CEMAsuite User Manual
Contents
1. essesssssssseereessssssseceressssssseceressssssreccresssessseeeressssssseeeresssssssees 8 2 6 1 Customizing Primer3 Settings cccscesssssscccccccceessseceeccccceseeessseeecececesecesssseeeeeeeenees 9 2 Hybridization Stability Estimation ccccsccccccccseesssseeecccccssecessessecsceesseeeesseeeeeceeeuees 9 3 Hybridization Considerations Data Analysis cccccccssssssccccccceeaeeeseeececeeeeauaeeseseeeeeeeeaaeeenees 12 De POU ISS MOO LEO is dacactes ect casas E S E A E A O E teres tases 16 The protein MSA file failed to load or loaded incorrectly cceeeeeeseeceeeeceeeeeseeeeeeeeeeeeaas 16 The CDS file failed to load or loaded Incorrectly ceecceecccccceeeeeeeeeeeceeeeeeeaeeeeeeeeeeeeeaaas 16 The Efetch utility 1s not Working proper ly cccceccccccccccccessseseeeccceeceeeeseeeeeceeseeeeseeeseeeeeeeeeeaags 16 The Primer Design tab is not functioning properly cc ceeeeeesssseeeeeccceeeeeeeeeeeeaasseeeeseeeeees 16 General Tunc tionality errors are OCCU osade a E oo ediuaunctasmieuntcnedanetionst 16 The program is not launching when I double click the CEMAsuiteV2 Jar file 0 17 My CDS was not found using the Entrez Efetch utility but I want to include it a 17 IROL OL ETI CES iia accrc tae sales T eee eee eral eee eee 18 This file contains detailed information on performing user actions in CEMAsuite This file should be accompanied by2. 6 0 kcal mol when applying the Owczarzy et al correction This value was then used as the default individual primer warning threshold The mean value of the weaker oligos for successful amplifications was 9 5 0 7 kcal mol and 5 9 0 7 kcal mol when applying the Owczarzy et al and SantaLucia amp Hicks corrections respectively To see the effects of the overall binding ability of the primer set the sum of the two binding energies AG Ta AGr Ta AGr Ta was investigated and a histogram plot of those results are shown below Figure 11 It was observed that this value approached approximately 16 kcal mol before failures became prevalent when applying the Owczarzy et al correction This value was used as the default primer set warning threshold The mean sum binding energy for successful amplification was 24 1 2 kcal mol and 16 1 2 kcal mol when applying the Owczarzy et al and SantaLucia amp Hicks corrections respectively The results from this investigation allowed us to set up a benchmark for the notification system which warns the user when a primer set is unlikely to amplify within its intended region of a template sequence within the MSA 12 The results of the data analysis are presented here so that the user may make an informed decision regarding their specific primer design criterion 13 eantaLucia amp Hicks Owezarzy et al 10 Percent o _ Is 5 20 15 10 5 QO 55 10 20
3. and S Henikoff CODEHOP COnsensus DEgenerate Hybrid Oligonucleotide Primer PCR primer design Nucleic Acids Res 2003 31 13 p 3763 6 Rozen S and H Skaletsky Primer3 on the WWW for general users and for biologist programmers Methods in molecular biology Clifton N J 2000 132 p 365 386 Geer L Y et al The NCBI BioSystems database Nucleic Acids Res 2010 38 Database issue p D492 6 Allawi H T and J SantaLucia Thermodynamics and NMR of Internal G T Mismatches in DNA Biochemistry 1997 36 34 p 10581 10594 Allawi H T and J SantaLucia Thermodynamics of internal C T mismatches in DNA Nucleic Acids Research 1998 26 11 p 2694 2701 Allawi H T and J SantaLucia Nearest Neighbor Thermodynamics of Internal A C Mismatches in DNA Sequence Dependence and pH Effects Biochemistry 1998 37 26 p 9435 9444 Allawi H T and J SantaLucia Nearest Neighbor Thermodynamic Parameters for Internal G A Mismatches in DNA Biochemistry 1998 37 8 p 2170 2179 Peyret N et al Nearest Neighbor Thermodynamics and NMR of DNA Sequences with Internal A A C C G G and T T Mismatches Biochemistry 1999 38 12 p 3468 3477 SantaLucia J A unified view of polymer dumbbell and oligonucleotide DNA nearest neighbor thermodynamics Proceedings of the National Academy of Sciences 1998 95 4 p 1460 1465 SantaLucia J and D Hicks THE THERMODYNAMICS OF DNA STRUCTURAL MOTIFS Annual Review of Biophysics and
4. 15 10 5 D dG kcal mol dG kcal mol O More Stable O Less Stable 10 PETES Injssazone Figure 10 Histograms of the AGi T values from data taken from literature and calculated using the hybridization algorithm within CEMAsuite Detection value was based off of considerations listed within literature The colored lines on the plot represent the normal density of each sample population 14 SantaLucia amp Hicks Owczarzy et al Percent 0 ae 30 se 20 10 0 40 30 20 10 OQ 10 40 30 20 10 0 10 dG kcal mol dG kcal mol Figure 11 Histograms of the sum of the forward and reverse AG T 4 values from data taken from literature and calculated using the hybridization algorithm within CEMAsuite Detection value was based off of considerations listed within literature The colored lines on the plot represent the normal density of each sample population 15 5 Troubleshooting The protein MSA file failed to load or loaded incorrectly The problem is likely that the file parser failed to recognize your sequence identifiers or your sequence identifiers possessed white space Please alter the identifiers to a 1 word form and do not include the underscore character unless it is part of an accession The CDS file failed to load or loaded incorrectly The problem is likely that the file parser failed to recognize your sequence identifiers or your sequence identifiers Ensure you have the proper fasta fo
5. Biomolecular Structure 2004 33 1 p 415 440 SantaLucia J Jr and P N Method and system for predicting nucleic acid hybridization thermodynamics and computer readable storage medium for use therein World Intellectual Property Organization 2001 WO 01 94611 p Appendix Bommarito S N Peyret and J S Jr Thermodynamic parameters for DNA sequences with dangling ends Nucleic Acids Research 2000 28 9 p 1929 1934 Owczarzy R et al Predicting Stability of DNA Duplexes in Solutions Containing Magnesium and Monovalent Cations Biochemistry 2008 47 19 p 5336 5353 de Roda Husman A M et al The use of general primers GP5 and GP6 elongated at their 3 ends with adjacent highly conserved sequences improves human papillomavirus detection by PCR Journal of General Virology 1995 76 4 p 1057 1062 Ishii K and M Fukui Optimization of Annealing Temperature To Reduce Bias Caused by a Primer Mismatch in Multitemplate PCR Applied and Environmental Microbiology 2001 67 8 p 3753 3755 18 18 19 Snijders P J F et al The Use of General Primers in the Polymerase Chain Reaction Permits the Detection of a Broad Spectrum of Human Papillomavirus Genotypes Journal of General Virology 1990 71 1 p 173 181 Yamamoto S and S Harayama PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains Applie
6. should be in clustal format as shown in Figure 2 For best results using the Fetch CDSs feature the sequence identifiers should include proper accession identifiers For more information please read under 2 3 Obtaining amp Assigning Coding Sequences CDSs Additionally it is recommended to use global protein alignments not local alignments CLUSTAL O 1 1 0 multiple sequence alignment gi 399912868 gb AFP55294 1 gi 40863 emb CAA38872 1 gi 119454523 gb EAW35671 1 MDSKSNLGSTORNNNN NEQADQKANLWLA AESANPERKNERMVNPNNESNSM gi 1100794 emb CAA83241 1 MTLDNNQELTYR NSOSLGOPGESLA VNSSNPENHSGLNEGONNDSKKI gi 16330088 ref NP _ 440816 1 MTLNNDLPLNNIGETGSGLNDGTEGLDDLFSSSIVDNEPLE ALVETPTFAS gi 399912868 gb AFP55294 1 PNEQSWSSDIVPSNAAKTIKVIGVGGSGGNAINRMIDSEVSGVEFWAVNTDAQALTOSKAS gi 40863 emb CAA38872 1 MFEPMELTNDAVIKVIGVGGGGGNAVEHMVRERIEGVEFFAVNTDAQALRKTAVG gi 119454523 gb EAW35671 1 PHREESWSTDIIPSNAAKTKVIGVGGSGGNAVNRMIESEVSGVEFWAVNTDAQALAOSKAL gi 1100794 emb CAA83241 1 SVENNRIGEIVPGRVANIKVIGVGGGGGNAVNRMIESDVSGVEFWS INTDAQALTLAGAP gi 16330088 ref NP 440816 1 PSPNLKRDOIVPSNIAKIKVIGVGGGGCNAVNRMIASGVTGIDFWAINTDSQALTNTNAP x KKKKKKKK K KK ee eo Ke e Kee Ke ee KKK Oe KKK Figure 2 Clustal file format example To save this view select the FILE menu and choose SAVE PROTEIN MSA This saves the current output in the PROTEIN MSA tab to an html file which will appear identical to the output shown under the PROTEIN MS
7. A tab 2 3 Obtaining amp Assigning Coding Sequences CDSs 2 3 1 Obtaining Coding Sequences Using Fetch Utility If the protein accessions are available through the National Center for Biotechnology Information NCBI database 5 then the CDSs can be automatically fetched via their Efetch utility see CEMAsuite Screenshots Screenshot 3 In order for this to perform properly the sequence identifiers e g gi 399912868 gb AFP55294 1 in the protein MSA file must contain a valid NCBI accession number e g gi 399912868 or accession number which can be related to the NCBI database e g arpss294 1 for GenBank A gi 399912868 gi 40863 gi 119454523 MDSKSNLGSTORNNNN NEQADQKANLWLA AESANPERKNERMVNPNNESNSM gi 1100794 MTLDNNQELTYR NSQOSLGOPGESLA VNSSNPEFNHSGLNEGONNDSKKI gi 16330088 MTLNNDLPLNNIGETGSGLNDGTEGLDDLFSSSIVDNEPLE ALVETPTFAS B AFP55294 1 CAA38872 1 EAW35671 1 MDSKSNLGSTORNNNN NEQADOQKANLWLA AESANPFRKNERMVNPNNESNSM CAA83241 1 MTLDNNOELTYR NSQSLGOPGFSLA VNSSNPFNHSGLNEGONNDSKKI NP 440816 1 MTLNNDLPLNNIGFTGSGLNDGTEGLDDLFSSSIVDNEPLE ALVETPTFAS Figure 3 Example sequence identifier formats which are accepted Identifiers can be of the form found in Figure 2 or in A or B of this figure or some combination of the three For each CDS found the length is checked and compared to the protein sequence length So the CDS length must include all codons possibly
8. CEMAsuite Version 2 2014 Courtney E Lane Michael G Benton Cain Department of Chemical Engineering Louisiana State University Baton Rouge LA USA Contents Dis CTL ET A EE E E EE EE E E EEA EA i LL BC TO a E E E ocece eaueosanaaaceugedasessaneeocect i DO E E A E E E A E E E meen teeta 3 L EN a E E AEE E E E 3 PAE E O T E E A E A A O E 3 22 Importimi a Protein MSA File ascetics anenecncusnasenetseneeccessaulecssensscansoosessiseerecasseesseasseereecee 3 2 3 Obtaining amp Assigning Coding Sequences CDSS esssssssssseressssssssseerrsssssssserersssssssees 4 2 3 1 Obtaining Coding Sequences Using Fetch Utility cccccceceesesseeeeeeeeeeeeeaas 4 2 3 2 Importing amp Assigning Coding Sequences from a File cc eeceeccceccecsssseeeeeeeeeeeeeaes 5 2 4 Generating a Codon Equivalent Multiple Alignment CEMA ssssessssssssesseeeeeresssssssssss 5 2 5 Scoring CEMA Position Quality ccccssssssssseecccccceeessseeesccccsseeesssseececessseceesseeeeseeeenees 6 Fok S Conna On Percent 1enUty easan enn ren E EEE 6 2 5 2 Scoring on Runs of Complete Conservation c cccccccccccccesssesseecceeeeeeaeeeseeseceeeeeeaas 7 2 9 3 O COMMNe On Oli aL COG CAC asaisa se ererseceaasevesinasvent can ertagenacenstronanenasnacientcenensegsgateestes 7 2 5 4 Scoring on Identity Runs amp Potential Degeneracy ce eeecceccceceeeeeeeeeeeeceeeeeeeaes 8 2 6 Consensus Primer Design
9. W WO a aj S Oo tO co j b Co be l OO0O0OO0O0OO0O0OC0O0OOPrPRPRBRPRORrRPN tO Oy WW Wo CCCTTTACG 5 Figure 9 Primer template annealing algorithm First all possible single degeneracy permutations of a degenerate primer are created Next each of these single degeneracy primers are used to simulate annealing to the template region The stability of each potential hybridization conformation is estimated for each single degeneracy primer via nearest neighbor thermodynamics see text for details The result is a Gibbs free energy value kcal mol at the specified conditions as indicated by the numbers in parentheses next to each possible conformation Statistical analysis is then performed on the most stable conformation of each single degeneracy primer template hybridization and returned as the output values associated with that particular degenerate primer template hybridization 11 Primer template hybridizations where an individual primer s AG T gt Individual Primer Warning value 6 kcal mol by default will be flagged in yellow background Primer template hybridizations where the total AG T gt Primer Set Warning value 16 kcal mol by default will be flagged with a red background see CEMAsuite Screenshots Screenshot 7 as they may be unlikely to amplify based on our data analysis see 3 Hybridization Considerations Data Analysis See CEMAsuire Screenshots Screenshot 9 for adjusting these warning t
10. a CEMAsuite Screenshots file to for presenting example output results https sourceforge net projects cemasuite 1 Overview 1 1 Background The polymerase chain reaction PCR is a staple molecular biology tool It is used in a variety of applications including genetic screening DNA quality control molecular cloning DNA sequencing gene expression and many others Unfortunately PCR usually requires information for the desired sequence to be amplified which is not always available In many applications it can be beneficial to design PCR primers using a multiple sequence alignment MSA The primers can be designed within a conserved region of the target sequence so that the annealing ability of the primers remains relatively unchanged across multiple templates This usually allows for a single reaction condition across all template sequences and can greatly increase the likelihood of a successful amplification of an unknown targeted sequence Currently there are two primary methods of designing primers across multiple sequences 1 Use directly aligned target DNA sequences for consensus primer design This is a readily available method and is best suited for cases when 1 the target DNA is not a coding sequence 11 there are many sequences available large sample size n 111 all of the sequence information for the experimental samples are known e simple rapid e if the sequences represent coding e potential to produce l
11. d and Environmental Microbiology 1995 61 3 p 1104 9 19
12. en many sequences are available within the alignment OR when the amplification of unknown sequences will likely not be attempted This scoring method can be used to filter out the regions where runs of perfect matches will not occur The regions of high quality are key regions to investigate for the 3 end of the primer 2 5 3 Scoring on Potential Degeneracy CEMA positions are scored on identity and then adjusted based on the potential degeneracy of the consensus codon positions according to 18 translation tables which can be selected in the TRANSLATION TABLE combo box of the CEMA tab hover over them for a brief description In other words if the consensus codon within the alignment is CGT and we want to score based off of the standard translation table codes for Serine CGN AGR MGN then the quality of C s position will be divided by 2 the quality of G s position will be divided by 1 and the quality of T s position will be divided by 4 Figure 7 Plot of quality from highlighted region of Figure 4 scored using the Potential Degeneracy method and the standard translation table The CDS residue for each position is printed on top of the bar while the conserved amino acid is printed below the bars Note that positions of 100 conservation denoted by capital letters possess quality scores of less than 100 This method is most useful when there are few sequences available within the alignment as it attempts to filter out regi
13. equence for this CEMA and the quality scores assigned to each position and design high quality consensus primers Selective degeneracy can be added by the user until the calculated stability of each primer template pair is acceptable This application also attempts to condense the vast amounts of information usually associated with MSAs into formats which are intuitive and discernable Gather Score Consensus Selectively Generate Add Protein MSA Coding CEMA Consensus Primer Sequences Sequence Design Degeneracy Figure 1 CEMAsuite usage flow diagram Alternatively CEMAsuite can be used to quickly design primers by inspection 2 User Manual This program is intended for users with moderate experience in PCR and primer design All of the following examples will be performed on the same set of sequences and primers 2 1 Installation Once downloaded and moved to the desired directory the program jar format can be launched from the file dist CEMAsuiteV2 jar For proper functioning do not move the files within this folder It is recommended to create a shortcut to this jar file for future use and a shortcut icon is included within the files 2 2 Importing a Protein MSA File Importing an alignment clustal format can be done by selecting FILE IMPORT ALIGNMENT or by pressing Ctrl I The resulting alignment should be displayed under PROTEIN MSA tab see CEMAsuite Screenshots Screenshot 1 Protein MSA files
14. esign since it has no influencing factors to remain conserved meaning the use of primers based on these regions on an unknown DNA sequence may result in a loss of primer annealing ability Indirectly speculate the possible sequences of nucleic acids based on a MSA of the coding sequence protein product Again there are applications available that can perform this task This method of design requires the target DNA be a coding sequence and is best suited when 1 genes of interest are homologous globally 11 genes of interest contain local homologous regions conserved domains 111 there are few sequences available small sample size n iv attempting to detect homologues in a group organisms with high biodiversity e robust e usually high degeneracy primers are produced e 20 possible permutations for a given position e loss in specificity in an amino acid sequence e products are typically less predictable in length heterogeneous sizes e may require further conformation such as DNA sequencing The likelihood of a position appearing conserved due to random chance alone in a protein multiple sequence alignment is far less than that of a DNA MSA The analogous probability that the entire column will match the first sequence due to random chance alone can be described by the equation P 20 1 Thus we can be more confident that a conserved amino acid in a MSA is conserved due to evolutionary pressure than a conserved nucleic acid
15. hreshold values If the primer set proves unsatisfactory select Custom on the primer set combo box which allows the user to edit individual base sites to incorporate degeneracy as appropriate see CEMAsuite Screenshots Screenshots 8 amp 9 To save this view select the FILE menu and choose SAVE HYBRIDIZATION OUTPUT This saves the current output in the HYBRIDIZATION tab to a text file 3 Hybridization Considerations Data Analysis In order to obtain some information on exactly what constituted a good primer set based on our hybridization algorithms 94 data points were obtained through literature and subjected to the stability analysis at the specified conditions 16 19 For these calculations the annealing temperature Ta of the thermal cycles was used as the input temperature and positive detection was taken as it was cited within the literature as were failures Overall there were 29 observations of failures and 65 observations of strong successful amplification The stability of the individual primers was analyzed first these were sorted based on the relative stabilities of the oligos within the set i e one deemed more stable and one deemed less stable The resulting AGi T values were binned into 1 kcal mol bins and plotted on a histogram which is shown below Figure 10 It was observed that strong amplification began to fail when the weaker of the two oligos had a AG Ta value approximately
16. in a MSA CODEHOP exemplifies the implementation of this methodology 3 This application utilizes protein MSAs and codon frequency tables to generate moderate to high degeneracy primers Because codon frequency tables are used in lieu of actual CDS information it is quite possible that the consensus template sequence corresponding to a given primer was not represented as accurately as possible An additional downside to this method of design is the amount of noise produced in its output This application typically results in numerous primer sets to sift through before the user obtains their desired primer set 1 2 Goal CEMAsuite was developed in an attempt to find a compromise between the two methodologies mentioned above Its intent is to aid in the design of a sort of minimum degeneracy primer set which is robust enough for the assay while allowing the user to quickly balance the specificity and sensitivity of their primer set This application addresses this problem by starting with a protein MSA where the likelihood of true conservation is higher and generating a codon equivalent multiple alignment CEMA using the coding sequence of each protein sequence within the protein MSA Next it enables the user to quickly and effectively score each position within the alignment using one of multiple scoring algorithms in order to assign a quality to each position The Primer3 4 primer design program can then accept both the consensus s
17. including the stop codon Leps 3x Lprotein 1 or Leps 3XLprotein If this criterion is not met a message box will appear If all CDSs have been located successfully they will be displayed under the CDS tab in the same order as the protein MSA input file CDSs can be exported to a fasta format file by selecting FILE EXPORT CDSS At this point it is possible to generate paired accession tables as comma Separated values csv files 2 3 2 Importing amp Assigning Coding Sequences from a File If the protein and or coding sequences are not located in the NCBI database or for any other reason the CDSs can be imported from a fasta format file The file must contain exactly the same number of sequences as the protein MSA however the order does not matter Once the file has been located and parsed the order for pairing can be assigned manually see CEMAsuite Screenshots Screenshot 2 For each CDS found the length is checked and compared to the protein sequence length So the CDS length must include all codons possibly including the stop codon Leps 3x Lprotein 1 or Leps 3XLprotein If this criterion is not met a message box will appear If all CDSs have been located successfully they will be displayed under the CDS tab in the same order as the protein MSA input file At this point it is possible to generate paired accession tables as comma separated values csv files 2 4 Generating a Codon Equivalen
18. matically with each other To save this view select the FILE menu and choose SAVE CEMA This saves the current output in the CEMA tab to an html file with a similar format as the visualized output in the program or to a text based cema file If primers are present in the text areas they will be highlighted in the html file 2 6 1 Customizing Primer3 Settings As of version 2 0 8 the user can alter the Primer3 settings file These values can be edited through SETTINGS gt PRIMER3 EDIT CURRENT SETTINGS once these setting values have been edited by the user they will remain set to that value until restored or altered again One of four specific default Primer3 settings files can be specified through SETTINGS P PRIMER3 SELECT DEFAULT SETTINGS This file will contain all of the settings desired to return upon a call to SETTINGS PRIMER3 RESTORE DEFAULT SETTINGS Note that this will overwrite the current setting values as well 2 7 Hybridization Stability Estimation One of the key elements of CEMAsuite 1s the ability to anneal the primers to each template and output an estimated Gibbs free energy for the designated conditions This allows the user to pinpoint cases where the primer set is likely to fail and improve the primers as they see fit It is recommended that the conditions set are the actual PCR reaction conditions and the annealing temperature of the thermal cycles The algorithm for the thermodynamic parameter esti
19. mation first locates the primer template region columns with the least mismatches throughout ALL sequences for each primer Next it simulates annealing for each primer template pair in this region i e iterates down through the columns for new templates utilizing the nearest neighbor parameter estimation methods outlined in 6 14 In order to account for the entropic dependence on the cation concentration two methods of adjustment have been included 12 15 If a primer is degenerate each permutation of that degenerate primer is simulated individually and the most stable conformation is used to populate the mean min max Gibbs free energy values The mean value is the average of the most stable conformation of all permutations of a primer annealing The minimum value is the most stable conformation of the most stable permutation of a degenerate primer The maximum value is the most stable conformation of the least stable permutation of a degenerate primer This part of the algorithm is outlined in Figure 9 What this algorithm does What this algorithm does not do e Iterates through all combinations of regions of e Perform higher order structural analysis hairpins matching and mismatching within a primer template asymmetric loops etc pair and calculates a AG T value in order to find e Iterate outside the bounds of the set of columns for the most stable conformation which the primer has the least number of e Iterates through every p
20. ns untested If this program is run on any other type of machine then primers can be designed using other software or inspection The most convenient alternative would be to use Primer3plus online and export the consensus sequence with the quality information via see CEMAsuite Screenshots Screenshot 5 FILE EXPORT CONSENSUS amp QUALITY from CEMAsuite If using Primer3plus it is recommended to keep the PRIMER_ MAX_NS_ACCEPTED value set to zero as the consensus sequence output will mask gaps as generic n bases for input into the primer design software For the inputs please reference the Primer3 documentation One notable input is the minimum end quality Briefly the MIN END QUALITY input will set a threshold on the minimum quality score allowed in the 3 end of the primer If using an alternative method of primer input simply design the primers then select CUSTOM under the SET combo box on either the CEMA or HYBRIDIZATION tabs to input the primers as text see CEMAsuite Screenshots Screenshot 4 Once primers values have been entered into the FORWARD PRIMER and REVERSE PRIMER text fields they should appear highlighted in the score plot and on the top row of the CEMA see CEM lt Asuite Screenshots Screenshot 6 Primers should be entered 5 to 3 and the reverse primer should be the reverse complement of the template sequence The primer text fields on both the CEMA tab and the HYBRIDIZATION tab update auto
21. nt covered within the following score examples Regions depicted in score examples are highlighted in yellow above 2 5 1 Scoring on Percent Identity CEMA positions are scored simply on the normalized frequency of the consensus nucleotide throughout the sequences This method is most useful when many sequences are available within the alignment OR when the amplification of unknown sequences will likely not be attempted This is the default scoring method Figure 5 Plot of quality from highlighted region of Figure 4 scored using the Percent Identity method The CDS residue for each position is printed on top of the bar while the conserved amino acid is printed below the bars 2 5 2 Scoring on Runs of Complete Conservation CEMA positions are scored on identity and then adjusted based on the number of consecutive completely conserved positions within the location The score adjustment value is controlled via the RUN WEIGHT slider of the CEMA tab The run weight should always be a positive integer less than or equal to 100 ETT I q iwasi iai L Figure 6 Plot of quality from highlighted region of Figure 4 scored using the Identity Runs method and a run weight of 10 The CDS residue for each position is printed on top of the bar while the conserved amino acid is printed below the bars Note that positions of 100 conservation denoted by capital letters possess quality scores of less than 100 This method is most useful wh
22. ons of low conservation AND high potential degeneracy This is a method which can be useful for the cases where the primers will be used to try to amplify on organisms with unknown target sequences 2 5 4 Scoring on Identity Runs amp Potential Degeneracy CEMA positions are scored using each of the 3 scoring methods described above This method can help to discover regions of high conservation from IDENTITY RUNS with low potential degeneracy from POTENTIAL DEGENERACY s T oas os os uiu oo agge Figure 8 Plot of quality from highlighted region of Figure 4 scored using the Runs amp Degeneracy method with a block weight of 10 and the standard translation table The CDS residue for each position is printed on top of the bar while the conserved amino acid is printed below the bars Note that positions of 100 conservation denoted by capital letters possess quality scores of less than 100 2 6 Consensus Primer Design While CEMAsuite does include a compiled Primer3 executable it should be noted that the scored plot can greatly speed up the process of primer design by inspection if desired CEMAsuite utilizes a compiled Primer3 4 executable v2 3 6 for primer design and for which it provides a specialized interface the PRIMER DESIGN tab It should be noted that the functionality of this tab is only applicable to Windows and Mac OS based machines possibly other UNIX based operating systems as well but this remai
23. ow degeneracy DNA then codon information reading primers frame is lost e only 4 possible permutations for a given position in a DNA sequence A C G T This method of consensus primer design has been implemented by applications such as PrimaClade and PriFi 1 2 These applications are proficient in returning low degeneracy consensus primers for nucleic acid MSAs however the methods used to align sequences risks losing vital codon grouping information since the alignments may not be designed with a focus on coding sequences Because the codon information is lost for coding sequences in these methods prediction of possible permutations of a given residue position is limited to the residues observed within the alignment column unlike the known potential translations of a codon position This can lead to a potential loss in robustness for a given primer set Direct nucleic acid multiple sequence alignments may not be ideal for consensus primer design targeting coding sequences For example a random sequence position 2 within an alignment of n sequences the probability that all residue positions within the column will match the first residue position due to random chance alone can be described by the equation P 4 1 Such an event would represent a sort of Type I error where the conservation would be present but due only to random chance and not from evolutionary pressures This sort of conservation is undesirable in probe d
24. peated for newer versions Also an allow permission prompt which performs this task automatically will be issued for Mac OS computers upon initialization General functionality errors are occurring Please ensure that ALL files have remained within the same directory and relative location as they were originally distributed If you would like to create a shortcut to the executable please to do to the CEMAsuiteV2 jar file 16 The program is not launching when I double click the CEMAsuiteV2 Jar file This is a popular problem for Windows 7 You can either include the path for all java programs https docs oracle com javase tutorial essential environment paths html to open with a double click or alternatively you can launch the program from the command prompt by entering the proper directory the directory where the CEMAsuiteV2 jar file is located and commanding the following java jar CEMASuiteV2 jar My CDS was not found using the Entrez Efetch utility but I want to include it CDS gathering could have failed for many reasons The most common problem is the use of a partial CDS where the CDS includes a couple of extra nucleotides Even if this is not your problem the easiest way around this 1s detailed below 1 Load desired protein alignment use Efetch utility to gather all CDSs possible a Make a note of the accessions which fail to be properly obtained 2 Export the CDSs using FILE EXPORT CDSS and save the incomple
25. rimer template pair and mismatches in the alignment performs the calculation above within the set of columns for which the primer has the least number of mismatches e Iterates the calculation above for each permutation of a degenerate primer e Adjusts the entropy parameter for the presence of cations based on 1 of 2 methods in all cases Once a primer set has been decided upon the user can input the desired parameter values into the HYBRIDIZATION tab and click SUBMIT to view the stability of each primer template pair see CEMAsuite Screenshots Screenshot 7 10 5 CGGAGGTGGTGGNAATGC 3 3 GCCGTCACCCCCTTTACG 5 5 CGGAGGTGGTGGAAATGC 3 Peet Serr yy Se ea Boece es P d bed thle LP ssdddlds ESETERE 3 GCCGTCACCCCCTTTACG 5 Bae a Reve 8 i IIl 2 65 J OY Oy WO 5 CGGAGGTGGTGGCAATGC 3 It lt It I I NR O WO O 5 CGGAGGTGGTGGNAATGC 3 SPTP Pe tilt ttdl 3 GCCGTCACCCCCTTTACG 5 eD WO D DA 0 OOO CC OFF FF F RN Oy oo co Mean 3 83 CCCTTTACG 5 Min 5 34 Max 2 95 W WO e A Iil eee ee 3 59 OY Oo 02 N 5 CGGAGGTGGTGGGAATGC 3 I I I I I j NO co O O OY WO J J o De o e oooooorrFN N W tO Oy Ww WO CCCTTTACG 5 5 CGGAGGTGGTGGTAATGC 3 Prd ssddddetts I I 2 57 Oy Oy UI co W
26. rmat for the file The Efetch utility is not working properly Please make sure to review 2 3 1 Obtaining Coding Sequences Using Fetch Utility and if the problem is not cannot be resolved try obtaining the coding sequences manually and importing them through a file The Primer Design tab is not functioning properly The primer3 executable called by this program has only been tested on Windows and MacOS based machines If you are not on such a machine this functionality may be lost but the program will default to the UNIX based compiled binary please see 2 6 Consensus Primer Design for alternative methods of primer design If you are on a MacOS based machine and experiencing mac primer3 core Permission Deniederrors you need to allow permission to execute the primer3_core file UNIX and UNIX like systems generally will not execute a program unless it is marked with permission to execute To allow permissions From your finder go to Applications gt Utilities gt Terminal and type in the following chinod U lt Director CENA SUE Release s dlSt y esoueCes PEIMers Male primers cOre Alternatively you can type in the chmod u x command and drag and drop the file specified above This command only changes the permissions associated with the file it does not change the security controls associated with the entire volume This action should only need to be performed once for that user NOTE this may need to be re
27. t Multiple Alignment CEMA Once the protein and CDSs have been imported and matched the CEMA can be generated using the ALIGN button under the CEMA tab Once complete the alignment will be displayed under the CEMA tab see CEMAsuite Screenshots Screenshot 4 A CEMA is similar to a traditional DNA MSA but it includes 2 additional lines of sequence information The first is at the top and is the amino acid at the corresponding position in the protein alignment These characters only appear where the protein alignment clustal input file designated a conserved position i e columns with a or under them The second additional line is the consensus sequence with residues appearing in columns lacking gaps only this sequence can be used for primer design 2 5 Scoring CEMA Position Quality Once a CEMA consensus sequence has been successfully generated each column position within it can be assigned a quality score which is displayed at the bottom of the main window see CEMAsuite Screenshots Screenshot 4 There are 4 methods of quality scoring which are available and can be selected using the SCORING METHOD combobox located in the CEMA tab AFP55294 1 ae TPVOEAFRYADDVLROGVOGISDIIT CAA38872 1 ene LT ISLLDAFGAANDVLKGAV OGIAELIT EAW35671 1 s CAA83241 1 e e SVENNRIGEIVPGRV NP 440816 1 PSPNLKRDQIVPSNI TPLQEAFRVADDILROGVQGISDIII e ckx Keke ks ekEKKe tek Figure 4 Region of example alignme
28. te collection 3 Manually gather the erred accessions and edit the CDSs manually so they reflect ONLY and EXACTLY the coding portions of the related protein sequences in the alignment In other words 3 nucleotides per amino acid residue with an allowance of a full stop codon a The use of the DNAToolkit FILE DNATOOLKIT should be quite useful for this step of the process Especially for quickly editing a sequence into the required FASTA format 4 Insert the corrected sequences into the FASTA format file previously exported using the Efetch utility a Order is not extremely important as you can match them manually during import b NOTE if you want the organism taxonomic information retained and handled ensure the taxon is enclosed in brackets 1n the identifier e g gt XYZO00000 1 geneA GenusB speciesC 5 Import the newly completed CDS collection FASTA file a You may consider overwriting the previous FASTA file with the accessions in proper order automatically assigned once paired imported 17 References 10 11 2 13 14 15 16 17 Fredslund J et al PriFi using a multiple alignment of related sequences to find primers for amplification of homologs Nucleic Acids Research 2005 33 suppl 2 p W516 W520 Gadberry M D et al Primaclade a flexible tool to find conserved PCR primers across multiple species Bioinformatics 2005 21 7 p 1263 1264 Rose T M J G Henikoff
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