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read 00readme located in ReAS_2.02/code
Revision as of 14:05, 15 April 2010
To simplify, this page assumes eukaryotic genomic DNA repeat finding.
Repeat finding can be divided into two tasks, depending on availability of repeat library:
A) Library exists for a given (or possibly closely related species)
B) you construct such library de novo.
Task A is usually a prerequisite step for genome annotation and even blast searches. For newly sequences genomes one should start with B (constructing species specific repeat library).
Detecting known repeats
Most comonly used: Repeatmasker
- web site: http://www.repeatmasker.org/
- current version (checked on 2010-03.22): 3.2.8
- documentation: http://www.repeatmasker.org/webrepeatmaskerhelp.html
- Online web server 
- command line
You have to have a FastA file (it can be multiple FastA). Type:
You will get a file: your_sequence_in_fasta_format.masked --- name tells all
species options (choose only one):
-m(us) masks rodent specific and mammalian wide repeats -rod(ent) same as -mus -mam(mal) masks repeats found in non-primate, non-rodent mammals -ar(abidopsis) masks repeats found in Arabidopsis -dr(osophila) masks repeats found in Drosophilas -el(egans) masks repeats found in C. elegans
De novo repeat library construction
For programs recommendations based on test see: Saha et al. Empirical comparison of ab initio repeat finding programs (2008)
For an extensive review listing tens of programs: Lerat E.Identifying repeats and transposable elements in sequenced genomes: how to find your way through the dense forest of programs (Nov 2009)
Keep in mind that resulting libraries should be further screened for gene families. There are border cases, where genome may contain thousands of modified copies of a gene, ranging from seemingly functional copies, through pseudogenes, gene fragments and single exons (i.e Speer family in rodents).
One has to have at least a draft of the genome or multiple genomic sequences.
command line only, requires compilation
current version (2010-03): 1.05
- PPT presentation presenting algorithm: http://bix.ucsd.edu/repeatscout/repeatscout-ismb.ppt
- publication (PDF)De novo identification of repeat families in large genomes 2005
- build frequency table
build_lmer_table -sequence input_genome_sequence.fas -freq output_lmer.frequency
output_lmer.frequency file can be still quite large (1.7Gb for 900Mb fasta file)
- create fasta file containing all kinds of repeats
RepeatScout -sequence input_genome_sequence.fas -output output_repeats.fas -freq output_lmer.frequency
- RAM usage (RepeatScout): > 17Gb for 800Mb genomic sequence.
- 9.6h Xeon E7450 @ 2.40GHz
The output (output_repeats.fas) is a fasta file with headers (>R=1, >R=232 etc.). It contains also trivial simple repeats (CACACA...), tandem repeats
- filter out short (<50bp) sequences. Remove "anything that is over 50% low-complexity vis a vis TRF or NSEG.". Perl script.
It does require trg and nseg to be on the PATH, or setting env variables TRF_COMMAND and NSEG_COMMAND pointing to their location
filter-stage-1.prl output_repeats.fas > output_repeats.fas.filtered_1
this prints tons of messages
- run RepeatMasker on your genome of interest using filtered RepeatScout library
RepeatMasker input_genome_sequence.fas -lib output_repeats.fas.filtered_1
This is a very long step (36h for 800Mb draft genome) when run in such default mode. See discussion for this page for possible, but so far untested speedups.
Output used for the next step: input_genome_sequence.fas.out
- filtering putative repeats by copy number. By default only sequences occurring > 10 times in the genome are kept
cat output_repeats.fas.filtered_1 | filter-stage-2.prl --cat=input_genome_sequence.fas.out > output_repeats.fas.filtered_2
Fast (< 1min ). You can modify the filter using i.e. "--thresh=20" (only repeats occurring 20+ times will be kept)
Piler (with lastz)
- lastz from http://www.bx.psu.edu/miller_lab/ (tested version from 2010-Jan-12)
- piler from http://www.drive5.com/piler/ (tested version 1.0)
Also read the manual of pals, in case you need to understand the piler's gff input format: http://www.drive5.com/pals/pals_userguide.htm Both programs install easily on Ubuntu 9.10.
- fasta file. Fasta identifiers should not contain spaces/special characters (stay with alpha-numeric ones)
- things to keep in mind:
- default fasta output of i.e. Newbler needs to be fixed
lastz my_fasta_input.fa[multiple] my_fasta_input.fa --output=my_fasta_lastz_output.csv \ --format=general:name2,zstart2,end2,score,strand2,name1,zstart1,end1,identity --notrivial --ambiguousn --markend
Converting lastz output to gff piler input
Python script (still in the testing phase)
Draft! See: http://www.drive5.com/piler/piler_userguide.html
piler -trs my_fasta_lastz_output.gff -out my_fasta_lastz_output.trs
This is pre-assembly repeat finding method.
- get ftp://ftp.genomics.org.cn/pub/ReAS/software/ReAS_2.02.tar.gz
- unpack it in a suitable directory
tar xfvz ReAS_2.02.tar.gz; cd ReAS_2.02/code
- open N_matchreads.cpp and add line below i.e. after "#include<time.h>":
- compile ReAS
make; make install
You will get binaries + perl modules in ReAS_2.02/bin
- Put them on $PATH (bash)
read 00readme located in ReAS_2.02/code for more detailed instructions.
To run on 8 CPU cores with otherwise default settings with already selected set of reads:
nohup reas_all.pl -read 454_subset_4repeat_search.fas -pa 8 -output 454_subset_4repeat_search.fas.cons_reas 2 > nohup.reas1.txt &
For pages on similar topics visit: Wikiomics@OpenWetWare
- Church DM, Goodstadt L, Hillier LW, Zody MC, Goldstein S, et al. 2009 Lineage-Specific Biology Revealed by a Finished Genome Assembly of the Mouse. PLoS Biol 7(5): e1000112. doi:10.1371/journal.pbio.1000112