SortMeRNA User Manual - Bonsai Bioinformatics
Contents
1. 1 Evguenia Kopylova evguenia kopylova lifl fr 2 Laurent Noe laurent noe lifl fr 3 Helene Touzet helene touzet lifl fr 2 Installation 2 1 Required g compiler version Tested platforms Ubuntu Linux OS 12 04 and Mac OS X 10 6 8 SortMeRNA uses features of the C 11 standard which are available for GCC compiler versions 4 3 and later To check your compiler version type g version For compiler versions less than 4 3 please follow Subsection 2 1 1 for Ubuntu Linux OS or Subsection 2 1 2 for Mac OS X to configure or install a new compiler 2 1 1 Ubuntu Linux OS instructions 1 Check whether you have other g compiler versions installed 4 3 or later type g then press the tab key gt gt g followed by the tab key 2 If the list output in Step 1 does not include a g compiler of version 4 3 or later install one SortMeRNA was tested with g 4 4 g 4 5 and g 4 6 gt sudo apt get install g 4 4 3 Determine the full directory path of where g 4 4 was installed in the example below the path is usr bin g 4 4 but others are also possible under different system configurations gt which g 4 4 usr bin g 4 4 4 a To select g 4 4 only for compiling SortMeRNA the user must specify the full path of the compiler in the Makefile change the line gt CC g to gt CC usr bin g 4 4 the full directory path from Step 3 above 4 b To set g 4 4 as a de2. rrna rrna silva bac 16s database id85 fasta 27026 rrna silva bac 23s database id98 fasta 68170 rrna silva euk 28s database id98 fasta 10 4 2 2 Example 3 sortmerna on multiple databases with output of accepted reads directed to one single file gt sortmerna n 3 db sortmerna rRNA_databases silva bac 23s database id98 fasta sortmerna rRNA_databases silva bac 16s database id85 fasta sortmerna rRNA_databases silva euk 18s database id95 fasta accept rrna 454 SRR106861 filtered fasta log bilan a 3 v WARNING option other has been left blank no output file for rejected reads Welcome to SortMeRNA Copyright C 2012 Bonsai Bioinformatics Research Group LIFL Universit Lille 1 CNRS UMR 8022 INRIA 2012 The size of the reads file lt 33862846 gt bytes will be executed in 1 partial section s of size lt 33862846 gt bytes Partial section 1 Time to mmap reads and set up pointers 0 2184s Begin analysis of rRNA_databases silva bac 23s database id98 fasta Time to load the Burst trie 1 4594s Begin parallel traversal Time of parallel traversal of automata 14 5262s 13 Begin analysis of rRNA_databases silva bac 16s database id85 fasta Time to load the Burst trie 2 2632s Begin parallel traversal Time of parallel traversal of automata 17 3602s Begin analysis of rRNA_databases silva euk 28s database id98 fasta Time to load the Burst trie 1 9980s Begin parallel trave
3. 4950 nonrrna fast a 50 Case 2 We want to accept all pairs with at least one read that hits paired in gt sortmerna gt cat bilan Results total reads non rRNA rRNA rRNA set6 databas gt grep c gt rrna fasta nonrrna fast n 1 db set6 database fasta I paired end 5000 reads fasta accept rrna other nonrrna log bilan paired in a 1 log 5000 50 4950 99 e fasta 99 gt rrna fasta nonrrna fasta 5000 a 0 Case 3 We want to reject all pairs with at least one read that hits paired out gt sortmerna n 1 db set6 database fasta I paired end 5000 reads fasta accept rrna other nonrrna 15 log bilan paired out a l gt cat bilan log Results total reads 5000 non rRNA 50 rRNA 4950 rRNA 99 set6 database fasta 99 gt grep c gt rrna fasta nonrrna fasta rrna fasta 4900 nonrrna fasta 100 5 SortMeRNA parameters There are two parameters in SortMeRNA which the user can moderate for performance The length of the sliding window s the seed and the threshold ratio r of matching windows to the rRNA database for a read to be accepted Both of these paramaters and their default values are discussed in detail in Section 2 Parameter Setting of the supplementary file The user may adjust the threshold parameter r with the sortmerna command line option r new ratio To adjust the length of the sliding window
4. reads in fasta or fastq format and filters out the reads matching to at least one of the rRNA databases The user has an option to output the accepted reads into a single file default or into multiple files sorted by the closest matching database add the flag bydbs The indexed part of the databases created by buildtrie is loaded into sortmerna independently The user can adjust the amount of memory allocated for loading the reads through the command option m By default m is set to be high enough for 1GB If the reads file is larger than 1GB then sortmerna internally divides the file into partial sections of 1GB and executes one section at a time Hence if a user has an input file of 15GB and only 1GB of RAM to store it the file will be processed in partial sections using mmap without having to physically split it prior to execution Otherwise the user can set m high enough to store all 15GB in RAM 4 2 1 Example 2 sortmerna on multiple databases with output of accepted reads sorted by database gt sortmerna n 3 db sortmerna rRNA_databases silva bac 23s database id98 fasta sortmerna rRNA_databases silva bac 16s database id85 fasta sortmerna rRNA_databases silva euk 18s database id95 fasta sccept rrna bydbs 454 SRR106861 filtered fasta log bilan a 3 v WARNING option other has been left blank no output file for rejected reads Welcome to SortMeRNA Copyright C 2012 Bonsai Bioinfor
5. s the user must provide the option L new length even integer when indexing an rRNA database using the buildtrie executable 16
6. will be used L length of the sliding window the seed default 18 F search only the forward strand R search only the reverse complementary strand default both strands are searched h help There are eight rRNA representative databases provided in the sortmerna rRNA_databases folder All databases were derived from the SILVA SSU and LSU databases release 111 and the RFAM databases using the tool UCLUST Additionally the user can index their own database 4 1 1 Example 1 buildtrie gt buildtrie db sortmerna rRNA_databases silva bac 16s database id85 fasta Burst trie s built in 36 7594s Writing Burst trie forward to silva bac 16s database id85 bursttrief dat Writing Burst trie reverse to silva bac 16s database id85 bursttrier dat Done The indexed databases ex silva bac 16s database id85 bursttrief dat will be stored in the directory some path to sortmerna automata the path stored in variable SORTMERNADIR which was established in Step 1 4 of Subsection 2 2 or Subsection 2 3 and later retrieved by the command sortmerna explained in the following section 4 2 Filter reads against the indexed rRNA database command sortmerna The executable sortmerna filters rRNA reads against an indexed rRNA database To see the man page for sortmerna gt sortmerna h To run SortMeRNA type in any order after sortmerna I illumina reads file name fasta fastq 454 roche 454 reads file name f
7. SortMeRNA User Manual Evguenia Kopylova evuguenia kopylovaQlifi fr February 2013 version 1 5 Contents 1 2 Introduction 3 Installation 3 2 1 Required g compiler version aoao ea 3 2 1 1 Ubuntu Linux OS instructions 2 0 00 0004 3 2 1 2 Mac OS X instructions using MacPorts 04 4 2 2 Install from source code Leo 6 2 3 Install from precompiled code 2 2 0 0002 ee ee 7 Databases 8 How to run SortMeRNA 8 4 1 Index the rRNA database command buildtrie 1 122211 8 4 1 1 Example l BUNdYTIE1 0 no wood saw aw WR WA wie a tis 9 4 2 Filter reads against the indexed rRNA database command sortmerna 10 4 2 1 Example 2 sortmerna on multiple databases with output of accepted reads sorted by database L A 11 4 2 2 Example 3 sortmerna on multiple databases with output of accepted reads directed to one single file LLL 12 15 4 2 3 Example 4 sortmerna on paired end reads 1 241 11 14 SortMeRNA parameters 16 1 Introduction Copyright C 2012 Bonsai Bioinformatics Research Group LIFL Universit Lille 1 CNRS UMR 8022 INRIA 2012 SortMeRNA is a software designed to filter metatranscriptomic reads data It takes as input a file of reads fasta or fastq format and an rRNA database file and sorts apart the accepted reads and the rejected reads into two files specified by the user For questions amp help please contact
8. asta fastq n number of databases to use must precede db db rrnas database name s One database ex 1 n 1 db path1 database1 fasta Multiple databases ex 2 n 2 db path2 database2 fasta path3 database3 fasta OPTIONS The list of OPTIONS can be left blank the default values will be used accept accepted reads file name other rejected reads file name default no output files are created bydbs output the accepted reads by database default concatenated file of reads log overall statistics file name default no statistics file created paired in put both paired end reads into accept file paired out put both paired end reads into other file default if one read is accepted and the other is not separate the reads into accept and other files f ratio of the number of hits on the read read length default Illumina 0 25 Roche 454 0 15 F search only the forward strand R search only the reverse complementary strand default both strands are searched a number of threads to use 10 default 1 m m x 4096 bytes for loading the reads into memory ex m 4 means 4 4096 16384 bytes will be allocated for the reads note maximum m is 1020040 default m 262144 1GB v verbose default deactivated h help version version number The command sortmerna takes as input a list of rRNA databases in fasta format and a set of Illumina or Roche 454
9. ath to sortmerna 4 Open the bashrc or profile file in any editor and add the line gt export SORTMERNADIR some path to sortmerna automata gt export PATH PATH some path to sortmerna 5 Run the bashre or profile file to add the variable SORTMERNADIR and update the variable PATH in the list of environment variables gt source bashrc or gt source profile 6 Check that SORTMERNADIR has been added gt echo SORTMERNADIR some path to sortmerna if it has not been added this path will be empty 7 Check that PATH has been updated with the additional directory search path gt echo PATH 8 Check the path of the executables 2 3 6 gt which sortmerna buildtrie some path to sortmerna sortmerna some path to sortmerna buildtrie To begin using SortMeRNA type buildtrie h or sortmerna h Install from precompiled code Download sortmerna_linux_64 tar gz or sortmerna_linux_32 tar gz or sortmerna_mac_64 tar gz from http bioinfo lifl fr RNA sortmerna Extract the source code package into a directory of your choice must have permissions to read and write gt tar zxvf sortmerna_linux_64 tar gz gt cd sortmerna_linux_64 SortMeRNA indexes a database by writing its contents to the automata folder which is initially located in the directory sortmerna_linux_64 automata It is essential to set the SORTMERNADIR environmental variable to the path w
10. ef NR v 111 244077 23s silva arc 16s database id95 fasta 95 96 7 3845 SILVA SSU Ref NR v 111 10919 23s silva euk 18s database id95 fasta 95 96 7 4512 SILVA SSU Ref NR v 111 31862 26s 28s 238 silva bac 23s database id95 fasta 98 99 4 3055 SILVA LSU Ref v 111 19580 16s 26s 28s silva arc 23s database id95 fasta 98 99 5 164 SILVA LSU Ref v 111 405 16s 26s 28s silva euk 28s database id95 fasta 98 99 1 4578 SILVA LSU Ref v 111 9321 18s rfam 5s database id98 fasta 98 99 2 59513 RFAM 116760 rfam 5 8s database id98 fasta 98 98 9 13034 RFAM 225185 The tool UCLUST was used to reduce the size of the original databases id members of the cluster must have identity at least this id with the representative sequence average id average identity of a cluster member to the representative sequence Remark The user must first index the fasta database by using the command buildtrie and then filter reads against the database using the command sortmerna 4 How to run SortMeRNA 4 1 Index the rRNA database command buildtrie The executable buildtrie indexes an rRNA database To see the man page for buildtrie gt buildtrie h This program builds a Burst trie on an input rRNA database file in fasta format and stores the material in binary files under the folder automata buildtrie db path to rrnas database file name fasta OPTIONS The list of OPTIONS can be left blank the default values
11. fault compiler do not edit the Makefile but run the commands gt which g find the path of the g command usr bin g gt sudo rm usr bin gt remove the symbolic link of gt gt sudo ln s usr bin g 4 4 usr bin g create a new symbolic link from g to gt 4 4 gt g version should now be 4 4 5 The user may now run the make command in Step 2 of Section 2 2 2 1 2 Mac OS X instructions using MacPorts 1 Check whether you have other g compiler versions installed 4 3 or later type g then press the tab key gt g followed by the tab key 2 If the list given by Step 1 does not include a g compiler of version 4 3 or later install one SortMeRNA was tested with gcc43 gcc44 gcc45 gcc46 and gcc47 gt sudo port selfupdate gt sudo port upgrade outdated gt sudo port install gcc44 3 a To select gcc44 only for compiling SortMeRNA the user must specify the full path of the compiler in the Makefile change the line gt CC g to gt CC opt local bin g mp 4 4 the full directory path of the installed compiler 3 b To set gcc44 as a default compiler do not edit the Makefile but run the commands gt which g find the path of the g command opt local bin g gt sudo rm opt local bin g remove the symbolic link of gt gt sudo ln s opt local bin g mp 4 4 N create a new symbolic link from g opt local bin
12. g to gt mp 4 4 gt g version should now be 4 4 4 The user may now run the make command in Step 2 of Section 2 2 If linking errors occur such as library not loaded or undefined symbols for architecture try to upgrade your current compiler version to establish correct links gt sudo port n upgrade force gcc44 2 2 Install from source code 1 Download sortmerna tar gz from http bioinfo lifl fr RNA sortmerna 2 Extract the source code package into a directory of your choice must have permissions to read and write and build the executable files sortmerna and buildtrie gt tar zxvf sortmerna tar gz gt cd sortmerna gt make note The g compiler version must be 4 3 or later for make to work Please see Subsec tion 2 1 to configure or install a new compiler if the command which g returns a version less than 4 3 3 SortMeRNA indexes a database by writing its contents to the automata folder which is ini tially located in the directory sortmerna automata It is essential to set the SORTMERNADIR environmental variable to the path where the automata folder is found so that the pro gram may read and write from it The user may move the automata folder to a workspace with larger memory separately from the directory sortmerna To find the path of the automata folder go into the directory where it is located and type gt pwd some p
13. here the automata folder is found so that the program may read and write from it The user may move the automata folder to a workspace with larger memory separately from the directory sortmerna_linux_64 To find the path of the automata folder go into the directory where it is located and type gt pwd some path to sortmerna_linux_64 Open the bashrc or profile file in any editor and add the line gt export SORTMERNADIR some path to sortmerna_linux_64 gt export PATH PATH some path to sortmerna_linux_64 Run the bashre or profile file to add the variable SORTMERNADIR and update the variable PATH in the list of environment variables gt source bashrc or gt source profile Check that SORTMERNADIR has been added gt echo SORTMERNADIR some path to sortmerna_linux_64 if it has not been added this path will be empty 7 Check that the PATH has been updated with the additional directory search path gt echo PATH 8 Check the path of the executables gt which sortmerna buildtrie some path to sortmerna_linux_64 sortmerna some path to sortmerna_linux_64 buildtrie 9 To begin using SortMeRNA type buildtrie h or sortmerna h 3 Databases SortMeRNA comes prepackaged with 8 databases representative database id average id seq origin seq filtered to remove silva bac 16s database id85 fasta 85 91 6 8174 SILVA SSU R
14. matics Research Group LIFL Universit Lille 1 CNRS UMR 8022 INRIA 2012 11 The size of the reads file lt 33862846 gt bytes will be executed in 1 partial section s of size lt 33862846 gt bytes Partial section tt 1 Time to mmap reads and set up pointers 0 2164s Begin analysis of rRNA_databases silva bac 23s database id98 fasta Time to load the Burst trie 1 4488s Begin parallel traversal Time of parallel traversal of automata 14 2974s Begin analysis of rRNA_databases silva bac 16s database id85 fasta Time to load the Burst trie 2 2975s Begin parallel traversal Time of parallel traversal of automata 17 3430s Begin analysis of rRNA_databases silva euk 28s database id98 fasta Time to load the Burst trie 2 0302s Begin parallel traversal Time of parallel traversal of automata 19 0678s Total number of reads found in current section 95206 Time to output reads to file 1 7558s Total number of reads found 95206 The option log bilan will create an overall statistics file called bilan log gt cat bilan log Time and Date Settings r 0 15 L 18 reads file SRR106861 filtered fasta Results total reads 105873 non rRNA 10667 rRNA 95206 12 rRNA 89 92 silva bac 23s database id98 fasta 64 39 silva bac 16s database id85 fasta 25 53 silva euk 28s database id98 fasta 0 009445 Now we can manually check the number of reads matched per database gt grep c gt
15. rsal Time of parallel traversal of automata 19 4148s Total number of reads found in current section 95206 Time to output reads to file 0 9978s Total number of reads found 95206 Now we can manually check the number of reads matched for all databases gt grep c gt rrna rrna fasta 95206 4 2 3 Example 4 sortmerna on paired end reads This example illustrates three cases of output for paired end reads default paired in and paired out We use the set6 database fasta found under Section 3 2 of the Supplementary file also avail able online at bioinfo lifl fr RNA sortmerna material php As for the reads 5000 Illumina paired end reads were simulated using MetaSim on set6 database fasta The reads were then arranged to have 2475 pairs of rRNA and the other 25 pairs where exactly one read is rRNA and the other is not this is possible if one of the reads covers a low complexity region on the rRNA sequence Remark The statistics in the log file will always give the true number of reads classified as rRNA Case 1 We don t care to keep the paired end order in the output default gt sortmerna n 1 db set6 database fasta I paired end 5000 reads fasta accept rrna other nonrrna log bilan a 1 14 gt cat bilan Results total reads non rRNA rRNA rRNA set6 databas gt grep c gt log 5000 50 4950 99 e fasta 99 gt rrna fasta nonrrna fasta rrna fasta
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