbose mode This means that some more information about the processing will be printed to stdout during the run This includes a long list of switched on or switched off options l
16. verlaps all mintsd 5 maxtsd 20 motif tgca motifmis 0 vic 60 longoutput args index chr02 19970727 fsa seed 30 xdrop 5 mat 2 minlenltr 100 maxlenltr 1000 mindistltr 1000 maxdistltr 15000 similar 90 0 overlaps all mintsd 5 maxtsd 20 motif tgca motifmis 0 vic 60 longoutput predictions are reported in the following way s ret e ret l ret s 1LTR e 1LTR 1 1LTR TSD 1 TSD cLTR 1 crLTR TSD 1 TSD m rLTR sim LTRs where s starting position e ending position 1 length m motif ret LTR retrotransposon 1LTR left LTR seq nr mis 2 m 1LTR ins 3 s rLTR del 3 3 cLTR right LTR TSD target site duplication sim similarity seq nr sequence number 259532 265448 5917 259532 259863 332 gtaat 5 tg ca 265117 265448 332 gtaat 5 tg ca 99 40 0 29632 35590 5959 29632 29963 332 ataat 5 tg ca 35259 35590 332 ataat 5 tg ca 99 70 0 220996 226911 5916 220996 221329 334 ggaat 5 tg ca 226575 226911 337 ggaat 5 tg ca 97 33 0 3 2 Predictions on the entire S cerevisiae genome As target sequences file we use some multiple FASTA file chrAl1 19971001 f sa gz which con tains all 16 chromosomal sequences of the release before Oct 1st 1997 probably used by Kim et al in their comprehensive survey of retrotransposons 6 First we create the enhanced suffix array We invoke gt suffixerator with options tis suf lcp des since LTRharv
17. vest is written in C and it is based on the GenomeTools library 5 LTRharvest is called as part of the single binary named gt LTRharvest runs on an enhanced suffix array index which is stored on files This index needs to be constructed by the program suffixerator which is also part of the GenomeTools binary gt 2 Usage Some text is highlighted by different fonts according to the following rules e Typewriter font is used for the names of software tools e Small typewriter font is used for file names e Footnote sized typewriter font with a leading is used for program options e small italic font is used for the argument s of an option 2 1 The options of LTRharvest Since LTRharvest is part of gt LTRharvest is called as follows gt ltrharvest index indexname options where indexname denotes the enhanced suffix array index of the target sequence s constructed by the program Suffixerator An overview of all possible options with a short one line description of each option is given in Table 1 All options can be specified only once 2 2 Input options index indexname Specify the name of the enhanced suffix array index The index must comprise the tables suf lcp des and tis An example of how the enhanced suffix array index is constructed with the program Suffixerator can be found in section 3 Table 1 Overview of the LTRharvest options sorted by categories Input options index specify the name of the e
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