Manual to Online FISH Probe Optimization Tool
Contents
1. its ends to allow PCR mediated cloning of the intermediate sequence Unique regions longer than the max_uniq_pcr are divided into multiple templates and a PCR primer pair is designed to clone each of them Section 10 The min_uniq_pcr and max_uniq_pcr values should be selected according to the type of experiment planned If FISH probes are generated by nick translation of random priming the overall length of the FISH probe target should be in the order of tens of kilobases for reliable fluorescence detection A PCR product less than a few kilobases in length would contribute rather insignificantly to the desired overall length of the FISH probe Hence the min_unig_pcr is by default set to 4000 If the unique regions on the other hand are very fragmented and no contiguous regions of sufficient length are identified this default value might need to be decreased The max_uniq_pcr value depends mainly on the feasibility of the PCR mediated cloning To save time and cost lower number of longer PCR products to cover a given region is desirable However the length of the PCR amplicons is limited by the efficiency of the PCR and the subsequent cloning The upper limit is set by default to 8500 For CyDNA based FISH probes in which individual FISH probe molecules are di rectly synthesized in a PCR reaction with E10 polymerase the min_pcr_size and max_pcr_size values limit the length of the produced FISH probes In the experi ments described in Chapte2. 19 TECHNICAL DETAILS OF THE SOFTWARE AND HARDWARE 25 INTRODUCTION STANDARD DESIGNER ADVANCED DESIGNER PROTOCOLS ADVANCED DESIGNER 1 Advanced designer searches for specific FISH probes within the provided DNA sequence using the settings from your uploaded configuration file You may modify and use the configuration file obtained as a result of the standard designer query or the default configuration file can be downloaded in text format and PDF format Note that the PDF version cannot be uploaded It is only meant for viewing Only unformatted raw text files can be uploaded Before proceeding to the next page copy the captcha password protecting the server againgst automatic web attacks Upload Configuration File conf vy Browse Type in the CAPTCHA password shown right HINT y Please copy the grainy captcha code which you see on the right Itis important to use captcha to distinguish your genuine inquiry from automatic robots repeatedly submitting the forms and overloading the server 2010 Jakub Nedbal iakub nedbal aooalemail com Figure 18 1 Advanced Designer shows typical layout used throughout the web site The page is introduced by a short description of its content It contains a file upload dialog button for the user to upload a configuration file The safety password image at the bottom right to protect the page from automated attacks At the bottom is a submission button leading to the
3. PCR product for unique similar FISH probe production Figure 12 1 Additionally combinations of 2 to 5 best PCR products are selected from the pool Figure 12 1p only two best FISH probes are displayed This offers the user the alternative to decide on the number of FISH probes that must be produced to a label sufficient amount of DNA for adequate fluorescence signal 13 Restricted Sequences The user is given the option to select a range of positions within the query which should be excluded from the analysis It becomes desirable if the DNA template for the PCR reaction BAC or plasmid is available for only part of the query and the cloning primers must be selected only form this template The range of sequences restricted from the search is specified by the restrict_sequence parameter obeying the following format The start and end positions of the restricted sequence within the query are separated 14 GRAPHICAL OUTPUT 14 i JEN 0 50k 100k 150k 200k 250k 300k a Single unique repetitive FISH probe binds five similar sites 0 50k 100k 150k 200k 250k 300k b Two best unique repetitive FISH probes extend the labeled length of the five similar sites Figure 12 1 Unique Similar FISH Probes The optimized unique similar FISH probes are made of sequences highlighted by the empty blue rectangles Their binding sites also span all sequences similar to each probe which are emphasized by the filled blue rectangles displayed in the same
4. a Human immunoglobulin heavy chain constant region genes as they are ordered and oriented on human chromosome 14 Their order is contra intuitive because first to the left is the IgA amp 2 gene while the last one is IgM u b The same query sequence with genes was horizontally mirrored to obtain the expected order 15 TEXTUAL REPORT 19 File ending Content primer tat Summary of all primer sequences unique_ segs tat Primer sequences for unique FISH probes complete PCR product sequences restriction endonuclease site presence nonunique_ segs tat Primer sequences for all unique similar FISH probes complete PCR product sequences restriction endonuclease site presence nonunique_ segs N tat Selection of primer sequences for the N best unique similar FISH probes complete PCR product sequences restriction endonuclease site presence XXX primers tat Detailed analysis for each primer pair including alternative primer pairs gencont Configuration file generated in the analysis log Log file of the analysis Table 2 Textual reports The analysis report is provided in a number of files whose names and contents are summarized in this table 15 Textual report Text reports are generated alongside the graphical output These files contain all in formation about the cloning PCR primers designed for the production of specific FISH probe templates Their contents are summarized in
5. are introduced here 14 1 Similar Sequence Distribution The background of the graphical output is composed of a map splitting the query into unique sequences similar sequences and unique similar sequences They are further distinguished by a darker tint if they are part of a similar region Figure 9 1p Each sequence type can be assigned a different color by the parameters unig_color sim_color tint_uniq_color tint_sim_color and unigq_sim_color The colors are specified by comma separated vector of its RGB components ranging from 0 to 255 The background map can be enabled and disabled by the plot_area parameter 14 2 Sequence Grouping into Regions The contiguous unique regions of the minimum required length see Section can be highlighted by rectangles spanning each region by setting the plot_uniq_reg pa rameter They are label Uxx where xx is an integer distinguishing one from another The color of the rectangle is specified by unig_pcr_color parameter Similarly the contiguous similar regions marking rectangles can be enabled by the plot_sim_reg parameter They are labeled Sxx where xx are the distinguishing integers Their color is determined by the sim_pcr_color parameter Distribution of regions in the default sequence is displayed in Figure 8 1 14 3 PCR Products The positions of the PCR products for unique FISH probe production Section are displayed if plot_uniq_pcr is set Figure 11 1 Their positions in the query are h
6. of Jena Germany The core of the software runs in GNU Octave 3 2 4 It calls NCBI Blast 2 2 18 2 performing the sequence alignment Primer3 2 2 2 beta searching for optimal primer pairs Emboss 5 0 0 for restriction endonuclease site search and Gnuplot 4 2 generating the graphical output The website user interface is operated by Apache 2 2 9 web server with CGI scripts handling the user input values executed by Perl 5 10 0 The individual web pages are written in HTML with JavaScripts controlling their dynamic features such as the hint buttons Emails are sent through Exim 4 69 email server by mutt 1 5 18 email client Other common programs supplied with Debian GNU Linux distribution are used for scheduling data parsing and file handling 19 1 Task Scheduling The script runs in an indefinite loop Every 60 seconds check for a new query submission is performed In such case the loop is temporarily interrupted and the query processing described throughout the Section is initiated At the start of each day results older than one week are removed and the genome databases are updated if their new version is available at the Ensembl project FTP sita ftp ftp ensembl org pub current_fasta January 2011
7. tags are surrounded with explanatory text Each XML tag consists of a parameter and a value organized in the following format lt parameter gt value lt parameter gt The user may vary any of the values if desired and the algorithm will adopt to this change The parameters in the configuration file are grouped into those defining Mega BLAST alignment parameters search parameters for the unique and unique similar FISH probes graphical output parameters and primer optimization parameters The default configuration file with annotations describing in detail each entry is included in Abstract If any XML tags are missing from the configuration file they are corrupted or the user specified values are out of range or otherwise unacceptable they are replaced http www ncbi nlm nih gov blast fasta shtml December 2010 5 SEQUENCE ALIGNMENT 5 by hardwired default values of which the user is notified These modified tags are appended to the end of the configuration file which is returned to the user at the end of the analysis Anything distinguished by a fixed width typeface throughout the rest of this chapter refers to a parameter used in the configuration file 5 Sequence Alignment The supplied sequences are aligned with the genome of the specified organism by Mega BLAST algorithm It is initiated with default parameters except for the disabled low complexity sequence filtering and the expectation value lowered from 10 to
8. temperature 68 primer_salt_corrections Primer salt corrections 0 Breslauer 2 1 SantaLucia 2 Owczarzy primer_gc_clamp Primer GC clamp 2 primer_min_gc Primer minimum GC content 30 primer_max_gc Primer maximum GC content 70 primer_max_tm_diff Maximum difference between primer 3 melting temperatures Table 1 Primer parameters in the configuration file The details of each param eter can be found in the Primer3 release 2 2 2 README file parameter is available in the Primer3 release 2 2 2 README fild 10 4 Restriction Endonuclease Recognition Site Search Each PCR product is scrutinized for the presence of selected restriction endonuclease recognition sites Their absence determines the optimal restriction sites for incorpo ration into the cloning PCR primers The user may select a comma separated list of names from the Restriction Enzyme Database and include it through the enzyme parameter for analysis 11 PCR Products for Unique Regions Designing FISH probes for unique sequences is straightforward For unique regions with length between min_pcr_size and max_unigq_pcr a primer pair is identified near 11 PCR PRODUCTS FOR UNIQUE REGIONS 12 MEE EEN e Ph 0 50k 100k 150k 200k 250k 300k Figure 11 1 Unique FISH Probes The optimized unique FISH probes are made of sequences highlighted by the empty pink rectangles They bind unique sequences which are present only in a single copy in the entire genome
9. user 18 WWW INTERFACE 23 Similar and Unique Regions in Target Region Human CH pl E E si Pi 0 50k 100k 150k 200k 250k 300k 106 34M 105 99M Position on Chromosome 14 of Homo Sapiens a bit_score 0 High stringency for unique FISH probes Similar and Unique Regions in Target Region Human CH HE EAE E Ea J tn 0 50k 100k 150k 200k 250k 300k 106 34M 105 99M gt Position on Chromosome 14 of Homo Sapiens b bit_score 200 Compromise value Similar and Unique Regions in Target Region Human CH OREO gooo a iid a agg 0 50k 100k 150k 200k 250k 300k 106 34M 105 99M Position on Chromosome 14 of Homo Sapiens c bit_score 1000 High stringency for unique similar FISH probes Figure 17 1 FISH Probe dependence on bit_score Highly specific unique FISH probes are obtained with low bit_score values a Highly similar unique FISH probes are obtained with higher bit_score values c Bit_score of 200 offers a compromise between both extremes b It reflects the average minimum cut off value of 260 bases for similar sequences which is similar to the size of the FISH probe molecules 19 TECHNICAL DETAILS OF THE SOFTWARE AND HARDWARE 24 for viewing and download 18 1 Data Safety The design of the web site offers a compromise between data safety on one side and the usability with simplicity on the other The user stays anonymous without the need to provide any login details or further inform
10. values The letter sizes used for the annotation and their type face can also be defined through the configuration file 14 7 Output Format The graphical output is produced as an image in EPS vector format which allows scaling without resolution limits and even manual editing of the content Optionally it can be converted into a bitmap formatted image using the graph_format parameter and selecting one of the following options dpng djpeg dtiff or the other formats listed in the configuration file dpi_res determines the resolution of the bitmap file in DPI 14 8 Output Mirroring Sometimes the genes contained in the query sequence are organized on the chromosome in a reversed order compared to the intuitive expectation For instance the human IgM immunoglobulin heavy chain constant region gene is functionally upstream of the other constant region genes but it is localized closer to the end of the chromosome To overcome this confusing arrangement the displayed sequence can be mirrored by setting the rev_xaxis to 1 instead of the default 0 14 GRAPHICAL OUTPUT 0 50k 105 99M yio 0 50k 106 34M 100k 150k 200k 250k Position on Chromosome 14 of Homo Sapiens a rev_xaxis 0 mei mi 100k 150k 200k 250k Position on Chromosome 14 of Homo Sapiens b rev_xaxis 1 18 E 300k 106 34M 300k 105 99M Figure 14 2 Output mirroring enabled by rev_xaxis parameter mirrors the content of the graphical output
11. 0 01 The filtering undesirably removes some repetitive sequences which potentially could represent suitable FISH probe targets Lowering the expectation value cutoff saves processing time by increasing the stringency of search for similar sequence in other words more dissimilar sequences are omitted from the alignment result Further details of the sequence alignment are discussed later in this chapter in Sections 16 and 6 Similar and Unique Sequences Each alignment contains information about the bit score the starting and ending po sitions in the query and the starting and ending positions of the similar hit in the genome Alignments obtained from all sequence databases e g chromosomes are pooled together and sorted according to their bit scores details in Section 17 The alignment with the highest score specifies the position of the query sequence within the genome and is not considered in the further analysis The remaining alignments are mapped onto the query by marking any sequence between each alignment start and end positions as a similar sequence The similar sequences are those which are found in the query and one or more similar copies identified by Mega BLAST elsewhere in the genome Section 5 This way the target region becomes divided into stretches of 7 MERGING ADJACENT SIMILAR SEQUENCE STRETCHES 6 unique and similar sequences as illustrated in Figure 7 1p which displays the unique sequences in white while the simi
12. Manual to Online FISH Probe Optimization Tool Jakub Nedbal 17 January 2010 ver 1 0 CONTENTS 1 This manual describes the details of the FISH probe optimization algorithm starting with the processing of the user supplied data and finishing with the output report generation It will help unfamiliar users understand the principle of the algorithm and help them understand its functions and ways to modify them by changing the default configuration file Contents 1 Introduction 2 2 Interaction with the User 3 3 Sequence for Analysis 4 4 Configuration File Import 4 5 Sequence Alignment 5 6 Similar and Unique Sequences 5 7 Merging Adjacent Similar Sequence Stretches 6 8 Region Length Filtering 6 9 Histogram of Unique Similar Sequence Repeats 8 10 PCR Product Search 9 10 1 PCR Templates Selection 2 0 0 0 000002 2 eee 9 10 2 Neighboring Template Overlap 2 2000 10 10 3 Primer Search Parameters 2 2 a a a a ee 10 10 4 Restriction Endonuclease Recognition Site Search 11 mn 1 PCR Products for Unique Regions 11 1 INTRODUCTION 2 12 PCR Products for Unique Similar Regions 13 13 Restricted Sequences 13 14 Graphical Output 14 14 1 Similar Sequence Distribution 2 200484 15 14 2 Sequence Grouping into Regions 20 00 000 15 14 3 PCR Products ss e d ios i nae ees kee eS we BES 15 14 4 Reference Sequences 2 aa ee 16 14 5 Horizontal Axis 2 e
13. Table 2 They provide details on the primer sequences their positions in the query sequence the complete PCR prod ucts sequences which are necessary for verification of any plasmids produced using these primers and the presence of restriction endonucleases recognition sites within the PCR products 16 Mega BLAST Alignment Parameters The query sequence is aligned to the genome using the Mega BLAST algorithm It was introduced in the Section 5 without providing any details which would disrupt the flow of the Chapter and they will be introduced in the following Sections with the 16 MEGA BLAST ALIGNMENT PARAMETERS 20 assumption that the reader is already familiar with the algorithm and its requirements The genome sequence used for the alignment is obtained from the Ensembl project The quality of the genome assembly varies among the species Some of the less inves tigated genomes are assembled into a single contiguous database not accounting for the individual chromosomes In others it is assembled into sequences of the individual chromosomes mitochondrial and nonchromosomal DNA The nonchromosomal DNA databases contain assembled sequences that could not have been ordered or oriented onto one of the chromosomes so far The most studied genomes also include patch and haplotype sequences The patch sequences contain newly sequenced parts of the genomes and amended sequences obtained by improved techniques which might in the fut
14. ated while a new web page is send to the user s web browser to display regularly updated information about the progress of the data processing Once the analysis is successfully finished a report is posted on the website and the user receives a notification email If any processing error occurs the user is also informed 3 SEQUENCE FOR ANALYSIS 4 3 Sequence for Analysis The user must provide at least one query sequence to be aligned with the genome for identification of the specific FISH probe targets Additionally a reference sequence can be provided This may consist of multiple shorter sequences of genes regulatory sequences or any other interesting parts of the query They will be aligned to the query sequence and their positions will be displayed in the graphical output for easier inter pretation of the identified FISH probes positions Both the query sequence and the optional reference must conform with the standard DNA FASTA format In simplified words the FASTA format consists of a string of bases ACGT preceded by a line initi ated by the greater than character gt followed by the name of the sequence Multiple sequences with their names can be concatenated into one file 4 Configuration File Import The user has the option to provide a custom configuration file or rely on the default one The configuration file allows modification of large range of parameters of the analysis It is based on the XML format in which XML
15. ation and the results are accessible exclusively to the user for one week following their completion None of the data submitted to the web server is publicly available yet it could be potentially viewed by a third party exerting a concentrated hacking effort Privacy Protection Means Each user submission to the web server is given a unique 11 to 16 digit tag without which the data cannot be accessed by anybody else It consists of an ever increasing 10 digit number distinguishing the queries by the time of their submission This is followed a randomly generated 1 to 6 digit number separated by a dash The user submitted query including the DNA sequences configuration files and the email address are kept on the server only for a short time before it is safely stored for analysis The email address is only kept during the analysis and destroyed after sending the notification email The results are irrecoverably destroyed a week after finishing the analysis The web server communicates with the Internet browser of the user through a non encrypted connection which is the common case for similar research tools intended for the scientific community 19 Technical Details of the Software and Hardware The analysis software and the web server operates on a single computer featuring AMD Athlon II X2 240e processor and 4 GB of RAM installed with Debian 5 0 GNU Linux operating system The server is physically located at King s College London with
16. aximum allowed length of the PCR template If the scrutinized region is longer than this value it is first divided into the lowest possible number of templates of length within the allowed range The re gion division depends on the value of the pcr_overlap parameter which defines the requirement for overlap between the neighboring PCR products and its extent 10 PCR PRODUCT SEARCH 10 10 2 Neighboring Template Overlap The primers are sought only in the terminal tails of the selected PCR templates Ini tially the length of these tails is limited to 100 bases If no primers could be identified within the tails they are incrementally extended by another 100 bases and the search is repeated until a suitable primer pair is found or the absolute value of the pcr_overlap parameter is reached If no primers could be found within the extended tails the se quence search window is shifted by the absolute value of the pcr_overlap parameter The search for primers again starting with only 100 bases long tails is repeated until successful PCR primer pair is identified or the end of the region is reached The length of the terminal tails into which the primers localize is purposely minimized to lessen the gaps between individual PCR products The search window and the terminal tails are always chosen such that both PCR primers stay within the studied region to ensure specificity of the resulting FISH probes If the per_overlap value is negative the PCR pri
17. considered unique These unique regions of sufficient length are selected for the design of FISH probes Any parts of the query which are found in the genome similar If several similar sequences in multiple similar copies are referred to as overlap or are separated by a gap of a maximum permitted length they are grouped into a single similar region These similar regions are analyzed for the presence of a subclass of unique similar sequences Such sequences are found in multiple similar copies within the genome but all of them localize to the same similar region and make suitable repetitive FISH probe targets Both unique and unique similar regions are partitioned into segments of user defined length range for production of for single copy unique and repetitive unique similar FISH probes PCR primers with user determined characteristics are designed to enable either PCR mediated cloning of the FISH probe templates or direct synthesis of the FISH probes 2 Interaction with the User A successful query submission through the web page results in a project directory being produced on the web server In this directory the query sequence for analysis is saved along with the selected target species the user s email address a default or user modified configuration file and an optional file with the reference sequences such as genes This project directory content is imported and the analysis of the input files is initi
18. d unique similar FISH probes as long as it does not mask presence of similar sequences found elsewhere in the genome which would lead to production of nonspecific unique similar FISH probes Therefore the bit_score can be increased only if it does not result in longer overall stretch of unique similar FISH Figure J The implications of changing the bit_score values should be understood and taken into account when designing the FISH probes Repeating the algorithm with several bit_score values might be desirable before deciding which is the optimal set of FISH probes to be used All FISH probes used in the experiments presented in this thesis were designed with the bit_score value of 200 18 WWW Interface The user interface of the FISH probe optimization algorithm is achieved through an Internet pag The page has four main sections The Introduction explains the usage and applications of the algorithm while Protocols provide technical advice on cloning and FISH probe production The Standard Designer allows the user to upload a query sequence select a target species provide an email address and upload optional reference sequence file The Advanced Designer in addition requires upload of an user supplied configuration file Figure 18 1 Once analysis is successfully completed a new page with summary of the results is provided Figure and a notification email is sent to the user specified address The data is kept on the server for one week for the
19. e 16 14 6 Annotation of the Output wade 2d ek oe eS we we ae ES 17 14 7 Output FORMAT lt c s ss sac tdia bara k E ee ee ee eR 17 14 8 Output Mirroring cacy ep ae Bec oe Sw Bee ow e 17 15 Textual report 19 16 Mega BLAST Alignment Parameters 19 16 1 Genomes in Single Sequence o ooo a a e 20 16 2 Genomes with Patches and Haplotypes ooa a a aa aa 21 17 Mega BLAST Alignment Import 21 18 WWW Interface 22 18 1 Data Satety s lt sos a s skoa na REE EAE e aa Swe BEE a 24 19 Technical Details of the Software and Hardware 24 19 1 Task Scheduling arch ee aaa e 27 1 Introduction The website http www nanoimaging uni jena de fish is the user interface to the FISH probe search algorithm It gives the user the option to select a target species and input a DNA sequence to be analyzed for the presence of potential FISH targets 2 INTERACTION WITH THE USER 3 The sequence together with other optional parameters are analyzed and a report is generated It includes a comprehensive graphical presentation of the analyzed DNA sequence with suitable FISH probes highlighted alongside the relevant genes on a back ground map of unique and similar sequences Primer pairs necessary for the production of the FISH probes and their exact sequences are also delivered The user supplied DNA sequence referred to as the query is aligned to the genome of the selected species Query sequences found in only a single position in the entire genome are
20. e penalties for a mismatch gap occurrence and gap extension depend on a lookup table called the substitution matrix whose values are proportional to the natural likelihood of occurrence of these mutations insertions or deletions The raw score is dependent on the parameters of the substitution matrix and therefore raw scores obtained from alignments using different substitution matrices are not comparable The bit score unlike the raw score is normalized in respect to the substitution matrix values and therefore even bit scores obtained using different substitution matrices can be directly compared Both scores are closely related to the expectation value introduced in the Section 5 This is equivalent to the number of alignments with raw scores better than the current one which would occur in the query and database sequences by random chance 18 WWW INTERFACE 22 lower limit for the similar sequences in the human genome to 260 40 bases in length with the percentage similarity of 85 2 1 5 The length matches the FISH probe size which is preferably between 100 and 300 bases Lower bit_score value might be desirable for designing unique FISH probes Figure 17 1a The lower bit score threshold decreases the stringency of the search for the similar sequences making the resulting FISH probes more specific for their targets and less likely to bind other parts of the genome Higher bit_score on the other hand might increase the quality of the designe
21. ented in Figure D I with the unique similar sequence highlighted in the cyan color 10 PCR PRODUCT SEARCH 9 200 0 50k 100k 150k 200k 250k 300k Figure 9 1 Similar unique sequence histogram value represents the number of similar repeats of given sequence stretch within each similar region of the query se quence The histogram value is zero for sequences which are similar to any part of the genome outside the scrutinized similar region The unique similar sequences with non zero histogram bins are highlighted by cyan color in the sequence map 10 PCR Product Search The algorithm serves the main purpose of designing PCR primers to clone suitable sequences for specific FISH probe production The obtained PCR products are tiled across to the selected unique and unique similar sequences Sections 8 and 9 The permissible PCR product length and the overlap between neighboring PCR products can be defined in the configuration file 10 1 PCR Templates Selection Multiple parameters decide which sequences should be used for PCR cloning and sub sequent FISH probe production The templates for the PCR primer design are selected from the unique and unique similar regions Min_unig_pcr and min_sim_pcr parame ters define the minimum permissible length of the PCR template within these regions If the given region is not sufficiently long the PCR primers will not be designed Sim ilarly max_unig_pcr and max_sim_pcr limit the m
22. ighlighted by rectangles of color is defined by unig_pcr_color The naming strategy for the PCR products changes if the unique regions are displayed or not see previous 14 GRAPHICAL OUTPUT 16 Section When disabled plot_uniq_reg is 0 the PCR products are labeled with the letters of alphabet applied in an increasing order i e A Za z Otherwise they are given the name of the respective region and a letter if more than one PCR product is contained in a single region Similar labeling convention applies to the unique similar PCR products Section enabled by the plot_sim_pcr parameter Figure 12 1 Their color is determined by the sim_pcr_color parameter Unlike with the unique PCR products which are plotted side by side each unique similar PCR product is plotted on a separate line of the graphical output Figure 12 1p This provides space for marking the positions of sequences similar to each PCR product These similar sequences are highlighted by filled rectangles with the color defined by mistarget_color if plot_mistarget is set 14 4 Reference Sequences Apart from the analyzed query sequence the user may provide a second FASTA file containing one or more reference sequences such as genes their exons or regulatory sequences These reference sequences are plotted in their respective positions as empty rectangles with their color specified by the genes_color parameter Each sequence in the user supplied FASTA fild is preceded by i
23. lar ones in light gray 7 Merging Adjacent Similar Sequence Stretches As introduced in the Section the algorithm searches for unique similar sequences which are found within only a single similar region of the query sequence These unique similar sequences are suitable targets for repetitive yet unique FISH probes which bind to a number of places within the similar region but not elsewhere in the entire genome The boundaries of such similar region must be selected carefully in order for the al gorithm to correctly identify the unique similar sequences A set of unique similar sequences might be localized into a single limited region of a chromosome but if sepa rated by even a short stretch of unique sequence they would be interpreted as belonging to different similar regions and not considered being unique similar Therefore a toler ance for short unique sequence gap within a single similar region is introduced This allows merging of several similar sequences separated by short gaps into a single similar region The length of this permitted gap is specified by max_sim_gap parameter in the con figuration file It set to 1000 by default which suited the needs of the project introduced in Chapter In general this parameter is dependent on the distribution of the unique and similar sequences in each query and can only be determined empirically by the user Selecting max_sim_gap as large as the length of the entire query might be desi
24. mer search of the second and successive PCR products starts beyond the end of the previous PCR product such that neighboring PCR products never overlap This is particularly beneficial for the design of the CyDNA FISH probes which should not compete for the same overlapping bind ing sites On the contrary positive pcr_overlap gives rise to overlapping neighboring PCR products which are desirable for FISH probes generated by nick translation or random priming Such FISH probes are generated randomly and therefore are inher ently overlapping and competing for their binding sites anyway The overlap of the PCR products increases the span of the FISH probe binding sites by eliminating any gaps between the individual PCR products 10 3 Primer Search Parameters The primers search referred to in the previous paragraphs is conducted by the Primer3 program The user can vary the primer search parameters such as the permissi ble length and annealing temperatures of the primers by modifying the configuration file summarized in Table i The detailed information about each of each of these 11 PCR PRODUCTS FOR UNIQUE REGIONS 11 Configuration parameter Description Default primer_opt_size Primer optimum length 20 primer_min_size Primer minimum length 15 primer_max_size Primer maximum length 30 primer_opt_temp Primer optimum temperature 66 primer_min_temp Primer minimum temperature 58 primer_max_temp Primer maximum
25. next page Hint buttons reveal information dialogs to aid the user The menu bar at the top simplifies the page navigation 19 TECHNICAL DETAILS OF THE SOFTWARE AND HARDWARE 26 INTRODUCTION STANDARD DESIGNER ADVANCED DESIGNER PROTOCOLS QUERY FINISHED Your query has been processed Below you can see the result COMPLETE PROJECT The zip file provides a convenient way to download the complete project directory 129445061 1 139937 zip OUTPUT GRAPHICS Here you can download the output images with the sequence map the optional gene positions and the optimized FISH probe positions Each image comes in EPS vector graphic format and a bitmap format which by default is PNG To get a different output format or resolution modify the configuration file and resubmit the query 129445061 1 139937_1_1 eps 129445061 1 139937_1_2 eps Wi mapy mnt g W EFS AU N mo nn d a v th a 129445061 1 139937_1_1 png 129445061 1 139937_1_all png FILE WITH UNIQUE FISH TARGETS This is a list of all the optimized unique FISH probe targets 1294450611 139937 unique_segs txt Figure 18 2 Result output consists of clearly laid out graphical outputs and textual reports for download and viewing Only part of the result page is shown here for illustration 19 TECHNICAL DETAILS OF THE SOFTWARE AND HARDWARE 27 the Internet traffic redirected from the http www nanoimaging uni jena de domain server located at the University
26. r the goal was to obtain FISH probes around 500 bases long which was achieved by setting min_unig_pcr to 500 and max_unigq_pcr to 700 12 PCR PRODUCTS FOR UNIQUE SIMILAR REGIONS 13 12 PCR Products for Unique Similar Regions More complicated is the design of FISH probes for unique similar sequences These FISH probes bind multiple similar targets that are all located within a single simi lar region The unique similar sequences are defined by the non zero regions in the histogram of the unique repeats Section 9 The PCR product length is limited by the min_sim_pcr and max_sim_pcr param eters These values are selected as described in the previous Section 11 with the differ ence in mind that these FISH probes bind repetitive targets and thus bind sequences of overall length exceeding their own Unique similar FISH probes prepared from shorter PCR products can be tolerated because they give rise to stronger fluorescence signal compared to the unique probes The default min_sim_pcr value is 2000 Providing that any unique similar sequences are present within the query a large number of suitable PCR products is likely to be identified Each of these PCR products or its portions are similar to multiple sites within the same similar region The FISH probes obtained from these PCR products would also bind sequences exceeding their own length The PCR product which is similar to the longest overall pool of sequences is selected as the best
27. rable to detect the presence of any unique similar sequences in case none were identified using the default value The pay off for the large max_sim_gap is the loss of any unique sequences found in the gaps between the similar regions which could otherwise be potentially available as suitable FISH probe targets 8 Region Length Filtering Each region whether unique or similar might contain sequences potentially suitable as FISH targets The maximum permissible length of these target is determined by the 8 REGION LENGTH FILTERING 7 0 50k 100k 150k 200k 250k 300k a max_sim_gap 0 SSeS Sass Sia 0 50k 100k 150k 200k 250k 300k b max_sim_gap 1000 0 50k 100k 150k 200k 250k 300k c max_sim_gap 3000 Figure 7 1 Non contiguous similar region grouping Similar sequences separated by gaps consisting of unique sequences are merged into a single similar region if the gap length is less than the max_sim_gap value a With max_sim_gap set to 0 the similar sequences shown in light gray are fragmented without presence of any similar unique sequences b The default value of 1000 for max_sim_gap yields two similar regions S and S c max_sim_gap of 3000 results in merging most of the similar sequences into a single similar region S 9 HISTOGRAM OF UNIQUE SIMILAR SEQUENCE REPEATS 8 w tr ides 0 50k 100k 150k 200k 250k 300k Figure 8 1 Unique and similar regions of satisfactory lengths are highlighted by pink and blue rectangle
28. row as the respective FISH probe by a comma Semicolon separates several restricted sequence in each query and the pipe symbol separates restricted sequences if multiple queries are submitted simultaneously Restricted sequences are automatically defined for positions of the query sequence which contain other values than the standard bases ACGT An example of this would be the human T cell receptor B locus which is not fully sequenced and it contains two regions with sequences entirely consisting of undefined bases N Figure b 14 Graphical Output While the PCR cloning primers for FISH probe templates production are the most important results of the analysis a graphical output is crucial for understanding and interpreting the results It provides a highly customizable graphical representation of the query sequence with the distribution of unique similar Section 6 and unique similar Section 9 sequences The the binding sites for the optimized FISH probes Sections 11 and are emphasized along with the optional reference sequences such as genes Section 14 4 The configuration file parameters allow the user to choose 14 GRAPHICAL OUTPUT 15 which features should be displayed enable their annotation and modify their colors By default all the options are enabled and a standard colors are provided The detailed description of each parameter is described in the available configuration file Only the most important ones
29. s respectively FISH probes are sought within these regions length of each region The fluorescence intensity obtainable from each FISH probe is proportional to the length of its target and therefore only sufficiently long targets can justify the labor intensive and expensive FISH probe production A length threshold for the regions is therefore introduced to only use the sufficiently long ones It is determined by the min_uniq_length and min_sim_length parameters by default set to 4000 and 30000 for the unique and similar regions respectively The selection of the regions of fulfilling length is illustrated in Figure 8 1 9 Histogram of Unique Similar Sequence Repeats Each similar region is divided into unique similar sequences which occur in one or more similar repeats found exclusively in the similar region and those which are similar to sequences localized to other parts of the genome To identify them a histogram is generated which displays the number of similar sequence repeats within each similar region of the query It has a bin for each base of the query sequence The value in each bin corresponds to the number of similar repeats of its surrounding sequence found the same similar region The histogram bin value is zero if the surrounding sequence is similar to any genomic sequence not contained in the similar region The non zero histogram bins define the unique similar sequence positions in the query An example of such histogram is pres
30. time and to avoid undesired introduction of duplicated or similar sequences The user has the option to enable them by setting include_patch and include_haplo to 1 17 Mega BLAST Alignment Import This Section discusses an important bit_score parameter which determines the thresh old between similar and unique sequences It is linked to the alignment introduced above Section 16 The distribution of the similar and unique sequences detected in the query will depend on the setting of this threshold It also influences the specificity of the pro duced FISH probes which is partially determined by the presence of similar sequences throughout the genome There is no direct characterized link between the sequence similarity obtained by Mega BLAST and the FISH probe s tolerance for mismatches and therefore it must be determined empirically To obtain it it was assumed that the similar sequence stretch should be at least the length of the FISH probes and exceed similarity of 85 The Mega BLAST delivers positions of the similar sequences sorted by their bit scord value which reflects the length and the number of mismatches and gaps between the two compared sequences The minimum required bit score for an alignment to be processed is defined by the bit_score value By default it is set to 200 This sets the Bit score is calculated from the raw score being the sum of scores of all mismatches gaps and extensions of the compared sequences Th
31. ts name which is displayed inside the rectangle Figure 14 2 The sequence names can take advantage of the advanced for matting options offered by TEX which for example allows to display Greek letters by typing a backslash followed by their English name i e mu or sigma Underscore 14 oP ie your text would define subscripts while the power symbol super script The line defining the sequence name for the IGHG3 gene would then look the following gt gamma_ 3 and it would display y3 in the graphical output 14 5 Horizontal Axis The readability and arrangement of the query features are enhanced by the horizontal axis divided into multiple intervals It can be enabled and disabled by the plot_axis http www ncbi nlm nih gov blast fasta shtml December 2010 14 GRAPHICAL OUTPUT 17 0 50k 100k 150k 200k 250k 300k Figure 14 1 Horizontal Axis simplifies visual alignment of features in the resulting graphical output parameter Its text annotation position below 1 or above 0 the axis is determined by axis_topbottom The axis normally starts with 0 but can be offset by any value by axis_offset if desired 14 6 Annotation of the Output Depending on the plot_labels value the graphical output can be annotated with a title bearing the user provided name of the query the chromosome number and the studied species and the X axis and histogram would be labeled with their limiting
32. ure be incorporated into the main chromosomal assemblies However certain parts of the genome are highly susceptible to changes between different organisms of the same species This prevents the determination of the invariable genome sequence for that species Typically this occurs with polymorphic genes such as the natural killer cell immunoglobulin like receptor Section the major histocompatibility complex MHC or the color pattern of butterflies The haplotype databases contain such alternative sequences of the variable loci acquired from different organisms and The type and quality of the genome assembly influences the function and perfor mance of the algorithm and is discussed in the next two paragraphs 16 1 Genomes in Single Sequence The genome sequences assembled into a single file not accounting for the individual chromosome are divided number of sequence database files to speed up the alignment to such genomes The genome fractioning is done at random positions which would impede alignment if the query sequence would overlap with one of these breaks Due to the size of each genome and the very small number of such artificial breaks it is highly unlikely to ever occur ftp ftp ensembl org pub current_ fasta 17 MEGA BLAST ALIGNMENT IMPORT 21 16 2 Genomes with Patches and Haplotypes The patches and haplotypes for the chromosomal sequences are typically not used for the alignment to save computation
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