RNA-MATE user manual - Expression Genomics Laboratory
Contents
1. BC_length 5 Indicate the length of barcode sequence raw_BC data cxu post_mapping_data S0014_ 20080526 _2 PolysomeAndAtlas_microRNA_Atl as_R3 csfasta This file lists each tag s barcode sequence which must belong to the barcodes listed in the configure file Eg BC1 BC10 It is not necessary to list this parameter if all tags are from same barcode Adaptor Parameters name_Adaptor Adaptor_BC1 Adaptor_BC2 Adaptor_BC3 Adaptor_BC4 Adaptor_BC5 Adaptor _BC6 Ad aptor_BC7 Adaptor_BC8 Adaptor_BC9 Adaptor_BC10 This parameter lists all adaptor names Each adaptor name must follow format Adaptor _ BC the BC must be consistent with the barcode names listed in name_BC All related adaptor sequences must be listed in this configure file See below example Adaptor_BC1 33020103031311231200032032222031220201003000312 Adaptor_BC2 33020103031311231200111122222031220201003000312 Adapt or_BC3 33020103031311231220200022222031220201003000312 Adaptor_BC4 33020103031311231230323122222031220201003000312 Adaptor_BC5 33020103031311231231013132222031220201003000312 Adaptor_BC6 33020103031311231201130012222031220201003000312 Adaptor_BC7 33020103031311231201221022222031220201003000312 Adaptor_BC8 33020103031311231221302222222031220201003000312 Adaptor_BC9 33020103031311231202020022222031220201003000312 Adaptor_BC10 33020103031311231202103312222031220201003000312 Adaptor_start 33020 The pipeli2. Congratulations you have just built a hairpin library and index suitable for use with miRNA MATE You will need both the index and cat files to pass to miRNA MATE in its configuration file Condensing the mature miRNAs by mapping distance and family relationship Making mature miRNA libraries is a little more involved because the sequences are small and miRNAs that cross map as a single entity need to be grouped Additionally it is beneficial to include miRNAs from other species to determine the level of background mapping noise versus miRNA expression however these additional miRNAs may well be identical to miRNAs in the species you are interested in and could therefore cause multi mapping The process has the following basic steps e create a temporary library of the mature miRNAs e split the miRBase sequences by length and match them to the temporary library ie themselves at a given number of mismatches e collect and cluster all the miRNAs that multi map and assign them to miRNA families e collect all related miRNAs and assign them to miRNA families e create the index and cat files as above for hairpin miRNAs e make separate and custom indices for the recursive matching and the isomiR matching Note Because the miRNAs are mapped at a specific number of mismatches the library you create here will only be valid for matching with that same number of mismatches Hence different libraries will be required each time matchin
3. MATE mv miRNA MATE tar gz home software cd home software tar xzvf miRNA MATE tar gz cd miRNA MATE Page 7 of 21 2 To install this package type the following sequence of commands perl Makefile PL make make install The final make install command may require that you be root or that you have permission to use the sudo command If you want to install it locally append your location to PREFIX on the first command eg perl Makefile PL PREFIX yourDir 3 Move f2m pl to the directory where mapreads is installed e g mv yourDir miRNA MATE bin f2m pl yourMapreadsDir Note If installed properly there should be four executable files and one schema directory f2m pl map mapreads mask_schemas_mapreads pl schemas Note The perl script f2m pl requires Path Class which you can put in the mapreads directory if you didn t install it globally The script mask_schemas_mapreads pl is also in the miRNA Mate package bin directory and can be copied to the mapreads directory 4 Make sure your perl path is set and includes the miRNA MATE lib directory export PERL5LIB yourDir miRNA MATE 1lib perl5 site_perl 5 8 8 5 Make sure weblogo is installed and accessible otherwise the final logo images can t be created miRNA MATE should now be ready to test Page 8 of 21 Testing miRNA MATE To ensure your installation is functioning correctly we have provided test data and comparison results withi
4. ength 29 miRNA_freq_filter 100 miRNA_start_offset 10 miRNA_end_offset 10 w_seq_length 50 recursive_map true recursivesmapipara 30 PO 29r IROS n2 Jeet 22 0 2 Del OZ An eOn DS r rw 2 2cle OZ lO 20m denial Vode Oe ey sO ye 0 NAGS jiehor parad 2 9 n2 IRIA Molle Qa Asli ZO a yrclk va aA lea Al nD dhe AG ae le ae Dale Ab AAW Ne Al Aig A es Ae ANS es hn Ihe alts 2 ll IRE nek expect_strand run_seqlogo data weblogo seqlogo f input F GIF h 10 w 30 k 0 o output c f 2m data cxu mapping_strategy matching f2m pl mapreads data cxu mapping_strategy matching mapreads script_editing data cxu mapping_strategy matching editing pl recursive_library data cxu MiRNA library discovery_miRNA_library padded cat recursive_library_index data cxu MiRNA library discovery_miRNA_library padded index once_library data cxu MiRNA library human_miRNA_editing_library padded cat once_library_index data cxu MiRNA library human_miRNA_editing_library padded index Figure 3 Example of a miRNA MATE configuration file Page 10 of 21 Configuration options Standard Parameters exp_name S0014 20080526_2 PolysomeAndAtlas_microRNA_Atlas Set the experiment name with this parameter output_dir data MiRNA 20080526_output Specify the full path of the output directory This directory must exist prior to running the pipeline raw_F3 data cxu post_mapping_data S0014_20080526_2 PolysomeAndAtlas_microRNA_Atl a
5. ference sequence w_seq_length 50 For visualization purposes we add the letter w on both sides of high frequency miRNA sequences whose adaptor sequences have been stripped This procedure makes these sequences the same length as non stripped sequences The length can be specified here Description of output files For all files listed below the string lt expname gt refers to the experiment name provided by the exp_name configuration parameter lt BC gt refers to one of the names given for the bar codes using the name_BC parameter lt run_type gt is either recursive or once lt direction gt is either positive tag matched in the expected orientation and negative tag matched on the reverse strand to the direction provided in the library file lt seqLength gt refers to the length of the input sequence and will vary according to the optional recursive strategy employed lt libName gt is the name of the library files This is used to prefix some raw mapping output files lt miRNA gt is the name of the matched miRNA or miRNA family found in the dataset and listed in the provided library files e lt expname gt lt BC gt log The log file for the run This file contains important information about the status of a run and should help diagnose any potential errors e lt expname gt lt BC gt lt runtype gt SIM lt direction gt freq A frequency file containing the frequency of occurre
6. for making the package available with the miRNA MATE system Page 2 of 21 Table of Content Th MIRNA MA TE Pipelihes prun nia i e a a A a EE E E E 4 INMIHODUCHON TORNO E EEEE tee enone cenn caus 4 PRY AT abiity seor a e E E a oe Gia ie coves a 6 Reguirements ecean a E bud gente E E E R 7 Installation instructions MIRNA MATE ccccccssccscccccecescsessssecececeeeceesesseseceseceesesessnsaeceseeeens 7 Testing MmiR NA MATE gnana e a o E A uncesaqdevadavensdsedaasdacenteccueeaeess 9 Configurations ids 2 cat a dats ad neuen Saale E E A E a OS 9 Configuration OPUOMS isi ceeien dea sevdyeastunynmen E E E E OEE STEEN SENEE 11 Descipton of output TES seser csnsdat anatase caaiiecuebiat an atass cevaatis ina eaten caadal aca doduacsals 15 Creating custom miRNA libraries os cciscecccccesaisesesendcovesveavareteusteeveslavasatassadedecaaaaessaddasgacaeedbaeneensee 16 Preprocessing the miRBase download files u 15 2 s2 c0d ccseeessacedceesee code censtecdvensczeeuecevicencebecseenscie 16 Finishing the hairpin libraries for mapping ceeesceeseneceececeeaeeceeneeceeneeceeaeeceeceeeeeteeeesaeees 17 Condensing the mature miRNAs by mapping distance and family relationship 18 Create a temporary library of the mature mMiRNAS seeessssssssrsesesressessresressessrssressessresrresreseresre 18 Split the miRBase sequences by length and matching them to the temporary library 19 Cluster all the miRNAs that
7. g is performed using a different mismatch number For example a IMMVA library allows 0 1 colour space errors counting valid adjacent errors as one error should not be used as a reference when matching is performed allowing 2MMVA 2 colour space errors counting valid adjacent errors as one error Allowing fewer mismatches would be fine Create a temporary library of the mature miRNAs This process is identical to the process described above for finishing the hairpin libraries 03_pad_mature_miRNAs pl f mature fasta o mature fasta padded 04_make_index pl f mature fasta padded o mature fasta padded index 05_concatenate_sequences pl f mature fasta padded o mature fasta padded cat h temp_miRNA_library_miRBasev15 Page 18 of 21 Split the miRBase sequences by length and matching them to the temporary library Mapreads will only match at one given length at a time but miRNAs in miRBase range from 16 35 nucleotides In order to match them all the miRNAs in the miRBase files need to be separated by length The following scripts will split the miRBase input files and convert them to colour space 1 This script takes the preprocessed mature sequences and splits them by length outputting a series of files called split_miRBase length 06_split_by_length pl f mature fasta 2 The next script takes the output of the above script and converts the nucleotide sequences to colourspace tags prepends a T and converts to col
8. ge Figure 3 Page 9 of 21 exp_name S0014_20080526_2 PolysomeAndAtlas_microRNA_Atlas output_dir data cxu MiRNA scripts Jun18 test raw_F3 data cxu post_mapping_data S0014_20080526_2 PolysomeAndAtlas_microRNA_Atlas_F3 csfasta raw_BC data cxu post_mapping_data S0014_20080526_2_ PolysomeAndAtlas_microRNA_Atlas_R3 csfasta maS ck aL ah ak ab a ab bal aah aL ababalaeal ab iba ababab ab abit abab abit ababal max_multimatch 5 raw_tag_length 35 Adaptor_start 33020 Adaptor_start_mismatch 1 max_adaptor_mismatch 5 name_Adaptor Adaptor_BC1l Adaptor_BC2 Adaptor_BC3 Adaptor_BC4 Adaptor_BC5 Adaptor_BC6 Adaptor_BC7 Adaptor BES Adaptor_BC9 Adaptor_BC10 Adaptor_BC1 33020103031311231200032032222031220201003000312 Adaptor_BC2 33020103031311231200111122222031220201003000312 Adapt or_BC3 33020103031311231220200022222031220201003000312 Adaptor_BC4 33020103031311231230323122222031220201003000312 Adaptor_BC5 33020103031311231231013132222031220201003000312 Adaptor_BC6 33020103031311231201130012222031220201003000312 Adaptor_BC7 33020103031311231201221022222031220201003000312 Adaptor_BC8 33020103031311231221302222222031220201003000312 Adaptor_BC9 33020103031311231202020022222031220201003000312 Adaptor_BC10 33020103031311231202103312222031220201003000312 name_BC BC1 BC2 BC3 BC4 BC5 BC6 BC7 BC8 BC9 BC10 BC_length 5 BC1 G00032 BC2 G00111 BC3 G20200 BC4 G30323 BC5 G31013 BC6 G01130 BC7 G01221 BC8 G21302 BC9 G02020 BC10 G02103 max_miRNA_l
9. iRNA MATE package This script takes multi line fasta files and converts the file to a one line fasta format sequence spans a single line with no line break This step makes is easier to work with the files but is not important for the mature sequences which are typically so small that they don t span multiple lines anyway 01_make_one_line_fasta pl f miRBasevl5 hairpin fa o hairpin fa one_line 01_make_one_line_fasta pl f miRBasev15 mature fa o mature fa one_line Run the script 02_change_U_to_T_and_remove_description pl provided in the miRNA MATE package This script strips the header information and converts the U uracil to T thymine This step is important If you fail to convert the Us to Ts the mapping fill fail later 02_change_U_to_T_and_remove_description pl f hairpin fa one_line o hairpin fasta Page 16 of 21 02_change_U_to_T_and_remove_description pl f mature fa one_line o mature fasta Select the species for mapping using the script 00_filter_species pl provided as part of the miRNA MATE package This final script selects the species that may be of particular interest This is best done for the hairpin library but not for the mature miRNA library Having all species in the mature library allows one to determine the specificity of matching but also allows the possibility of miRNA discovery leveraging the knowledge of other species This can be quite difficult to do in colourspace other
10. immond imb ug edu au miRNA MATE Page 6 of 21 Requirements This pipeline is written in perl and requires that you have version 5 8 8 of perl or later It is designed to run in a unix environment with a PBS queue manager although running the scripts using PBS is not required Required perl modules are available in CPAN e Parallel ForkManager e Path Class e Object InsideOut e Devel StackTrace e Class Data Inheritable The alignment section of this pipeline is dependant upon the mapreads tool This tool and its installation instructions are available from http solidsoftwaretools com gf project mapreads requires registration which is free Additionally the package weblogo is required and can be downloaded here http weblogo berkeley edu Finally you will need a library against which to map The software comes with a custom library generated from miRBase version 16 Future versions of miRBase can be made into libraries using the steps outlined in section Creating custom miRNA libraries All data for the libraries can be downloaded from miRBase here http www mirbase org ftp shtml Installation instructions miRNA MATE The instructions given below in courier font are examples of the commands needed to carry out the installation miRNA MATE source is downloaded as a single gzipped tar file 1 Move the tarball to the destination directory navigate to your chosen directory and decompress miRNA
11. instructions for doing so at the end of this manual miRNA MATE can be run in either of two modes Mode 1 Alignment against mirBase custom library only The first mode maps SOLiD sequencing reads against a custom library designed from miRBase Figure 1 Unaligned tags are trimmed and re aligned against the custom library The resulting output is a count of miRNAs found in the sequencing run Mode 2 Alignment against mirBase custom library and identification of isomer usage In the second mode following alignment against the custom library designed from miRBase SOLiD reads are translated into base space and analyzed for evidence of sequence variants from the canonical mirBase miRNA Figure 1 Sequence variants can be single base substitutions offset from the miRNA start position or length variation The isomiR usage is summarized in form of sequence logos Figure 2 Page 4 of 21 Read Configuration File wy Identify si PL Custom Library Custom Library Alignment I tag aligned tag aligned Discard Tag Yes Yes O ee base space E n d Summarize isomiR usage Create Sequence Logos End Figure 1 Outline of the miRNA Mate pipeline The pipeline can be run in two modes In the first mode only steps 1 3 are performed This mode maps SOLID sequnence reads to a custom library generated from miRBase The output contains counts for each miRNA sequence In the second mode the addit
12. ional steps 4 8 are run After alignment to the custom library sequence reads are translated into base space and searched for evidence of isomiR usage such as single base substitutions miRNA start offset or length variation Page 5 of 21 Ml Figure 2 Example of a sequence logo produced by miRNA MATE If the pipeline is run in mode 2 and the data are analyzed for evidence of isomiRs sequence logos are generated The consensus sequence of the mature miRNA and the star sequence can easily be identified The diagram shows the full length of the hairpin Alternatively only the mature miRNA sequence can be represented as a sequence logo W indicates that positions were padded to make the full length hairpin sequence OO Ze m m The output from miRNA MATE includes a list of miRNAs by their miRBase ID encountered in the sample the number of reads that support the canonical miRNA as represented in miRBase and the number of reads that support isomiRs such as length start or sequence isomiRs Table 1 Table 1 Example of the output summary file produced by miRNA MATE miRNA TOTAL CANONICAL ISOMIR diff_length diff_start diff_sequence hsa let 7b 7 0 7 7 7 6 hsa let 7c 7 0 7 7 7 4 hsa let 7d 2 0 2 2 2 0 hsa let 7e 2 0 2 2 2 0 hsa let 7g 6 0 6 6 6 4 hsa let 7i 2 0 2 2 2 0 Availability All source code documentation and associated files described in this manual are freely available for download from http gr
13. miRNA MATE user manual Version 1 0 January 2011 Contact miRNA mate expressiongenomics org Institute for Molecular Bioscience The University of Queensland St Lucia QLD 4072 Page 1 of 21 License This software is copyright 2011 by the Queensland Centre for Medical Genomics All rights reserved This License is limited to and you may use the Software solely for your own internal and non commercial use for academic and research purposes Without limiting the foregoing you may not use the Software as part of or in any way in connection with the production marketing sale or support of any commercial product or service or for any governmental purposes For commercial or governmental use please contact licensing qcmg org In any work or product derived from the use of this Software proper attribution of the authors as the source of the software or data must be made The following URL should be cited http grimmond imb ug edu au miIRNA MATE This package is distributed in the hope that it will be useful but WITHOUT ANY WARRANTY without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE Applied Biosystems Software components distributed with this package Mapreads carry their own license agreements located at the following URLs http solidsoftwaretools com gf project mapreads We thank and acknowledge the contributions of the developers of the above package as well as Applied Biosystems
14. multimap and assign them to miRNA families 0 0 19 Finishing the mature miRNA libraries for matching eesseeeseseeseesessresressersresresseseresrreseeseresee 20 Description OF modules iioii i a i t ETETE REER E EET Eana 21 Page 3 of 21 The miRNA MATE pipeline Introduction miRNA MATE is a package designed specifically for mapping and summarizing miRNA and isomiR data from SOLiD sequencing data sets MicroRNAs can be particularly challenging to map because of the limited information content in a 22nt molecule Given that we have to allow errors when mapping and that miRNAs are very small it is difficult to align a miRNA tag to a genome unambiguously To get around this we create a library of known miRNAs to match against The generally accepted reference repository for miRNAs is miRBase http www miRBase org Even when considering only known miRNAs there are significant problems with multi mapping because miRNAs are often very closely related For example miR 19a differs from miR 19b by only a single nucleotide Until sequencing error becomes negligible we are unlikely to be able to distinguish these miRNAs from each other The solution that we have adopted is to collapse the miRNAs into families which include known family members but also miRNAs from different families that are likely to mis map miIRNA MATE comes with a custom library generated from miRBase 16 Alternatively a new custom library can be made and we give
15. n the miRNA MATE package in the test directory The test data directory includes e miRNA_mate_input csfasta e miRNA _mate_example conf e miRBasev16 hairpin hsa padded cat e miRBasev16 hairpin hsa padded index e results the directory of results for information about the files in here see the section Description of output files INPORTANT Edit the configuration file so that it refers to the appropriate directories that you have set up on your system You can then run the pipeline using the following command nohup perl path mirna_mate pl c miRNA_mate_example conf amp Where path is the full path to miRNA Mate pl Testing will run miRNA MATE in approximately 15 minutes assuming your PBS queue is empty Once the run has completed the results should be compared to those provided in the results folder In particular the log file should be inspected for any errors and the freq files should be compared for any differences If there are no differences other than time stamps and directory names between your results and the packaged results then the installation is working correctly Configuration The configuration file is a text file containing all the required parameters to run miRNA MATE In this file directory listings must end with a there must be no other punctuation at the end of the lines and there should be no empty lines in this file An example of the configuration file is given on the next pa
16. nce for each miRNA or miRNA family in the data set e lt expname gt lt BC gt lt runtype gt SIM lt direction gt summary A summary file of the miRNAs isomiRs length and start differences for all identified miRNAs in the input data set e lt expname gt lt BC gt lt runtype gt SIM lt direction gt PreSummary sorted An intermediate file used to create the summary file described above e lt expname gt lt BC gt lt runtype gt SIM lt direction gt sorted ID An intermediate file used to create the frequency file described above e lt expname gt lt BC gt lt runtype gt SIM lt direction gt start_end sorted Another intermediate file used when creating the summary and frequency files listed above e lt expname gt lt BC gt mersN lt csfasta gt If the parameter recursive_map is set to true these files contain the csfasta colour space fasta files of the input data truncated to the specified length Only tags which do not map at the longer length are included in subsequent shorter length csfasta files e lt libname gt lt expname gt lt BC gt lt seqLength gt csfasta ma N M Raw mapping output files including STDOUT STDERR flag files shell scripts etc N tag length and M number of mismatches These files are collated to produce summarized mapping output files such as the collated file listed below e lt expname gt lt BC gt lt runtype gt collated The mapping resul
17. ne will strip the adaptor sequences from the raw tags and then map each tag To improve the speed for finding adaptor sequences the program first searches a short sequence identical to the start of the adaptor sequences If a match is found then the rest of the adapter sequence is compared Page 12 of 21 Adaptor_start_mismatch 1 max_adaptor_mismatch 5 Specify these parameters to set the maximum mismatch value for searching the adaptor start sequence and the maximum mismatch value for searching the whole adaptor sequence Mapping Parameters once_map_para 21 2 1 20 2 1 These parameters define the lengths at which matching will occur for tags after the adapter sequences have been stripped off the number of mismatches permissible between the tag and the reference sequence and whether or not to treat valid adjacent errors as a single mismatch These are dot separated parameters with the format of length mismatches valid_adjacent length defines the length of the tag to match at mismatches defines the number of mismatches allowed and valid_adjacent is set to 1 if valid adjacent errors are to be treated as a single mismatch or 0 if they are not In the above example tags of length 21 will be mapped using the parameters 2 mismatches and treating valid adjacent mismatches as a single mismatch NOTE Mapping schemas must be available to do the mapping at the specified length and number of mismatches or else the pipeline
18. nical sequences will need to be created with the appropriate amount of mismatches incorporated see section Creating custom miRNA libraries Parameters for analyzing isomiRs These parameters only apply if miRNA MATE is run in Mode 2 where miRNA sequences are searched for evidence of isomiRs such as miRNAs that differ by a single base or whose start position is offset compared to the canonical miRNA script_editing data cxu mapping_strategy matching editing pl This parameter lists the full path to the script that is used to retrieve the base sequence and information regarding the number of mismatches when comparing to the reference file run_seqlogo data weblogo seqlogo f input F GIF h 10 w 30 k 0 o output c This parameter not only lists the full path to the script but also allows the user to set multiple input parameters The input and output files will be replaced automatically when running this script as part of miRNA Mate max_miRNA_length 29 After adapter sequences have been stripped tags shorter than this length are treated as miRNA sequences miRNA _ freq filter 100 miRNA sequences are only reported if they appear more than this number of times miRNA _start_offset 10 miRNA _end offset 10 Page 14 of 21 We append letter N to each end of the reference sequences in the miRNA library These two parameters indicate how many N s are appended on each side of the miRNA re
19. ourspace There are no arguments for this script it searches the current working directory 07_convert_to_colourspace_with_initial_T pl 3 The next script takes these csfasta files and submits the matching jobs to the queue You need to specify the temporary mature miRNA library created above eg mature fasta padded cat as well as the mismatch parameters 08_match_miRs pl 1 mature fasta padded cat p data matching mapreads s data matching schemas m 2 a 1 This matching step does not take very long since there is a tiny number of samples mapping against a tiny search space At most this script will take about 2 minutes to finish at which point all of the matching results should be concatenated into a single file The directory can then be cleaned up by removing the temporary files cat 2 1 gt mapping_results 2MMVA rm split_miRbase Cluster all the miRNAs that multi map and assign them to miRNA families The next step is to identify those miRNAs that are cross mapping and thus have the potential to allow mismapping to occur miRNAs are assigned to miRNA families based on their match positions and names 1 Identify clusters of miRNAs with similar matching positions This script decodes the matching positions using the specified index file and clusters those miRNAs together that have similar matching positions one line per miRNA cluster Page 19 of 21 09_make_miRNA_clusters pl f mature fasta padded inde
20. prevents errors in miRNA MATE image generation scripts from complaining because there is no data And that s it Congratulations you should now have successfully made a mature miRNA library suitable for use with miRNA MATE Description of modules For module descriptions please use the embedded PerlDoc by typing perldoc lt ModuleName pm gt Page 21 of 21
21. rary 2MMVA padded index KR 05_concatenate_sequences pl f miRBasevl5_mature_miRNA_library 2MMVA padded o miRBasevl5_mature_miRNA_library 2MMVA padded cat h miRBasev15_mature_library_all_miRNAs However the long IDs in the index file will cause havoc with miRNA MATE unless these are condensed Two more indices need to be generated specifically for miRNA MATE The first will be for recursive matching and the second will be for isomiR once only mapping 12_reannotate_index_file pl f miRBasevl5_mature_miRNA_library 2MMVA padded index p hsa Page 20 of 21 The only difference between the two indices is that the recursive index has duplicate names whereas the isomiR index has unique names Figure 4 iRNA family__miR169_1 0 40 iLRNA family__miR169_2 42 82 iRNA family__miR169_3 84 124 LRNA family__miR169_4 126 166 LRNA family__miR169_5 168 209 2MMVA padded index recursive hsa LRNA famil LRNA famil LRNA famil y__miR169 y__miR169 y__miR169 0 42 84 40 82 124 LRNA family__miR169 166 LRNA family__miR169 209 Figure 4 The top panel shows an example of an index file for isomiR once only mapping while the bottom panel shows an example of an index file for a recursive mapping Note the duplicate names in the bottom panel used for recursive mapping This allows the recursive matching to add together all the counts for the family as a single number and
22. s_F3 csfasta Specify the full path of the csfasta file to be mapped raw_tag_length 35 This parameter defines the longest length of the tags contained in the csfasta file mask 11111111111111111111111111111111111 This setting allows you to ignore particular bases in the tag when computing the number of mismatches consider this base 0 do not consider this base The length of the mask should equal the length of the longest tags max_multimatch 5 Defines the maximum number of positions to be reported for multi mapping tags The higher this number the more disk space is required to store the data and the slower the program will run Recommended size for most applications is 10 expect_strand This defines the strandedness of the data For example libraries made with the SREK protocol or other direct ligation protocols will have tags that are sequenced in the sense strand relative to the expressed gene Libraries made with the SQRL protocol will have tags that are sequenced in the antisense relative to the expressed gene Barcode Parameters name_BC BC1 BC2 BC3 BC4 BC5 BC6 BC7 BC8 BC9 BC10 This parameter lists all barcode names All related barcode sequence must be listed on this configure file when more than one barcode are used for whole samples See below example Page 11 of 21 BC1 G00032 BC2 G00111 BC3 G20200 BC4 G630323 BC5 G31013 BC6 G01130 BC7 G01221 BC8 G21302 BC9 G02020 BC10 G02103
23. ts for either recursive or once mapping runs Page 15 of 21 lt expname gt lt BC gt lt runtype gt lt miRNA gt FASTA file containing aligned sequences for the specified miRNA used to create the weblogo lt expname gt lt BC gt lt runtype gt lt miRNA gt gif Weblogo image file for this miRNA lt expname gt lt BC gt NT lt N gt Csfasta file containing all adaptor trimmed sequences of N length lt expname gt lt BC gt lt runtype gt SIM lt direction gt These files list the single match SIM tags in both the positive and negative directions lt expname gt lt BC gt lt runtype gt SIM lt direction gt bc sorted These files provide details about the exact location of matched tags on miRNA hairpins the offset relative to the canonical miRNA location as well as the tag name sequence reference sequence and number of mismatches The sorted file is sorted by reference miRNA sequence Creating custom miRNA libraries While miRNA MATE comes with a custom library for mapping miRNAs new custom libraries can be made following the steps outlined here The process requires access to the UCSC genome browser and perl scripts provided as part of the miRNA MATE package Preprocessing the miRBase download files 1 Download miRNA sequences from miRBase The files mature fa and hairpin fa are required Reformat the files using the script 01_make_one_line_fasta pl provided in the m
24. will fail In this example the schemas required are schema_21_2_adj schema_20_2_adj Mapping schemas are available from http solidsoftwaretools com f2m data cxu mapping_strategy matching f2m pl mapreads data cxu mapping_strategy matching mapreads These parameters define the full paths of the scripts programs required to run miRNA MATE once_library data MiRNA library human_miRNA_editing_library padded cat once_library index data MiRNA library human_miRNA_editing_library padded index These parameters define the miRNA libraries and their associated index files that are used in miRNA MATE Recursive Mapping Parameters recursive_map true Page 13 of 21 recursive_map_para 30 1 0 29 1 0 28 1 0 27 1 0 26 1 0 25 1 0 24 1 0 23 1 0 22 1 0 21 1 0 20 1 0 19 1 0 18 1 0 17 1 0 recursive_library data MiRNA library discovery_miRNA_library padded cat recursive_library_index data MiRNA library discovery miRNA_library padded index If you set recursive mapping to true you must list recursive mapping parameters in the recursive_map_para parameter These parameters define the lengths at which matching will occur recursively The naming convention is the same as for the once_map_para When recursively mapping a tag which fails to map at length N is then truncated and remapped at a smaller length The amount of truncation is specified in the above parameters NOTE miRNA MATE libraries for mature cano
25. wise due to the mapping specificity issues discussed above The following example takes the hairpin fasta file and extracts only the human hsa hairpins into an output file called hairpin fasta hsa 00_filter_species pl f hairpin fasta o hairpin fasta hsa s hsa This is all the preprocessing that needs to happen before continuing with the library construction Hairpin libraries are very straightforward to finish from this point next section but mature miRNA libraries need a little more processing the section after next Finishing the hairpin libraries for mapping There are three scripts required to finish off the hairpin library ready for matching l The first script pads the ends of the hairpin miRNA with a string of 10 N s This prevents short tags mapping across the boundaries of hairpins when they are all concatenated for the final library 03_pad_mature_miRNAs pl f hairpin fasta hsa o hairpin fasta hsa padded The second script makes an index file so that miRNA MATE can deconvolve the matching positions and return a miRNA ID Important you must use the padded file to create the index or the coordinates will be wrong 04_make_index pl f hairpin fasta hsa padded o hairpin fasta hsa padded index The final script concatenates the miRNAs ready for matching with mapreads 05_concatenate_sequences pl f hairpin fasta hsa padded o hairpin fasta hsa padded cat h miRBasev15_human_hairpins Page 17 of 21
26. x m mapping_results 2MMVA o mapping_results 2MMVA clusters The next script then takes these clusters and makes fasta sequences from them removing duplicate sequences as well as those that are fully contained within another sequence 10_create_new_fasta pl f mature fasta m mapping_results 2MMVA clusters o mapping_results 2MMVA clusters fasta miRNAs with similar names e g has mir 27a and has mir 27b generally have similar sequences and these families might be missed when mapping at a particular threshold especially if the miRNA contain insertions or deletions Experience shows however that shadow matching to these family members will occur in large enough data sets To remove this shadow matching members of the same family that might be more distantly related are collapsed This next script performs pattern matching on the miRNA name to retrieve all family members and output the complete miRNA library 11_final_cluster_by_family pl f mapping_results 2MMVA clusters fasta o miRBasevl5_mature_miRNA_library 2MMVA Finishing the mature miRNA libraries for matching Like the temporary matching libraries and the hairpin libraries the same three basic scripts need to be run on the complete miRNA library 03_pad_mature_miRNAs pl f miRBasev1l5_mature_miRNA_library 2MMVA o miRBasevl5_mature_miRNA_library 2MMVA padded SS 04_make_index pl f miRBasev1l5_mature_miRNA_library 2MMVA padded o miRBasevl5_mature_miRNA_lib
Download Pdf Manuals
Related Search