PingPongPro v1.0
Contents
1. PingPongPro can generate browser track files These files can be loaded into the UCSC Genome Browser http genome ucsc edu IGV https www broadinstitute org igv home or other genome browsers for visualization of ping pong signatures and ping pong regions PingPongPro must be launched with the switch b if browser tracks should be generated For example pingpongpro i alignments bam T 1000 b 4 How PingPongPro works 4 1 Detecting ping pong signatures The ping pong cycle produces short RNA molecules which are complementary at their 5 ends over a length of exactly ten nucleotides In piRNA Seq data these molecules appear as stacks of reads on opposite strands which overlap by ten nucleotides 5 7 2 295 800 bp 2 295 900 bp 2 296 000 bp Poop pr Figure 1 Two examples of ping pong signatures The figure on the left shows the coverage around the genomic coordinate chr2L 2 295 900 which is inside the transposon copia of a piRNA Seq dataset from D melanogaster available at the NCBI Sequence Read Archive 10 via the SRA accession number SRR499784 7 There are three stacks of reads on the forward strand and two stacks on the reverse strand which stick out Four of them have been highlighted because they are likely to result from ping pong cycle activity Reads thought to belong to these stacks are colored red on the forward strand and blue on the reverse strand The stacks satisfy all the characteristics of2. A slicer mediated mechanism for repeat associated siRNA 5 end formation in Drosophila Science 2007 315 5818 1587 90 4 Kent WJ Sugnet CW Furey TS Roskin KM Pringle TH Zahler AM Haussler D The human genome browser at UCSC Genome Research 2002 12 6 996 1006 5 Li C Vagin CC Lee S Xu J Ma S Xi H Seitz H Horwich MD Syrzycka M Honda BM Kittler ELW Zapp ML Klattenhoff C Schulz N Theurkauf WE Weng Z Zamore PD Without Argonaute3 Aubergine bound piRNAs collapse but Piwi bound piRNAs persist Cell 2009 137 3 509 521 6 Li H Handsaker B Wysoker A Fennell T Ruan J Homer N Marth G Abecasis G Durbin R 1000 Genome Project Data Processing Subgroup The Sequence Alignment Map format and SAMtools Bioinformatics 2009 25 16 2078 9 7 Preall JB Czech B Guzzardo PM Muerdter F Hannon GJ shutdown is a component of the Drosophila piRNA biogenesis machinery RNA 2012 18 8 1446 57 8 R Development Core Team 2008 R A language and environment for statistical computing R Foundation for Statistical Computing Vienna Austria ISBN 3 900051 07 0 URL http www R project org accessed April 9 2014 9 Thorvaldsdottir H Robinson JT Mesirov JP Integrative Genomics Viewer IGV high performance genomics data visualization and exploration Briefings in Bioinformatics 2012 10 Wheeler DL Barrett T Benson DA Bryant SH Canese K Chetvernin V Church DM Dicuccio M Edgar R Federhen S Feolo M Gee
3. a typical ping pong signature They are all aligned at their 5 ends and the stacks on the forward strand overlap with the stacks on the reverse strand by ten nucleotides The characteristic overlap is marked by dashed lines The left most stack on the forward strand not highlighted is probably not to be attributed to the ping pong cycle because there is no stack on the reverse strand overlapping it by ten nucleotides forward strand M reverse strand ping pong signature stacks on opposite strands overlapping by 10 nt PingPongPro scans a piRNA Seq dataset for this pattern also known as ping pong signatures Ping pong signatures are not always as evident as in Figure 1 When the stacks are not that prominent they are hard to distinguish from random alignments of reads which only happen to overlap by ten nucleotides by chance PingPongPro therefore evaluates a total of three properties that are characteristic for true ping pong signatures PingPongPro v1 0 User Manual 3 e Stack height High stacks of reads are the most reliable indicator of a true ping pong signature e Independence of the local coverage High stacks are not necessarily the result of ping pong cycle activity In regions with good coverage the probability is increased that reads stack up on each other Conversely a relatively small stack likely is to be attributed to the ping pong cycle when there are hardly any oth
4. the FDR field in the output file ping pong signatures tsv see section 8 1 So the closer the score is to 1 the more likely it is a true ping pong signature and not a random alignment of reads the closer the score is to 0 the more unlikely it is a true ping pong signature Annotated transposons A bed file is generated for detected ping pong cycle activity within the annotated transposons that have been passed to PingPongPro via the parameter t Refer to http genome ucsc edu FAQ FAQformat html format1 for more information about the BED file format The file transposons bed contains the transposons given in the annotation file The score column reflects how confident PingPongPro is that there is ping pong cycle activity within the transposon The score is calculated as 1000 minus the value of the qValue field in the output file transposons tsv multiplied by 1000 So a score near 1000 indicates high confidence and a score near 0 indicates low confidence Predicted transposons If PingPongPro is run with the parameter T then it can generate browser tracks for predicted regions with ping pong cycle activity The browser track is named predicted_transposons bed and has the same format as the BED file for annotated transposons The only difference is that the content is based on the output file predicted _transposons tsv 8 4 Plots When PingPongPro is run with the parameter p it generates graphical plots It uses R Project for ren
5. the heights of stacks on the forward strand Most of the stacks have fractional heights because they are mostly made up of multi mapping reads The third row scores shows the scores that PongPongPro calculated for the ping pong signatures The heights of the bars range between 0 and 1 and represent the scores of ping pong signatures The fourth row Flybase transposons shows the location of annotated transposons Transposons are shaded according to their score Transposons with high scores are darker and transposons with low scores are brighter The last row RepeatMasker shows regions with repetitive elements The transposon is covered with ping pong signatures from start to end which is an indication that it is thoroughly suppressed through the ping pong cycle Ping pong signatures Three bedGraph files are generated for the detected ping pong signatures Refer to http genome ucsc edu FAQ FAQformat html format1 8 for more information about the bedGraph file format The file ping pong signatures read stacks on minus _strand bedGraph contains the heights of the stacks on the reverse strand of ping pong signatures The file ping pong signatures read stacks on plus_strand bedGraph contains the heights of the stacks on the forward strand of ping pong signatures The file ping PingPongPro v1 0 User Manual 13 pong signatures scores bedGraph contains the scores of ping pong signatures The scores are calculated as 1 minus the value of
6. F7 The compiled executable can then be found in seqan trunk build debug bin Debug 5 3 Additional pre requisites for plotting PingPongPro uses R Project http www r project org 8 to generate plots If you want PingPongPro to generate plots option p then install R Project Linux Instructions on how to install R Project on Linux can be found here http cran r project org doc manuals r release R admin html Installing R under Unix_002dalikes Make sure Rscript is in your PATH environment variable before running PingPongPro export PATH path to R_HOME bin PATH Windows Instructions on how to install R Project on Windows can be found here PingPongPro v1 0 User Manual 6 http cran r project org doc manuals r release R admin html Installing R under Windows Make sure Rscript is in your PATH environment variable before running PingPongPro set PATH C path to R_HOME bin sPATH 6 Command line options h help Print a short help about available command line options and exit Default off version Print information about the version of PingPongPro and exit Default off b browserTracks By default PingPongPro produces output in TSV format only When b is specified it creates additional browser track files which are suitable for display in common genome browsers such as the UCSC Genome Browser http genome ucsc edu 4 or the Integrative Genomics Viewer h
7. PingPongPro v1 0 User Manual Sebastian Uhrig suhrig students uni mainz de September 27 2014 Table of contents 1s What S NOW scssscssscasniasanisnsiansaxxtuncanaenesesinaiesens caasa aoni aaia nE 2 Introd ction eosisisssscsssiscniiriseresssisssodsdtssecds issen isinsi n iiaa 3 QUICK SLANE arinena E E EES 4 How PingPongPro works sssessssescseeccsecccoseccoeecoecccseecoeecoeccceeccoeecoeseceeeo 4 1 Detecting ping pong signatures sssssssesesssesessscsecssesecesesessseseseseseeeses 4 2 Finding transposable elements with ping pong cycle activity ce ee eeee eens Be installation esseen a a E E EE E TE E EE 5 1 Using pre compiled executables sosessosusssessossssesessenessosessessoseseesesseses 3 2 COMPILING TOM SOUICE srsirrsrresirisrs inistart s taten entener SENERE eee ewasesvecneeienes 5 3 Additional pre requisites for plotting cece cece cece eee ee eneeeeeeeeeeneeee 6 Command line OPTIONS sicssscccinacddecspawsdndeaasdacciaacdeaiapausdsthendsecdigacsdacinaesind 7 Accepted input formats sis cess cceceses cece cesses cceses awe Ses ee cease ea ce seca eeede eae sess 7 1 High throughput sequencing data ccccecescccccceeesssccccceeenssssccsceeeeeeees 7 2 Annotations of transposable elements sccesesccccscscresscarcesesccecscsevess 8 1 Ping pong SIGMAUUNES nce exclu ncsiete ceuduesnniesnsudvaenteaudecetiasdieatiwcivexeiandsaxeed 8 2 Ping pong
8. The chromosome or scaffold which the transposon is located on start Start coordinate of the transposon zero based end End coordinate of the transposon half open i e the coordinate points to the first base pair after the end of the transposon pValue P value indicating the confidence that there is ping pong cycle activity within the region of the transposon qValue P value corrected for multiple testing using the false discovery rate procedure by Benjamini and Hochberg 1995 pingPongReads The number of reads that overlap by ten nucleotides within the region of the transposon after the reads have been weighted by PingPongPro s scoring system normalizedPingPongReads RPKM normalized value of pingPongReads i e the value of pingPongReads divided by the length of the transposon in kbp and divided by the total number of reads in the input file per million This figure should be suitable to compare the ping pong cycle activity between transposons and between samples 10 discardedPingPongReads The number of reads which overlap by ten nucleotides within the region of the transposon but which have been discarded by PingPongPro s scoring system The sum of pingPongReads and discarded PingPongReads equals the unweighted number of reads which overlap by ten nucleotides within the region of the transposon 11 strandRatio The unweighted number of ping pong reads o
9. a transposon Gene Transfer Format file extension gtf Refer to http genome ucsc edu FAQ FAQformat html format4 for information about the Gene Transfer Format PingPongPro parses gt files in the exact same way as files of the General Feature Format Tab separated values file extension tsv PingPongPro parses tsv files in the exact same way as csv files with two exceptions Fields must be separated by exactly one tab And fields cannot optionally be enclosed in double quotes Example rj Bti0015567 i chr2L 20740303 20748120 PingPongPro v1 0 User Manual 10 8 Output 8 1 Ping pong signatures PingPongPro outputs the list of all stacks which overlap by ten nucleotides in a tab separated file named ping pong signatures tsv The following table lists the meanings of the columns in the file Table 2 Output of ping pong signatures in tsv format Field name Description The chromosome or scaffold which the ping pong 1 contig signature is located on The coordinate of the 5 end of the stack on the Ponitid forward strand zero based Estimated fraction of signatures that have the same combination of properties but that are not true ping pong signatures Lower values indicate higher confidence that the signature is not due to 3 FDR coincidence For example if there are 100 putative ping pong signatures and 18 stacks with the same combination of properties but with a d
10. and the time it took to run each step to standard error Default off 7 Accepted input formats 7 1 High throughput sequencing data PingPongPro scans high throughput sequencing data given via the parameter i for signs of ping pong cycle activity Accepted file types for the high throughput sequencing data are the Sequence Alignment Map SAM format and the Binary Compressed Sequence Alignment Map BAM format See http samtools sourceforge net for more information on these file formats Only single end alignments can be processed by PingPongPro Paired end alignments are treated like single end alignments The SAM BAM input file does not need to be sorted or indexed 7 2 Annotations of transposable elements PingPongPro can check a list of transposable elements given in parameter t for ping pong cycle activity It accepts various formats The format is determined by the file extension In general only text files are accepted Every line represents the coordinates of a single copy of a transposon Lines that cannot be parsed are discarded silently The chromosome names in the annotation file must be identical to those in the file with high throughput sequencing data PingPongPro v1 0 User Manual 9 BED file format file extension bed Refer to http genome ucsc edu FAQ FAQformat html format1 for information about the BED file format PingPongPro only uses the fields chrom 1 chromStart 2 chromEnd 3 name 4 an
11. cycle activity per transposon scccceeesesccccccceesssscccceseeneses 8 3 Browser WACKS vatccsrsewlnrecarivewaneucddseveias dadasvavesacwadsencesyevadeeniivdidedsavediwnss 22 PingPongPro v1 0 User Manual 1 What s new v1 0 This is the initial release of PingPongPro 2 Introduction Piwi interacting RNAs piRNAs are a class of small non coding RNAs predominantly active in the germ line There they limit the detrimental effect of transposons on the genome They do so by forming a complex with Piwi proteins The complex binds and then cleaves mRNA molecules of active transposons The resulting mRNA fragments induce the production of more piRNAs thus reinforcing the anti transposon response in a feed forward loop called the ping pong cycle 2 3 A key question in ping pong cycle research is identification of piRNAs which are amplified through the cycle PingPongPro is a command line tool for detecting ping pong cycle activity in piRNA Seq data 3 Quick start Pre compiled binaries of PingPongPro can be downloaded from here http sourceforge net projects pingpongpro files PingPongPro takes a file with aligned reads in SAM or BAM format as input see http samtools sourceforge net for more information This file is passed to PingPongPro using the parameter i For example pingpongpro i alignments bam The program then writes a list of putative ping pong signatures to the file ping pong signat
12. d scores Plots for the scoring of transposable elements As explained in section 4 2 PingPongPro decides if a transposon shows signs of ping pong cycle activity based on the relative abundance of reads with an overlap of ten nucleotides compared to reads with other overlaps If the former is significantly higher the transposon is assigned a high score otherwise it is assigned a low score The file transposons z scores pdf contains one bar plot for every transposon that was given in the annotation file via the parameter t The file predicted transposons z scores pdf contains the same data for predicted transposons if the parameter T was specified The bar plots show the abundance of reads with an overlap of ten nucleotides within the region of the transposon compared to the abundance of reads with other overlaps The read counts have been weighted according to PingPongPro s scoring system and are normalized using z scores 9 References 1 Benjamini Y Hochberg Y Controlling the False Discovery Rate A Practical and Powerful Approach to Multiple Testing Journal of the Royal Statistical Society 1995 Series B Methodological 57 1 289 300 2 Brennecke J Aravin AA Stark A Dus M Kellis M Sachidanandam R Hannon GJ Discrete Small RNA Generating Loci as Master Regulators of Transposon Activity in Drosophila Cell 2007 128 6 1089 103 3 Gunawardane LS Saito K Nishida KM Miyoshi K Kawamura Y Nagami T Siomi H Siomi MC
13. d strand 6 All other columns are ignored The fourth field name serves as the identifier of a transposon Comma separated values file extension csv csv files are expected to have at least five fields The fields must be separated by exactly one comma Fields can optionally be enclosed by double quotes The following table lists the fields and their meanings All other columns are ignored Table 1 Fields in csv formatted transposon annotations Field name Description 1 identifier A name that uniquely identifies the transposon 2 strand The strand which the transposon is located on either or 3 chromosome The chromosome which the transposon is located on 4 start Start coordinate of the transposon zero based 5 lend End coordinate of the transposon half open i e the coordinate points to the first base pair after the end of the transposon Example FBti0015567 chr2L 20740303 20748120 General Feature Format file extension gff Refer to http genome ucsc edu FAQ FAQformat html format3 for information about the General Feature Format PingPongPro only uses the fields seqname 1 start 4 end 5 strand 7 and group 9 All other columns are ignored Notably the field feature 3 is not evaluated such that all lines in the file are checked for ping pong cycle activity regardless of their feature type The ninth field group serves as the identifier of
14. dering of plots So the program Rscript must be available in the PATH environment variable see section 5 3 In general PingPongPro first generates an R script with the name of the plot and the file extension R The script contains the data to be plotted and commands for the generation of the plot This script is then executed with Rscript which renders the plot as a PDF file Plots for the scoring of ping pong signatures As explained in section 4 1 PingPongPro scores putative ping pong signatures based on how likely it is that the signature is just a random alignment of reads which happen to overlap by ten nucleotides In order to estimate how often a signature with a certain combination of properties occurs by chance it counts how often such signatures occur with overlaps other than ten nucleotides The file ping pong signature z scores pdf contains one bar plot for every possible combination of properties height score independence of the local coverage adenine bias The bar plots show how often signatures with a certain combination of properties were counted with an overlap of ten nucleotides and how often with other overlaps The counts are normalized using z scores When the bar for an overlap of ten nucleotides is remarkably higher than other bars this means that this combination of properties is PingPongPro v1 0 User Manual 14 peculiar to true ping pong signatures Putative ping ping signatures with these properties get goo
15. er reads in the vicinity In conclusion stacks that are considerably higher than what can be explained by the local coverage i e independent of the local coverage are indicators of true ping pong signatures e Adenine bias piRNAs naturally have a preference for uracil at their 5 ends Since piRNAs amplified through the ping pong cycle are complementary over a length of ten nucleotides these piRNAs also show a preference for adenine at position ten Consequently stacks of reads with adenine at position ten are a third indicator of true ping pong signatures PingPongPro examines the properties of every putative ping pong signature In order to decide if this combination of properties is peculiar to ping pong signatures it counts how many stacks there are in the dataset which have the same combination of properties but which overlap by some other length than ten nucleotides If there are many then PingPongPro concludes that this combination of properties is easily satisfied by random alignments of reads and not something which is peculiar to ping pong signatures In this case the signature gets a bad score If on the other hand the signature has properties which are rarely seen in combination with arbitrary overlaps then the signature gets a good score The score is directly based on relative frequencies For example if PingPongPro finds 100 stacks with an overlap of ten nucleotides and 18 stacks with the same combination of propertie
16. files Extract it to segan trunk sandbox workspace tar xvzf pingpongpro source tar gz mv pingpongpro source sandbox workspace apps pingpongpro Compile PingPongPro cd seqan trunk build debug cmake make pingpongpro PingPongPro v1 0 User Manual 5 The compiled executable can then be found in seqan trunk build debug bin Windows Download the contribution package for SeqAn on Windows as explained here http trac seqan de wiki HowTo InstallContribsWindows Follow the instructions for installing the SeqAn API on Windows http trac seqan de wiki Tutorial GettingStarted WindowsVisualStudio Skip the Hello World example Instead create a sandbox for PingPongPro cd seqan trunk python util bin skel py repository sandbox workspace python util bin skel py app pingpongpro sandbox workspace Download the source code of PingPongPro from http sourceforge net projects pingpongpro files Extract it to seqgan trunk sandbox workspace with a program that is capable of extracting files from tar gz archives Then move all the files to the newly created sandbox move pingpongpro source sandbox workspace apps pingpongpro Rerun cmake to register the newly created application cd segan trunk build vs9 cmake Open the Visual Studio solution seqan trunk build release sandbox workspace apps pingpongpro seqan_ sandbox workspace apps pingpongpro sin In Visual Studio go to Build gt Build Solution
17. ifferent overlap then FDR has a value of approximately 0 18 18 divided by 100 The number of reads that make up the stack on 4 stackHeightOnPlusStrand the forward strand may be fractional if it contains multi hits The number of reads that make up the stack on 5 stackHeightOnMinusStrand the reverse strand may be fractional if it contains multi hits 8 2 Ping pong cycle activity per transposon If the parameter t or T is specified PingPongPro produces statistics about ping pong cycle activity within the regions of given t or predicted T transposons Statistics about given transposons are written to a tab separated file named transposons tsv statistics about predicted transposons are written to a tab separated file named predicted transposons tsv Both files have the same format Table 3 Output of ping pong cycle activity per transposon in tsv format Field name Description For annotated transposons the identifier of the transposon given in the annotation file for predicted transposons a dynamically generated identifier based on coordinates 1 identifier PingPongPro v1 0 User Manual 11 Field name Description strand The strand which the transposon is located on either or In the file for predicted transposons this field always has the value This is because PingPongPro currently does not try to guess the strand contig
18. information about the types of plots which are generated PingPongPro uses R Project for rendering of plots Make sure the program Rscript is available in the PATH environment variable see section 5 3 Default off PingPongPro v1 0 User Manual t transposons PATH PingPongPro can check a given list of transposons for ping pong cycle activity If PATH points to a file with coordinates of transposons then PingPongPro will produce statistics about ping pong cycle activity within the regions of the given transposons See section 7 2 for file formats that are accepted as input Section 8 2 describes the output that is produced This option can be specified multiple times All given files will be processed but only one output file is produced in which the transposons of all input files are merged Default none m T predict transposons RANGE If a list of annotated transposons is not available then PingPongPro can predict the regions of transposons or more generally speaking regions with ping pong cycle activity It does so by defining regions around ping pong signatures dynamically Ping pong signatures that are not more than RANGE base pairs apart from each other will be merged into a contiguous region Regions with less than two ping pong signatures or a shorter length than 30 base pairs are not reported Default 1000 V verbos When this option is given then PingPongPro reports its current progress
19. n the forward strand divided by the unweighted number of ping pong reads on the reverse strand PingPongPro v1 0 User Manual 12 8 3 Browser tracks When PingPongPro is run with the parameter b it creates browser track files which are suitable for display in common genome browsers such as the UCSC Genome Browser http genome ucsc edu or the Integrative Genomics Viewer https www broadinstitute org igv home 02 o Up aba od l la ada lh bt dete bel IL s il da ae 1 68965 height of rea Willd i lal 1s dba Wah b l ta l j il l Lh il l i 102 art nhh i al y jli 1000 alye SESESESESESISSSISSS ESSE ES EE ES SEED 39 gt 3292929299999 pEEEEEEEEEEEEEEEEEED TE EEE EEEEE EESEEYEESEEPEEEEE Rep no E ler ct aatMasker Figure 2 Visualization of ping pong signatures in the UCSC Genome Browser PingPongPro is able to generate output in formats suitable for visualization in genome browsers like the UCSC Genome Browser and IGV This figure is a screenshot of the UCSC Genome Browser It depicts ping pong signatures within the transposable element FBti0019102 with the genomic coordinate chr2L 1220184 1227592 in D melanogaster available at the NCBI Sequence Read Archive via the SRA accession number SRRO10960 5 The topmost row read stacks on strand shows the heights of stacks on the reverse strand the second row read stacks on strand shows
20. ping pong cycle activity PingPongPro v1 0 User Manual 4 5 Installation 5 1 Using pre compiled executables Pre compiled executables of PingPongPro do not require any installation They only need to be downloaded and extracted from a compressed archive Linux 32 bit and 64 bit binary distributions of PingPongPro are available for Linux at http sourceforge net projects pingpongpro files After downloading the respective tar gz file for your architecture extract it with tar tar xvzf pingpongpro linux tar gz Windows 32 bit and 64 bit binary distributions of PingPongPro are available for Windows at http sourceforge net projects pingpongpro files After downloading the respective zip file for your architecture extract it by right clicking the zip file and choosing Extract all from the context menu 5 2 Compiling from source PingPongPro builds on the SeqgAn library http www segan de You first need to download the SeqAn API before you can compile PingPongPro from source Linux Follow the instructions for installing the SeqAn API on Linux http trac seqan de wiki Tutorial GettingStarted LinuxMakefiles Skip the Hello World example Instead create a sandbox for PingPongPro cd seqan trunk util bin skel py repository sandbox workspace util bin skel py app pingpongpro sandbox workspace Download the source code of PingPongPro from http sourceforge net projects pingpongpro
21. r LY Helmberg W Kapustin Y Khovayko O Landsman D Lipman DJ Madden TL Maglott DR Miller V Ostell J Pruitt KD Schuler GD Shumway M Sequeira E Sherry ST Sirotkin K Souvorov A Starchenko G Tatusov RL Tatusova TA Wagner L Yaschenko E Database resources of the National Center for Biotechnology Information Nucleic Acids Research 2008 36 Database issue D13 21 PingPongPro v1 0 User Manual 15
22. s but with different overlaps then it estimates that about 18 of the putative ping pong stacks must be due to chance It cannot tell which of the putative ping pong stacks are the product of ping pong cycle activity and which are due to chance Therefore all of them are assigned a score of 0 82 100 minus 18 As a result only 82 of the reads of these signatures will be counted and the rest is discarded Ultimately this weighting of signatures ensures that only those reads are counted which are probably not to be attributed to coincidence 4 2 Finding transposable elements with ping pong cycle activity In order to decide if a given transposon is suppressed by the ping pong cycle PingPongPro counts the number of reads that are part of putative ping pong signatures within the region of the transposon The reads are weighted before counting as explained in the previous section PingPongPro then counts how many reads are part of stacks which overlap by some other length than ten nucleotides Again reads are weighted before counting If the transposon is ping pong regulated then the abundance of reads which are part of putative ping pong signatures should be considerably higher than the abundance of reads which are part of arbitrarily overlapping stacks The difference in abundance is normalized to a z score which in turn is converted to a p value The lower the p value the more confident PingPongPro is that the transposon shows signs of
23. ti hits are ignored during analysis of RNA Seq data because it is unclear where the read really originated from In piRNA Seq data however it would lead to a major fraction of the reads being lost if multi hits were ignored PingPongPro can handle multi hits in one of several ways weighted A multi hit is counted as the reciprocal of the number of alignments of the same read For example if a read has 5 alignments it is counted as one fifth unique No distinction is made between multi hits and unique hits Multi hits are counted as one full read This option is useful if the input data has already been filtered for multi hits such that all but one randomly selected multi hit have been deleted from the input file discard Multi hits are not counted at all This option can be used to compare the effect of multi hits on the output of PingPongPro Default weighted O Output PATH Specify the path to a directory where PingPongPro should put all output files The directory is created if it does not exist yet The parent directory must exist though Existing files are overwritten without warning If this option is omitted then PingPongPro writes all files to the current working directory It is therefore recommended to run PingPongPro from an empty directory if this option is not specified Default current working directory p plot This option instructs PingPongPro to generate plots Refer to section 8 4 for
24. ttps www broadinstitute org igv home 9 See section 8 3 for more information on the types of tracks that are generated Default off s min stack height NUMBER OF READS Ping pong signatures with one or both stacks smaller than NUMBER OF READS are omitted from the output They are still considered to find suppressed transposons however Default 0 i input PATH Specify the path to a file with high throughput sequencing data that PingPongPro shall scan for signs of ping pong cycle activity See section 7 1 for accepted file types On Linux you can use the special file dev stdin to make PingPongPro read data from standard input Specifying this option is mandatory Default none PingPongPro v1 0 User Manual 7 l min alignment leng L max alignment leng th th LENGTH LENGTH PingPongPro ignores alignments given in the input file that are shorter or longer than the typical length of a piRNA between 24 and 32 nucleotides Use the parameter 1 to define the minimum length and the parameter L to define the maximum length of alignments that should not be ignored by PingPongPro Default 24 for 1 and 32 for L m multi hits METHOD Very often reads of piRNAs map to several loci because the genome contains multiple copies of a transposable element that is targeted by a particular piRNA Usually such multi mapping reads mul
25. ures tsv The column FDR in this file indicates how confident PingPongPro is that a given ping pong signature is not just a random alignment of reads but truly a product of ping pong cycle activity FDRs close to 0 are good and FDRs close to 1 are bad In addition PingPongPro can find transposable elements that are suppressed through the ping pong cycle when run with the parameter T pingpongpro i alignments bam T 1000 PingPongPro produces a file named predicted_transposons tsv which contains a list of regions which are possibly suppressed by the ping pong cycle The column qValue indicates PingPongPro s confidence Again values close to 0 ideally lt 0 01 are good and values close to 1 are bad The argument 1000 tells PingPongPro to merge ping pong signatures within a window of 1000 bp into one region PingPongPro can check a list of transposable elements of interest for ping pong cycle activity The list must take the form of an annotation file in GFF GTF or BED file format see http genome ucsc edu FAQ FAQformat html for more information It is passed to PingPongPro using the t parameter pingpongpro i alignments bam t annotated transposons bed PingPongPro v1 0 User Manual 2 PingPongPro then generates a file named transposons tsv which assigns a q value to every transposon given in the file annotated _transposons bed The file is to be interpreted alike the file predicted _transposons tsv Lastly
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