SortMeRNA User Manual - Bonsai Bioinformatics
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1. 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 1 input file paired end reads are interleaved FASTQ paired end reads GSEQUENCE ID_1 1 ACTT QUALITY 1 1 GSEQUENCE ID 1 2 GTAC QUALITY_1 2 Figure 2 Paired end read format accepted by SortMeRNA 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 b oinfo lifl fr RNA sortmerna material php As for the reads 5000 Illumina 13 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 1og 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 gt cat bilan log Results total reads 5000 non rRNA 25 rRNA 4975 rRNA 99 5 set6 database fasta 99 5 gt2. SortMeRNA User Manual Evguenia Kopylova evguenia kopylova lifi fr May 2013 version 1 8 Contents 1 Introduction 3 2 Installation 3 2 1 Installfronrsourc code 4 l ee ee EEE RAS ee eb ee Ama 3 2 2 Install from precompiled code oaoa 5 2 95 Uninstall dua gaz ek he ees me Go eere eto qo etin a a n A Lo ie cree aqua eset 6 3 Databases 6 4 How to run SortMeRNA 7 4 1 Index the rRNA database command buildtrie 7 4 11 Example 1 buildtrie 2 s na a est VIRA wow wom k w wb 8 M 7 4 2 Filter reads against the indexed rRNA database command sortmerna 9 4 2 1 Example 2 sortmerna on multiple databases with output of accepted reads sorted by database 0 0000 pee eee ee 10 4 2 2 Example 3 sortmerna on multiple databases with output of accepted reads directed to one single file 0 20020 12 12 4 2 8 Example 4 sortmerna on paired end reads 1 input file paired end reads are interleaved u icu upper meer Dur de EAE UA Hu t 13 4 2 4 Example 5 sortmerna on forward reverse paired end reads 2 input files 15 5 SortMeRNA parameters 16 1 Introduction Copyright C 2012 2013 Bonsai Bioinformatics Research Group LIFL Universit Lille 1 CNRS UMR 8022 INRIA Nord Europe 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 accepte
3. a 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 BCcept 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 33862846 bytes Will be executed in 1 partial section s of size 33862846 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 12 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 traversal 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
4. d reads and the rejected reads into two files specified by the user For questions amp help please contact 1 Evguenia Kopylova evguenia kopylovaOlifl fr 2 Laurent Noe laurent noe lifl fr 3 Helene Touzet helene touzet lifl fr 2 Installation Figure 1 sortmerna vx directory tree sortmerna vx src include scripts tests rRNA_databases bacteria 16S fasta automata bacteria 16S bursttrief dat bacteria 16S bursttrier dat bacteria 16S kmer dat yr 2 1 Install from source code 1 Download sortmerna vx tar gz from http bioinfo lifl fr RNA sortmerna 2 Extract the source code package into a directory of your choice for quick installation without tests type Quick Installation no testing gt configure gt make gt make install with root permissions Go to Step 5 3 For installation with testing proceed by typing gt configure gt make gt make check 4 At this point two executables buildtrie and sortmerna will be located in the sortmerna vx directory as well as the indexed rRNA databases in sortmerna vx automata If the user would like to install the executables into usr local bin and the sortmerna vx directory into usr local sortmerna vx then type gt make install with root permissions gt make installcheck 5 SortMeRNA indexes a database by writing its contents to the automata folder see Figure 1 which is initially located in the directory sort
5. db pathi databasei 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 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 Ilumina or Roche 454 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 t
6. eads fastq outfile fastq Now the user may input outfile fastq to SortMeRNA for analysis 15 FASTQ forward reads GSEQUENCE ID 1 1 FASTQ reverse reads SEQUENCE_ID 1 2 ACTT GTAC h pair 1 2 QUALITY 1 1 QUALITY 1 2 SEQUENCE_ID 2 1 QSEQUENCE ID 2 2 GTTA CCAC s pair 2 de QUALITY 2 1 QUALITY 2 2 Figure 3 Forward and reverse reads in paired end sequencing format Similarly for unmerging the paired reads back into two separate files use the command gt bash unmerge paired reads sh merged reads fastq forward reads fastq reverse reads fastq 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 s the user must provide the option L new length even integer when indexing an rRNA database using the buildtrie executable 16
7. earch 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 l6s 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 fasta fastq n number of databases to use must precede db db rrnas database name s One database ex 1 n 1
8. grep c gt rrna fasta nonrrna fasta rrna fasta 4975 nonrrna fasta 25 Case 2 We want to accept all pairs with at least one read that hits paired in gt sortmerna n 1 db set6 database fasta I paired end 5000 reads fasta accept rrna other nonrrna log bilan paired in a 1 gt cat bilan log Results total reads 5000 non rRNA 25 rRNA 4975 14 rRNA 99 5 set6 database fasta 99 5 gt grep c gt rrna fasta nonrrna fasta rrna fasta 5000 nonrrna fasta 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 log bilan paired out a 1 gt cat bilan log Results total reads 5000 non rRNA 25 rRNA 4975 rRNA 99 5 set6 database fasta 99 5 gt grep c gt rrna fasta nonrrna fasta rrna fasta 4950 nonrrna fasta 50 4 2 4 Example 5 sortmerna on forward reverse paired end reads 2 input files SortMeRNA accepts only 1 file as input for the reads If a user has two input files in the case for the foward and reverse paired end reads see Figure 3 they may use the merge paired reads sh script found in sortmerna scripts folder to interleave the paired reads into the format of Fig ure 2 The command for merge paired reads sh is the following gt bash merge paired reads sh forward reads fastq reverse r
9. hre 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 path to sortmerna vx 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 path to sortmerna vx sortmerna path to sortmerna vx buildtrie 9 To begin using SortMeRNA type buildtrie h or sortmerna h If the user installed SortMeRNA in Step 3 then the public rRNA databases distributed with SortMeRNA have been indexed in SORTMERNADIR automata and the user may directly run the command sortmerna Otherwise the user must firstly index the databases with the command buildtrie before they can run the command sortmerna 2 3 Uninstall If the user installed SortMeRNA using the command make install then they can use the com mand make uninstall to uninstall SortMeRNA with root permissions 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 Ref NR v 111 244077 23s silva arc 16s database id95 fasta 95 96 7 3845 SILVA SSU Ref NR v 111 10919 23s
10. ll be executed in 1 partial section s of size 33862846 bytes Partial section 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 11 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 rrna rrna silva bac 16s database id8b fasta 27026 rrna silva bac 23s database id98 fasta 68170 rrna silva euk 28s database id98 fast
11. merna vx automata It is essential to set the SORTMERNADIR environmental variable to the path where 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 vx To find the path of the automata folder go into the directory where it is located we assume here it is located under the directory sortmerna vx and type gt pwd path to sortmerna vx Open the bashrc or profile file in any editor and add the line gt export SORTMERNADIR path to sortmerna vx If the user installed SortMeRNA in Step 2 or Step 4 go to Step 6 Otherwise the user must also include the path of the executable files in the PATH variable we assume buildtrie and sortmerna are located in the sortmerna vx directory gt export PATH PATH path to sortmerna vx 6 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 2 2 gt source profile Check the path of the executables if installed manually gt which sortmerna buildtrie path to sortmerna vx sortmerna path to sortmerna vx buildtrie Check that SORTMERNADIR has been added gt echo SORTMERNADIR path to sortmerna vx if it has not been added this path will be empty To begin using SortMeRNA t
12. o 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 Bioinformatics Research Group LIFL Universit Lille 1 CNRS UMR 8022 INRIA 2012 10 The size of the reads file 33862846 bytes Wi
13. 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 96 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 will be used L length of the sliding window the seed default 18 F search only the forward strand R S
14. ype buildtrie h or sortmerna h If the user installed SortMeRNA in Step 3 then the public rRNA databases distributed with SortMeRNA have been indexed in SORTMERNADIR automata and the user may directly run the command sortmerna Otherwise the user must firstly index the databases with the command buildtrie before they can run the command sortmerna Install from precompiled code Download the latest binary distribution of SortMeRNA from http bioinfo lifl fr RNA Sortmerna Extract the source code package into a directory of your choice gt tar zxvf sortmerna vx tar gz gt cd sortmerna vx SortMeRNA indexes a database by writing its contents to the automata folder see Figure 1 which is initially located in the directory sortmerna vx automata It is essential to set the SORTMERNADIR environmental variable to the path where 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 vx To find the path of the automata folder go into the directory where it is located we assume here it is located under the directory sortmerna vx and type gt pwd path to sortmerna vx Open the bashrc or profile file in any editor and add the line gt export SORTMERNADIR path to sortmerna vx gt export PATH PATH path to sortmerna vx Run the bas
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