SortMeRNA User Manual - Bioinformatics Software Server
Contents
1. 51 Number of seeds 2 Edges 4 as integer SW match 2 SW mismatch 3 SW gap open penalty 5 SW gap extend penalty SW ambiguous nucleotide SQ tags are not output Number of threads 1 OpenMP is not supported with your current C compiler Reads file SRR106861 fasta 2 3 Results Total reads 113128 By database aligned reads 104249 92 15 non aligned reads 8879 rRNA_databases silva bac 16s database id85 fasta 30 69 rRNA_databases silva bac 23s database id98 fasta 55 63 rRNA_databases silva arc 16s database id95 fasta 0 26 rRNA_databases silva arc 23s database id98 fasta 0 11 rRNA_databases silva euk 18s database id95 fasta 0 01 rRNA_databases silva euk 28s database id98 fasta 3 14 rRNA_databases rfam 5 8s database id98 fasta 0 01 rRNA_databases rfam 5s database id98 fasta 2 31 4 2 3 Filtering paired ended reads When outputting matching and non matching reads into FASTA Q files sometimes the situation arises where one of the paired ended reads matches and the other one doesn t For users who wish to keep the order of their paired ended reads we provide two options 1 the option paired in will put both reads into the file specified by accept 2 the option paired out will put both reads into the file specified by other And by default the reads will be split into two aligned and other files 4 2 4 Example 5 sortmerna on forward reverse paired end reads 2 inp2. bac 16s database id85 fasta Seed length 18 Pass 1 18 Pass 2 9 Pass 3 3 Gumbel lambda 0 602506 Gumbel K 0 328589 Minimal SW score based on E value 53 Loading index part 1 1 done 3 26 sec Begin index search done 27 78 sec Freeing index done 0 45 sec Begin analysis of rRNA_databases silva bac 23s database id98 fasta Seed length 18 Pass 1 18 Pass 2 9 Pass 3 3 Gumbel lambda 0 602275 Gumbel K 0 333737 Minimal SW score based on E value 53 Loading index part 1 1 done 2 04 sec Begin index search done 23 04 sec Freeing index done 0 31 sec 10 Begin analysis of rRNA_databases silva arc 16s database id95 fasta Seed length 18 Pass 1 18 Pass 2 9 Pass 3 3 Gumbel lambda 0 596068 Gumbel K 0 321832 Minimal SW score based on E value 52 Loading index part 1 1 done 1 21 sec Begin index search done 10 90 sec Freeing index done 0 17 sec Begin analysis of rRNA_databases silva arc 23s database id98 fasta Seed length 18 Pass 1 18 Pass 2 9 Pass 3 3 Gumbel lambda 0 596330 Gumbel K 0 324091 Minimal SW score based on E value 48 Loading index part 1 1 done 0 31 sec Begin index search done 8 73 sec Freeing index done 0 06 sec Begin analysis of rRNA_databases silva euk 18s database id95 fasta Seed length 18 Pass 1 18 Pass 2 9 Pass 3 3 Gumbel lambda 0 611988 Gumbel K 0 337232 Minimal SW scor
3. 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 increase m to map larger portions of the file The limit for m is given by typing sortmerna h 1024 off L L 2 3 off off 4 2 1 A guide to choosing parameters for filtering and quality of alignments In SortMeRNA version 1 99 beta and up users have the option to output sequence alignments for their matching rRNA reads in the SAM or BLAST like formats Depending on the desired quality of alignments different parameters choices must be set Table 1 presents a guide to setting parameters choices for most use cases In all cases output alignments are always guaranteed to reach the threshold E value score default E value 1 An E value of 1 signifies that one random alignment is expected for aligning all reads against the reference database The E value in SortMeRNA is computed for the entire search space not per read Table 1 SortMeRNA alignment parameter guide option speed description The first alignment reaching the E value threshold will be reported if a high scorin
4. SortMeRNA User Manual Evguenia Kopylova jenya kopylovQgmail com March 2014 version 1 99 beta Contents 1 2 Introduction 3 Installation 3 2 1 Install fromsourc G de sss oed ahua eee EEE RAS ee eb ee dm 3 2 2 Install from precompiled code oaoa 4 23 gt Uninstalls dua gaz a ARR ck ee he ete hot winter ann fe An nS cette Ba Bg ay 4 Databases 4 How to run SortMeRNA 5 4 1 Index the rRNA database command indexdb rna 5 4 1 1 Example 1 indexdb_rna using one database i 4441011 5 4 1 2 Example 2 indexdb_rna using all eight databases 1 1 1 11 1 6 4 2 Filter reads against the indexed rRNA database command sortmerna 7 4 2 1 A guide to choosing parameters for filtering and quality of alignments 9 4 2 2 Example 2 sortmerna using multiple databases and the fastest alignment option 10 4 2 3 Filtering paired ended reads 2 2 ee 13 4 2 4 Example 5 sortmerna on forward reverse paired end reads 2 input files 13 SortMeRNA advanced options 14 1 Introduction Copyright C 2012 2014 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 one or multiple rRNA database file s and sorts apart the accepted reads and the rejected reads into two files specified by the user S
5. cess will be added to the output files in order to avoid over writing output if the same aligned STRING base name is provided for different runs 15
6. dat writing nucleotide distribution statistics to index silva bac 16s stats done 4 1 2 Example 2 indexdb_rna using all eight databases Multiple databases can be indexed simultaneously by passing them as a no spaces allowed bj separated list to ref gt gt indexdb_rna ref rRNA_databases silva bac 16s database id85 fasta index silva bac 16s N rRNA_databases silva bac 23s database id98 fasta index silva bac 23s rRNA_databases silva arc 16s database id95 fasta index silva arc 16s rRNA_databases silva arc 23s database id98 fasta index silva arc 23s rRNA_databases silva euk 18s database id95 fasta index silva euk 18s rRNA_databases silva euk 28s database id98 fasta index silva euk 28s rRNA_databases rfam 5 8s database id98 fasta index rfam 5 8s N rRNA_databases rfam 5s database id98 fasta index rfam 5s LL LLL 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 gt sortmerna h usage sortmerna lt input gt lt output gt lt options gt parameter value description default lt input gt reads STRING FASTA FASTQ reads file mandatory ref STRING STRING FASTA reference file index file mandatory lt output gt aligned other fastx sam SQ blast log ex ref path to filei fasta path to
7. databases using the tool UCLUST Additionally the user can index their own database 4 1 1 Example 1 indexdb_rna using one database gt gt indexdb_rna ref rRNA_databases silva bac 16s database id85 fasta index silva bac 16s v Program SortMeRNA version 1 99 beta 11 03 2014 Copyright 2012 2014 Bonsai Bioinformatics Research Group LIFL University Lille 1 CNRS UMR 8022 INRIA Nord Europe SortMeRNA comes with ABSOLUTELY NO WARRANTY without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE See the GNU Lesser General Public License for more details Contact Evguenia Kopylova jenya kopylov gmail com Laurent Noe laurent noe lifl fr Helene Touzet helene touzet lifl fr Parameters summary K mer size 19 K mer interval 1 Maximum positions to store per unique K mer 250 Total number of databases to index 1 Begin indexing file rRNA_databases silva bac 16s database id85 fasta under index name index silva bac 16s Collecting sequence distribution statistics done 0 781479 sec start index part 0 1 3 building burst tries done 14 726437 sec 2 3 building CMPH hash done 22 519546 sec 3 3 building position lookup tables done 21 117368 sec total number of sequences in this part 8174 writing kmer data to index silva bac 16s kmer_0 dat writing burst tries to index silva bac 16s bursttrie_0 dat writing position lookup table to index silva bac 16s pos_0
8. e based on E value 51 Loading index part 1 1 done 1 76 sec Begin index search done 15 63 sec Freeing index done 0 27 sec Begin analysis of rRNA_databases silva euk 28s database id98 fasta Seed length 18 Pass 1 18 Pass 2 9 Pass 3 3 Gumbel lambda 0 611523 Gumbel K 0 335218 Minimal SW score based on E value 53 Loading index part 1 1 done 2 86 sec Begin index search done 19 54 sec Freeing index done 0 48 sec Begin analysis of rRNA_databases rfam 5 8s database id98 fasta Seed length 18 Pass 1 18 Pass 2 9 Pass 3 3 Gumbel lambda 0 617817 Gumbel K 0 340589 Minimal SW score based on E value 49 Loading index part 1 1 done 0 55 sec Begin index search done 5 71 sec Freeing index done 0 07 sec Begin analysis of rRNA_databases rfam 5s database id98 fasta Seed length 18 Pass 1 18 Pass 2 9 Pass 3 3 Gumbel lambda 0 616617 Gumbel K 0 341306 Minimal SW score based on E value 51 Loading index part 1 1 done 1 54 sec Begin index search done 7 62 sec Freeing index done 0 21 sec Total number of reads mapped incl all reads file sections searched 104249 Writing alignments done 5 14 sec Writing aligned FASTA FASTQ done 0 93 sec 11 Writing not aligned FASTA FASTQ done 0 08 sec real 2m30 574s user 2m26 740s sys Om2 420s The option log will create an overall statistics file gt
9. f a cluster member to the representative sequence Remark The user must first index the fasta database by using the command indexdb_rna and then filter reads against the database using the command sortmerna 4 How to run SortMeRNA 4 1 Index the rRNA database command indexdb_rna The executable indexdb_rna indexes an rRNA database To see the man page for indexdb_rna gt gt indexdb_rna h usage indexdb_rna lt input gt lt output gt lt options gt parameter value description default lt input gt ref STRING STRING FASTA reference file index file mandatory ex ref path to file1 fasta path to index1 If passing multiple reference sequence files separate them by 1 ex ref path to file1 fasta path to index1 path to file2 fasta path to index2 lt options gt fast FLAG suggested option for aligning 99 related species off sensitive FLAG suggested option for aligning 75 98 related species on tmpdir STRING directory where to write temporary files m INT the amount of memory in Mbytes for building the index 3072 L INT seed length 18 max_pos INT maximum number of positions to store for each unique L mer 250 setting max_pos 0 will store all positions v FLAG verbose h FLAG help There are eight rRNA representative databases provided in the sortmerna 1 99 beta rRNA_databases folder All databases were derived from the SILVA SSU and LSU databases release 111 and the RFAM
10. g feeling lucky Very fast alignment was found on the forward strand the reverse complementary strand will not be searched num alignments INT Very fast for INT 1 Same behavior as option feeling lucky Speed decreases for higher value INT Higher INT signifies more alignments will be made amp output Very slow for INT O All alignments reaching the E value threshold are reported this option is not suggested for high similarity rRNA databases due to many possible alignments per read causing a very large file output best INT Fast for INT 1 Only one high candidate reference sequence will be searched for alignments determined heuristically using a Longest Increasing Sub sequence of seed matches The single best alignment of those will be reported Speed decreases for higher value INT Higher INT signifies more alignments will be made though only the best one will be re ported Very slow for INT O AII high candidate reference sequences will be searched for alignments though only the best one will be reported 4 2 2 Example 2 sortmerna using multiple databases and the fastest alignment op tion gt gt time sortmerna ref rRNA_databases silva bac 16s database id85 fasta index silva bac 16s rRNA_databases silva bac 23s database id98 fasta index silva bac 23s rRNA_databases silva arc 16s database id95 fasta index silva arc 16s rRNA_databases silva a
11. gt cat aligned log Time and date SortMeRNA command lt command will be here gt Process pid 50199 Parameters summary Index index silva bac 16s Seed length 18 Pass 1 18 Pass 2 9 Pass 3 3 Gumbel lambda 0 602506 Gumbel K 0 328589 Minimal SW score based on E value 53 Index index silva bac 23s Seed length 18 Pass 1 18 Pass 2 9 Pass 3 3 Gumbel lambda 0 602275 Gumbel K 0 333737 Minimal SW score based on E value 53 Index index silva arc 16s Seed length 18 Pass 1 18 Pass 2 9 Pass 3 3 Gumbel lambda 0 596068 Gumbel K 0 321832 Minimal SW score based on E value 52 Index index silva arc 23s Seed length 18 Pass 1 18 Pass 2 9 Pass 3 3 Gumbel lambda 0 596330 Gumbel K 0 324091 Minimal SW score based on E value 48 Index index silva euk 18s Seed length 18 Pass 1 18 Pass 2 9 Pass 3 3 Gumbel lambda 0 611988 Gumbel K 0 337232 Minimal SW score based on E value 51 Index index silva euk 28s Seed length 18 Pass 1 18 Pass 2 9 Pass 3 3 Gumbel lambda 0 611523 Gumbel K 0 335218 Minimal SW score based on E value 53 Index index rfam 5 8s Seed length 18 Pass 1 18 Pass 2 9 Pass 3 3 Gumbel lambda 0 617817 Gumbel K 0 340589 Minimal SW score based on E value 49 Index index rfam5s Seed length 18 Pass 1 18 Pass 2 9 Pass 3 3 12 Gumbel lambda 0 616617 Gumbel K 0 341306 Minimal SW score based on E value
12. h 2 INT SW score negative integer for a mismatch 3 INT SW score positive integer for introducing a gap 5 INT SW score positive integer for extending a gap 2 INT SW score for ambiguous letters N s scored as mismatch FLAG search only the forward strand off FLAG search only the reverse complementary strand off INT number of threads to use 1 DOUBLE E value 1 m INT INT Mbytes for loading the reads into memory maximum m INT is 4096 v FLAG verbose advanced lt options gt see SortMeRNA user manual for more details passes STRING values for seed skip lengths for Pass 1 2 and 3 must be in the form INT INT INT respectively L is the seed length set in indexdb edges INT number or percent if INT followed by sign of nucleotides to add to each edge of the read prior to SW local alignment num_seeds INT number of seeds matched before searching for candidate LIS full_search FLAG search for all O error and 1 error seed matches in the index rather than stopping after finding a O error match lt 1 gain in sensitivity with up four fold decrease in speed pid FLAG add pid to output file names help h FLAG help version FLAG SortMeRNA version number The command sortmerna takes as input a list of rRNA databases must be indexed and a set of reads in fasta or fastq format and filters out the reads matching to at least one of the rRNA databases The user can adjust the amount of memory allocated for
13. index1 If passing multiple reference files separate them using the delimiter ex ref path to file1 fasta path to index1 path to file2 fasta path to index2 For alignments with sam or blast options feeling lucky or num_alignments or default best lt options gt paired_in paired_out match mismatch gap_open gap_ext STRING aligned reads base file name appropriate extension will be added STRING rejected reads base file name appropriate extension will be added FLAG output FASTA FASTQ file off for aligned and or rejected reads FLAG output SAM alignment off for aligned reads only FLAG add SQ tags to the SAM file off FLAG output BLAST like alignment off for aligned reads only FLAG output overall statistics off FLAG report the first alignment per read reaching E value off INT report first INT alignments per read reaching E value i num_alignments 0 signifies all alignments will be output INT report single best alignment per read reaching E value 2 from alignments of INT best candidate reference sequences ex best 2 find all alignments for the first 2 best matching reference sequences and report the the single best alignment best 0 signifies all highest scoring reference sequences will be searched FLAG both paired end reads go in aligned fasta q file off FLAG both paired end reads go in other fasta q file off INT SW score positive integer for a matc
14. ongest increasing subsequence LIS of the seeds positions between the read and the closest matching reference sequence By default this is set to 2 seeds passes INT INT INT In the primary seed search filter SortMeRNA moves a seed of length Z parameter of indexdb_rna across the read using three passes If at the end of each pass a threshold number of seeds defined by num_seeds did not match to the reference database SortMeRNA attempts to find more seeds by decreasing the interval at which the seed is placed along the read by using another pass In default mode these intervals are set to L L 2 3 for Pass 1 2 and 3 respectively Usually if the read is highly similar to the reference database a threshold number of seeds will be found in the first pass 14 edges INT The number or percentage if followed by of nucleotides to add to each edge of the alignment region on the reference sequence before performing Smith Waterman alignment By default this is set to 4 nucleotides full_search FLAG During the index traversal if a seed match is found with 0 errors SortMeRNA will stop searching for further l error matches This heuristic is based upon the assumption that 0 error matches are more significant than 1 error matches By turning it off using the full_search flag the sensitivity may increase often by less than 1 but with up to four fold decrease in speed pid FLAG The pid of the running sortmerna pro
15. ortMeRNA works with Illumina 454 Ion Torrent and PacBio data and can produce SAM and BLAST like alignments For questions amp help please contact 1 Evguenia Kopylova evguenia kopylova lifl fr 2 Laurent Noe laurent noe lifl fr 3 Helene Touzet helene touzet lifl fr Important This user manual is strictly for SortMeRNA version 1 99 beta 2 Installation Figure 1 sortmerna 1 99 beta directory tree sortmerna 1 99 be gt R Q include scripts cr n Q w pame S gt rRNA_databases bacteria 16S fasta sortmerna indexdb_rna i 2 1 Install from source code 1 Download sortmerna 1 99 beta tar gz from http bioinfo lifl fr RNA sortmerna 2 2 Extract the source code package into a directory of your choice enter sortmerna 1 99 beta and type gt configure gt make At this point two executables indexdb_rna and sortmerna will be located in the sortmerna 1 99 beta directory If the user would like to install the executables into their default installation direc tory usr local bin for Linux or opt local bin for Mac then type gt make install with root permissions To begin using SortMeRNA type indexdb_rna h or sortmerna h Databases must first be indexed using indexdb_rna 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 yo
16. rc 23s database id98 fasta index silva arc 23s rRNA_databases silva euk 18s database id95 fasta index silva euk 18s rRNA_databases silva euk 28s database id98 fasta index silva euk 28s rRNA_databases rfam 5 8s database id98 fasta index rfam 5 8s N rRNA_databases rfam 5s database id98 fasta index rfam 5s reads SRR106861 fasta feeling lucky sam fastx aligned accept other other log v LL LLL Program SortMeRNA version 1 99 beta 11 03 2014 Copyright 2012 2014 Bonsai Bioinformatics Research Group LIFL University Lille 1 CNRS UMR 8022 INRIA Nord Europe SortMeRNA comes with ABSOLUTELY NO WARRANTY without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE See the GNU Lesser General Public License for more details Contact Evguenia Kopylova jenya kopylov gmail com Laurent Noe laurent noeQlifl fr Helene Touzet helene touzet lifl fr Computing read file statistics done 2 31 sec size of reads file 35238748 bytes partial section s to be executed 1 of size 35238748 bytes Parameters summary Number of seeds 2 Edges 4 as integer SW match 2 SW mismatch 3 SW gap open penalty 5 SW gap extend penalty 2 SW ambiguous nucleotide SQ tags are not output Number of threads 1 OpenMP is not supported with your current C compiler 3 Begin mmap reads section 1 Time to mmap reads and set up pointers 0 11 sec Begin analysis of rRNA_databases silva
17. ur choice gt tar xvf sortmerna 1 99 beta tar gz gt cd sortmerna 1 99 beta To begin using SortMeRNA type indexdb_rna h or sortmerna h The user must firstly index the databases with the command indexdb_rna 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 silva euk 18s database id95 fasta 95 96 7 4512 SILVA SSU Ref NR v 111 31862 268 288 238 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 o
18. ut 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 2 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 3 The command for merge paired reads sh is the following gt bash merge paired reads sh forward reads fastq reverse reads fastq outfile fastq Now the user may input outfile fastq to SortMeRNA for analysis 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 13 FASTQ forward reads FASTQ reverse reads SEQUENCE_ID 1 2 GSEQUENCE ID_1 1 ACTT GTAC 4 pair 1 i QUALITY 1 1 QUALITY_1 2 SEQUENCE_ID 2 1 GSEQUENCE_ID_2 2 GTTA CCAC s pair 2 n QUALITY 2 1 QUALITY 2 2 Figure 2 Forward and reverse reads in paired end sequencing format FASTQ paired end reads GSEQUENCE ID_1 1 ACTT QUALITY_1 1 SEQUENCE_ID 1 2 GTAC QUALITY_1 2 Figure 3 Paired end read format accepted by SortMeRNA 5 SortMeRNA advanced options num_ seeds INT The threshold number of seeds required to match in the primary seed search filter before moving on to the secondary seed cluster filter More specifically the threshold number of seeds required before searching for a l
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