FAQ - Partek Inc.
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1. paired ends refers to the two ends of the same DNA molecule in the library when you sequence one end then turn it around and sequence the other end Partek will support paired end reads in the next beta version The software will automatically recognize the set of files that contains the paired end reads and import them Question What is paired gap Answer In Partek visualization we name the gap between a paired end reads as paired gap This is to distinguish from the splice junction between exons Question What source does Partek use for transcript annotation Answer Partek uses UCSC ref seq ID for transcript annotation Question Does Partek handle non human species sequencing data Answer Yes Partek handle human and non human species in next gen sequencing data analysis As long as the genome sequence data and the annotation file is available Partek Frequently Asked Questions 16 Question At the RNA seq dialogue at finding differentially expressed transcript we can choose if assay recognize sense strand or antisense What are the outcomes we can expect if we choose yes or no on this dialogue Answer It is depends on the sample preparation Some preparations will preserve strand information of the original transcript like Illumina s directional RNA seq or the SOLiD Whole Transcriptome Analysis Kit When cDNA was prepared from the RNA sample only 1 strand cDNA was synthesized On the other hand some preparation w2. any known transcripts and then visualize them on a track next to your predicted transcript Question It seems that the unexplained regions can come from a far away gene from where is currently located Can we know where this unexplained region was originally from Answer No that s the unexplained part It simply signifies that some RNA is being transcribed from this genomic location Question Can Partek detect fusion genes Answer Currently the system is designed to count reads into a predefined transcript space So Partek does not have an obvious mechanism to count the enormous possible combination of fusion genes that are possible Partek Frequently Asked Questions 17 Visualization Question How do I create a Venn diagram Answer Select View gt Venn Diagram and browse to the folder that contains the lists you want to include in the Venn diagram Select 2 or 3 list in the folder and click on the Venn diagram button Alternatively invoke a Venn diagram from the List Manager when selecting 2 or 3 lists You cannot create a Venn diagram from 4 or more lists but you can perform a union or intersection between multiple lists Question Partek is crashing when invoking a scatter plot or histogram chromosome view etc What can I do Answer The most common cause of this type of crash is out of date drivers Updating your graphics card drivers will usually solve this problem To get information on your current drivers s
3. applied in the chromosome view by a median sub sampling Region detection algorithms such as HMM or genomic segmentation have models that expect a level of noise in the data Smoothing may be useful for some analysis such as clustering Partek Frequently Asked Questions 6 Question What do the gray amp colored dots on the Copy Number Summary mean Answer The gray dots represent the raw copy number estimate at each marker These are represented as the values on the copy number spreadsheet The colored dots are the smoothed value of a variable number of copy number estimates across a given stretch of linear chromosome The sampling rate is disclosed in the visualization and is dependent on the size of the window and the resolution of the computer monitor Question How do I get cytobands on the Chromosome View Answer Please download the cytoband txt file from UCSC at http hgdownload cse ucsc edu downloads html On a copy number spreadsheet in Partek choose File gt Properties click on the Edit Genome button browse to the cytoBand txt file you downloaded for the Cytoband file entry Question How do I monitor the density of genetic markers from a segment result I would like to filter out segments that are close to the centromere Answer Right click on the Markers column header and insert a new column Right click on the Marker column header and copy paste to the new inserted column and rename the label as Density S
4. each isoform based on the proportions The reads mapped to the isoforms are then used to estimate the isoform proportions Question What is RPKM value Answer RPKM value is reads per 1k bases of exon model per million mapped reads It is defined in the paper Mapping and quantifying mammalian transcriptomes by RNA Seq Mortazavi et al Nature Methods 2008 http www nature com nmeth journal v5 n7 abs nmeth 1226 html Question How does Partek calculate differentially expressed transcripts Answer Partek uses Log likelihood ratio test to identify genes with different relative abundances of isoforms across samples In house method similar to the one discussed in the paper RNA seq An assessment of technical reproducibility and comparison with gene expression arrays Marioni et al Genome Research 2008 http genome cshlp org content 18 9 1509 full Question How does Partek calculate fold change of two transcripts between two contrast groups Answer The fold change was calculated as the ratio of RPKM values between two contrast groups Question What are paired end reads Does Partek support them If so where do we tell the software that these samples used paired end reads Answer When prepare samples for sequencing standard paired end libraries 200 500 bp or long insert 2 5kb mate pair libraries can be prepared to detect large and small insertions deletions inversions and other rearrangements The term
5. few SNPs for which each genotype is represented at least twice will allele ratio be calculated Omitted SNPs may also not display confident segregation clusters in the baseline files and were consequently omitted Question What is allelic imbalance Answer Allelic imbalance is a measure of how disproportionate the allele specific copy number AsCN estimates are relative to each other This is expressed in Partek as a proportion calculation where the proportion equals the difference of A and B over the sum of A and B For example if the alleles are present in equivalent abundance A 1 and B 1 the alleles are balanced and the proportion would be near zero If the alleles are in disproportionate abundance relative to each other then the proportion calculation would move close to 1 A and B alleles can also be referred to as max and min alleles Question How does Partek calculate whether a segmentation is considered within normal bounds Answer After determining the segmentation result two one sided t tests are performed on probes in each region detected Specifically the population of probesets within each region is compared to the upper and lower bounds set in the segmentation parameters You may set the minimum number of probes for each detected region as a parameter on the segmentation dialog Question Why are the max and min track of allele specific copy number estimates not both at one in normal regions Answer Observed allele num
6. Mean standard errors for both the REML and method of moments options Answer Model y X Zy e where y is response variable X is fixed effect design matrix Z is random effect design matrix B is fixed effect parameter y is random effect parameter iS error REML Let L contrast or LSMean coefficient matrix G variance of random effect parameter y R variance of residual effect parameter G estimate of G R estimate of R y A i XR X XR Z i CSCA BAPE E a oe ZR X ZR Z G7 Thus Standard error VLCL ki LGC J F y A rank L where N Rank X Z as denominator degrees of freedom N is the number of observations rank L as numerator degrees of freedom Method Moment It estimates the sum square as E SS By Cy Coba SSQ C ao SSQ C o gt SSQ C oa rank L o Partek Frequently Asked Questions 21 where C ML M is the inverse of the lower triangular Cholesky decomposition t 1 matrix of XZ XZ L SSQ trace C C For each effect in the model method moment determines the combination of these expected mean squares that produce an expectation that includes all the terms in the expected mean square of the effect of interest except the one corresponding to the effect of interest For example if the expected mean square of an effect A B is Var Error 3 x Var A Var A B Method Moment determines the combination of other expected mean squares in the model that h
7. aring two groups A and B if A gt B the ratio and fold change are both A B if A lt B the ratio is still A B but fold change is B A In less mathematical terms increases in gene expression are described as a fold change or a ratio of 2 But decreases are expressed as a fold change of 2 but a ratio of 0 5 A negative fold change is a ratio less than one Question How does Partek GS calculate fold change Why do my fold changes look unusual Answer Partek s ANOVA uses LS means to calculate fold change When the data is logged such as by RMA the resulting LS means are anti logged producing geometric means See Chapter 11 of the Partek User Manual for more detail on LS means and fold change calculations Question What is the best ANOVA model What factors should I include Answer It depends on the experiment you can look at the adjusted R square the bigger it is the better the model fits the data In general the factors should be included are the factors you are interested in factors contribute big variation etc If an experiment is being run with two categorical variables like time and dose it is often helpful to include the interaction between these factors in the ANOVA model Remember for paired designs to include the subject ID which matches paired samples together to avoid violating the assumption of independence in ANOVA For detailed information about your specific data you might want to ask for a statistician s advi
8. as expectation s Var Error 3 x Var A Standard error is JL XZ XZY L s If s Var Error as is often the case in balanced designs the F test is formed directly In this case the mean square of the effect of interest is used as the numerator the mean square of Error is used as the denominator and the degrees of freedom for the test are simply the usual model degrees of freedom When more than one mean square must be combined to achieve the appropriate expectation an approximation is employed to determine the appropriate degrees of freedom Satterthwaite 1946 Refer to Hocking 1985 for further theoretical discussion Project Question How do I save and open project Answer To save a partek project go to File gt Save Project specify a project file Partek project manager will go through each spreadsheet and save them into a single project file with the file extension ppjx To open a partek project go to File gt Open Project select the project file in the file browser Question What is the scope of Partek project What will be saved and what will not be save to a project Answer Partek project saves the tree structure of your spreadsheets and all spreadsheet data Original sample data files e g Affymetrix CEL files annotation files library files images and visualization graphics are not saved Copyright 2011 by Partek Incorporated All Rights Reserved Reproduction of this material with
9. ber lies on a distribution Since one allele is always observed to be greater than the other in regions of equal allele expression two close parallel lines are seen As the copy number estimates of each allele grow farther apart the assignments are affected less significantly by the noise of observed allele expression Question When should I use Allele Specific Copy Number AsCN vs Total Copy Number Answer AsCN was designed to analyze experiments using primary tumor samples We generally recommend that you perform both analyses if appropriate The AsCN workflow gives additional information but it may not create informative data depending on your experiment design Generally if you have paired tumor normal types of designs AsCN gives good estimates If you have unpaired mixed tissue tumors AsCN gives reasonably useful information If you have an unpaired analysis examining germ line changes pure tissue no heterozygosity in regions of interest ASCN does not produce useful information Only heterozygous calls are used for AsCN In paired analysis the genotypes from the normal are used For unpaired samples AsCN uses genotype calls of the study sample to determine when data is available If the study samples have pure deletions they will not have heterozygous calls leading to large stretches of missing data Partek Frequently Asked Questions 8 Question When should I use HMM vs Genomic Segmentation Answer HMM is very efficie
10. cant digits that might be computer floating point precision issue e Expression Console might be using sketch which is an approximation of quantile normalization While Partek always uses the full quantile normalization e Expression Console and Partek might use different library and or annotation files which may have different number of probes Different number of probes will result different answers in RMA e For Exon arrays to import core extended full probe sets Express Console uses ps file While Partek uses mps file in order to perform Alt splicing Anova That might result in different number of probe sets and different signal numbers Question How to import Expression Console results in Partek Answer Expression Console generates CHP files which can be imported in Partek Question After import Partek s QC box plot is different from Affymetrix Express Console box plot Why is that Answer Partek box plot shows the oe pe ta 50 75 and 100 percentile which correspond to min Q1 median Q3 and max While Affymetrix box plot shows Lower Extreme Lower Quartile Median Upper Quartile and Upper Extreme Partek Frequently Asked Questions 3 Question Why Partek GCRMA output is different from Bioconductor s Answer If you run the following 4 steps in R it should reproduce Partek s values 1 Sys setlocale LC_COLLATE C 2 data bgadj lt bg adjust gcrma data affinity source local 3 data expr l
11. ce Question How do I filter out low expressed genes Answer Select Filter gt Filter Columns gt Column Filter Manager and choose to Filter Exclude based on a Max that is less than a certain value Question If I had a parameter TYPE which could be A B C or a D and if I did an ANOVA on Type to compare against A and B I get a p value Type and p value A vs B I m assuming I should look at the p value A vs B column to identify differentially expressed genes between Type A and Type B Is this right Answer That is correct The overall p value tests HO A B C D Soa small p value for type could indicate a difference between A vs D C vs D etc The p value for A vs B only tests HO A B so it is the p value you should use to find the genes specifically different between Type A and Type B Partek Frequently Asked Questions 20 Question Is there a way to get more detailed information on the linear contrasts specifically standard error t or F statistic degree freedom For the contrast are they comparing the LSMeans Answer Yes a linear contrast compares the LSMeans The calculations of standard error t or F statistic or degree freedom are the same as LSMean except that L is the contrast coefficient matrix See Q2 For example Let L1 lsmean coefficient matrix of group L2 Ismean coefficient matrix of group2 Then the contrast coefficient matrix L L1 L2 Question How does Partek calculate the LS
12. deleted or amplified tissue the balance of alleles causes distributions to deviate from these values For example an allele ratio of 33 in the presence of 3 total copies indicates ABB and an allele ratio of 67 in the presence of 3 total copies indicates AAB Question How do I interpret allele ratio in context of total copy number data Answer Using allele ratio in combination with total copy number can help researchers have higher confidence in regions of amplification or deletion Allele ratio provides the best supporting evidence when changes tend to be clean integer changes of 2 to 3 or 2 to 1 The normal diploid tri mode distribution with peaks at 0 0 5 and 1 0 will change to a quad mode distribution 0 0 33 0 67 amp 1 0 with a gain to three copies or to a bi mode distribution 0 amp 1 0 with a loss to one copy Question What reference can I use for my publication Answer While the intensity normalization is performed differently the calculations are based on the following reference High resolution genomic profiling of chromosomal aberrations using Infinium whole genome genotyping Peiffer et al Genome Res 2006 Sep 16 9 1136 48 Epub 2006 Aug 9 Question I m using the Illumina platform how should I calculate allele ratio in Partek Answer Partek recommends that you import the B allele frequency from BeadStudio using the Partek plug in module to extract data from BeadStudio into Partek Genomics Suite Exo
13. e obscured by unwanted batch effects Question After I remove a batch effect from the data is it OK to perform a simpler statistical test which omits the batch factor e g a simpler ANOVA model or t test Answer No ANOVA already adjusts for batch effects and does not need the data to be adjusted Performing statistical tests on batch effect adjusted data will result in p values that are too small due to the failure to account for the degrees of freedom consumed during batch effect removal Question Is it OK to perform exploratory analysis such as PCA or cluster analysis on batch effect adjusted data Answer Yes Since these methods do not produce formal p values and cannot account for batch effects like ANOVA true biological effects will be more evident after unwanted batch effects are removed Partek Frequently Asked Questions 5 Question Does the batch remover actually change the expression values Answer Yes The values are adjusted to what they would have been had there been no batch effect Other signals and noise remain in the data Hierarchical Clustering Question How do I make a quick Hierarchical Cluster visualization Answer Use the Cluster based on significant genes function in the gene expression workflow If you d like to cluster using different parameters you can use the tmp spreadsheet generated from the workflow as input while selecting Tools gt Discover gt Hierarchical Clustering Ques
14. eally interested in the region between the two peaks rather than between the trailing edges of the peaks Question In CHIP seq workflow I set up the threshold of peak detection as 10 Why sometimes I see numbers smaller than 10 showed up in the spreadsheet column 8 number of reads that begin in the region for each sample Answer This is because you set directionally extend tags in your peak detection dialogue The peaks come from the extended reads and the read counts come from the original reads Question Will peak threshold setting affect the result How should I set up a proper threshold Answer You can always set the threshold conservative low and filter later based on p value An often used threshold is 10 Partek Frequently Asked Questions 13 Question In the detecting motifs dialog should the number of motifs always set to one Answer Because any given DNA binding protein has only one core binding motif one is probably a good choice If you are looking for two half sites then you would choose two Question In the detect motifs dialog the changes of minimum motif length and maximum motif length have great impact on the motif prediction Any recommendations to these two settings Answer Most binding sites are between 6 and 16 bases long The Partek default setting is 6 to 16 We don t recommend trying something less than 6 but more than 16 should be fine Questio
15. ed the error bars shown in the AS graph somehow indicate a significant difference between the two groups which is not true Moreover we note that the heights of the error bars are the same across all transcripts Answer The error bars on the AS graph are the standard errors of the Least Square Means LS Means for each probeset which are dependent on all the factors in the ANOVA model Due to the complexity of the ANOVA model and the least square estimates used to detect alternative splicing the standard error is estimated to be very small and consistent across probes The standard error may not be a good representation of the confidence of your data Gene Expression Question How do I create a gene list Answer Select Tools gt List Manager specify the criteria such as p value and fold change etc Select items to be included in the list name the list specify the location and select OK Tiling Question How does the annotation work in tiling datasets Answer Tiling probe ID represents its genomic location Question My tiling data analysis seems to be taking a long time what is happening Answer Tiling array analysis tends to be a computationally intensive task Partek Frequently Asked Questions 12 Next Generation Sequencing Import Criteria Question What is the criteria Partek uses to import my sequencing reads Answer Partek imports all reads that have been aligned A read is counted once even if it has multiple a
16. elect Help gt Graphics Information An information box similar to the one shown in Figure 1 will appear Graphics Information Renderer FireGL 3100 Pentium 4 SSE2 Vendor ATI Technologies Inc OpenGL Version 1 5 4583 Figure 1 Graphics Information for an ATI card For desktop computers you should check for updated drivers on the website of the manufacturer of the graphics card e If the vendor is ATI Technologies Inc check http ati amd com support driver html e If the vendor is NVIDIA Corporation check http www nvidia com content drivers drivers asp For laptop computers you should check the site of the manufacturer of the laptop Computer Specifications Question What single hardware investment will give the biggest performance boost to Partek Answer Faster hard drives Question What kind of computer should I buy to run Partek Answer For high density arrays we recommend a 2 gigahertz CPU and 2 gigabytes or RAM At Partek we use a machine with similar specs to benchmark most of our tutorial data It is likely a computer less powerful will still work especially if not using high density arrays Question What operating systems does Partek support Answer Partek will run on Windows Macintosh and Linux Linux seems to have a slight edge in performance particularly during import Partek Frequently Asked Questions 18 Question Does Partek leverage the benefits of multi core proces
17. elect the Density column and go to Transform gt Normalization amp Scaling choose to perform on selected column and select the Add Mul Sub Div page choose Divide by Column of the ength bps and select OK The resulting column is Marker Region length and you can filter regions based on this column to exclude small numbers e g 0 0002 for SNP5 which would indicate a centromere region Question Does Partek software use both CNV and SNP probes on SNP5 and SNP6 Answer Yes Partek uses both CNV and SNP probes for copy number analysis However two separate annotations files are required and automatically downloaded Question How do I visually combine or compare different array platforms within Partek e g 500K SNP6 Affymetrix Illumina A gilent Answer Import the data from different platforms into different spreadsheets and draw a chromosome view In the Plot Properties dialog choose the Profiles page to add a profile of each data from each platform Question Why allele ratio spreadsheet contains fewer number SNPs than assayed on the array Answer When Partek creates an allele ratio baseline from a reference population we only consider the SNP informative if it satisfies some quality criteria The most restrictive is that for each SNP 2 samples are required for each possible genotype Partek Frequently Asked Questions 7 This means that to cover the three possible genotypes in at least 6 samples are needed Only for those
18. he same probe model for a model based algorithm e g GCRMA Answer 1 First create a list of common probeset ID of the two platforms 2 Import the data from the array with greater density more genes On the Advanced Import Options page choose the Include From File option for Probe Filtering at the top of the dialog to include the list created 3 Choose GCRMA Background Correction option for Background Correction 4 Choose Quantile Normalization Save Distribution in the Quantile Normalization drop down list and specify a name for the file 5 Import this data in a spreadsheet 6 Import the second less dense data set by choosing the same setting for Probe Filtering and Background Correction 7 Choose Quantile Normalization Load Distribution and specify the saved distribution file 8 The data will be imported into the second spreadsheet The two spreadsheets should have same number of columns since they both are using the same gene list to filter during import the order of the gene column should be the same 9 Choose File gt Merge spreadsheet gt Append Row Batch Remover Question At a high level what is the batch remover doing Answer Removing the biases due to unwanted effects due to factors such as processing batches Question What analytical processes should I use with batch removed data Answer Batch remover is primarily designed for the visualization of data where the biological patterns might otherwise b
19. iMP BOWTIE TOPHAT Other Any tab delimited files containing Chromosome Start Position Strand and Sequence i e chrl 100 actgtactaactaga Partek Frequently Asked Questions 2 Question Can I import and analyze my raw sequencing data in Partek Answer Partek can align reads to the genome from fasta and fastq formats Partek uses the Bowtie program which is one of the fastest short read aligners available Question During import Partek asks me does reverse reads match forward strand How should I make the choice Answer The sequencing read can be written as it was read by the sequencer or be reverse complemented so that reads from the reverse strand match the forward strand sequence How the read is written depends on the aligner Partek will automatically make the correct choice if you specify the common aligners that we support If you want to specify it yourself or you are using other format of aligned files manually choose yes or no based on your knowledge Question Do I need to set any filter to the aligned reads if I only want to analyze uniquely mapped reads Answer No filtering is necessary because Partek only deals with those reads that are uniquely mapped Question I used both Partek and Affymetrix Expression Console to import the same CEL files with the same RMA algorithm I got different results Why is that Answer there could be several possible causes e Ifthe results match 6 7 signifi
20. ill reverse transcribe the mRNA into double stranded cDNA In this case the sequence was read from both sense and antisense strand and was not discriminated between them The biologists who prepare the sample should know that information from the kit they are using If you select yes on the genome browser you can see all the reads from a single transcript is either on sense or on antisense strand as indicated by two different colors comparing to choice no you can see mixed reads from both strand on the same transcript Question I ve noticed that Partek can display positive strand reads on one track and negative strand reads on another track How do I enable this in my analysis Answer As long as you make the right choice on the question of Can assay discriminate between sense and antisense strand Partek has the ability to automatically detect the reads that come from the positive strand or the negative strand and display and analyze strand specific sequencing result Question Are the unexplained peaks mapped reads How do we map the reads to the unexplained regions Answer Yes unexplained peaks are absolutely mapped reads They are mapping to the genome but not the transcriptome This shouldn t be confused with junction reads as that actually won t map into genome sequence And we don t map these reads to unexplained regions we run a peak finding algorithm to find a pile of reads in genomic space which does not map to
21. lignments Currently Partek only supports paired end reads that contain at most one alignment per end If both ends of a paired end read map to the same sequence then the ends are imported as one paired end read If the ends map to different sequences or if one of the reads is unmapped then the ends are imported independently if mapped as single end reads Question How does Partek count the number of alignments Answer Reads can be aligned to more than one location Since Partek only imports aligned reads the number of alignments will always be greater than or equal to the number of reads If the number of reads equals the number of alignments then each imported read is aligned to exactly one location A paired end read with both ends mapped to the same sequence is counted as one alignment If the ends are mapped to different sequences the ends are counted as two separated alignments If one end is unmapped the mapped end is counted as one single end alignment ChIP Seq Question How does Partek detect peaks in the CHIP seq work flow Answer Partek traverses the reads in order and locates coverage that is above the use defined threshold It then finds the endpoints of the regions by taking the median of the forward reads left endpoint and the median of the reverse strands right endpoint The reason that we refine the boundaries of the region from the median of the forward reads to the median of the reverse reads is because we are r
22. n Question Within an exon data table how do I get a gene symbol from a gene_assignment Answer Right click on the gene assignment column header to choose Split Column Use as a delimiter to split the column the second field contains the gene symbol Note Partek 6 4 and newer will automatically parse this information out Partek Frequently Asked Questions 11 Question How can I tell if an exon has been omitted from the analysis due to low intensity Note use must specify the option to exclude an exon due to low intensity Answer On the Alt splicing result spreadsheet right click on a row header a gene to draw a Gene View The exons that are translucent are the ones skipped during the computation Question Why are some probesets on my Exon Gene array not imported Answer Partek uses a meta probeset file mps to filter probesets which include only core extended or full By default only the core probesets are imported This can be changed in the Advanced dialogue during import Partek also does not import control probesets which do not have a cluster ID Some probesets are marked core extended full but they are not included in the core extended full mps file because they are more likely to splice abnormally though there are other possible reasons You might want to contact Affymetrix support for more detailed information Question While the actual expression levels between the two groups are strongly overlapp
23. n When using a known database it asks you to specify a 2 bit file but there is no dialogue box to input it into Answer Partek should automatically download the 2bit file However if it is not working you can also manually download the hg18 2bit file from http hgdownload cse ucsc edu goldenPath hg18 bigZips Within Partek go to Tools gt File Manager Where it says Please Specify Files For select Homo sapiens assuming you are running an experiment on human samples In the tab where it says Genome Sequence 2bit specify the location of your 2bit file RNA Seq Question How does Partek assign reads to Exonic Intronic or Intergenic regions Answer Given a list of transcripts each read can be assigned to one of three classes Exonic Intronic or Intergenic Exonic means the read came from the transcriptome Intronic means the read overlaps a gene but includes bases outside of the transcriptome Intergenic means the read came from a region outside of any gene The criteria to assign the reads are as follows Exonic A read is labeled exonic if any one of its alignments is completely contained within a transcript If the alignments are strand specific then the strand of the alignment must also agree with the strand of the transcript Intronic A read is labeled intronic if any one of its alignments overlaps a gene but none of the alignments are exonic If the alignments are strand specific then the strand of the alignment must als
24. notation files when you import Affymetrix CEL files Annotation information will be automatically associated with genomic data ILMN annotations are automatically handled and associated with using the Partek BeadStudio plug in Generally to associate a specific annotation file to a spreadsheet choose File gt Properties of the current spreadsheet use the Browse button to select the annotation file in the Choose chips and annotation files panel Detailed information can be found at the following link http www partek com Tutorials microarray User_Guides AddAnnotation pdf Question I am having difficulty importing my sample information what does the Add Column do Answer Add column will insert a column into the current spreadsheet Typically users should define the type as categorical and the attribute as factor when applying sample information into a genomic experiment Detailed information can be found at the following link http www partek com Tutorials microarray User_Guides CreatingSampleInfoFile pdf Question Is GC correction the same as GC RMA Answer No Partek s GC adjustment uses a model fit on all imported probes to remove the effects of GC content on probe level intensities If a user desires GC RMA then they may simply select that algorithm during import Question What aligners does Partek support Answer ELAND SOAP SOLiD Whole Transcriptome Analysis Pipeline WTAP produces max files MAQ SHR
25. nt as far as speed however it is not very accurate in terms of sensitivity or specificity HMM may be capable of detecting smaller regions than the segmentation algorithm however this comes at a cost of more false positives Generally genomic segmentation is easier to configure provides a p value to sort the resulting table and is easier to describe the regions of interest Question When I perform Find Regions in Multiple Samples why are some of the regions shorter in length than specified Answer In short it s due to how segments with differing overlaps are combined Here is an example of finding a region meet the criteria in 5 samples Region of amplification in sample 1 Region of amplification in sample 2 Region of amplification in sample 3 Region of amplification in sample 4 When you find regions in multiple samples Partek will combine all the segment breakpoints from all the samples Pe SPewe sew Sew Ses SeT eT ELT SLT Seecustustustuseuse s Ouse ese reese eee ese wees er tee When you find regions in at least 4 samples the result will look like the following graph Partek Frequently Asked Questions 9 Region of amplification in sample 1 A EE EE EEE EEE EE EE EEEE EEEE E Region o amplification in sample 2 Region of amplification in sample 3 Region of amplification in sample 4 Region of amplification in sample 5 The result will be over segmented If you choose T
26. o agree with the strand of the transcript Intergenic A read is labeled intergenic if none of its alignments overlap a gene See figure below for examples of exonic intronic and intergenic reads Partek Frequently Asked Questions 14 Exonic Reads Intronic Reads Intergenic Reads Question does Partek assign reads to different isoforms of a gene Answer We used an expectation maximization EM algorithm to probabilistically assign reads to known isoforms of a gene Similar methods have been used for identifying isoforms in this paper An expectation maximization algorithm for probabilistic reconstructions of full length isoforms from splice graphs Xing et al Nucleic Acids Research 2006 34 10 3150 3160 http nar oxfordjournals org cgi content full 34 10 3150 EM algorithm Input 1 Set of isoforms 2 Counts of the number of reads on each exon 3 Length of isoforms Output proportion of each isoform where the sum of the proportions is 1 Algorithm The E M algorithm is a way of solving the chicken and egg problem If you know relative proportions of isoforms you could assign the reads to each isoform accordingly If you knew the assignment of reads to isoforms you could get an estimate of the isoform proportions Partek Frequently Asked Questions 15 The algorithm works by first guessing the isoform proportions say 1 n where n is the number of isoforms Then reads are assigned to
27. ools gt Merge Adjacent Regions from the menu on this spreadsheet and choose Copy Number as Grouping Variable in the dialog you will get the result like the following Region of amplificati on in sample 1 JAAAAAAAAAAAAARAAAAABAAAAASAAAAAAAMURAAAAAAAARAABAABAAAAAAAAARAAAAAAAAAAAAAAAAAAAASAAAAAAAAAAAAAAAAAABAABAABAAAAASAAARAARAAAS Region of amplification in sample 2 Region of amplification in sample 4 Region of amplification in sample 5 I erevev7vev vevnPH ee rr e eV v PYTPYYV gt OV PY SHH HrYYTSSYU gt PPT PVOYUO PPT ee z Allele Ratio Question What is allele ratio Answer The allele ratio represents the proportion of A copy number to total A B copy number In normal diploids homozygous SNPs are expected to occur at O and 1 while heterozygous SNPs are distributed near 0 5 Partek Frequently Asked Questions 10 Question Why are intensities used as input and not allele calls Isn t this a single sample calculation based on genotype calls Answer The genotype call will not give the information necessary to calculate allele ratio The intensities of individual genomic markers are required Question Why does Partek use a baseline file for allele ratio calculation Answer Historical baseline data e g 270 HapMap samples allow Partek GS to estimate bias in A and B intensities that is used to reduce noise Question How does allele ratio change in regions of abnormal ploidy Answer In
28. out express written consent from Partek Incorporated is strictly prohibited Partek Frequently Asked Questions 22
29. sisisi Partek turning data into discovery Frequently Asked Questions These Frequently Asked Questions are some of the more common questions our customers ask Questions are divided into several categories for ease of use If you do not find the answer you are looking for please contact our Technical Support Team by phone at 1 314 878 2329 between 8 am 5 pm U S Central Time or by email support partek com at any time We strive to answer all support requests within 24 business hours Select a category from the list below e Import e Interplatform Comparison e Batch Remover e Hierarchical Clustering e Genomics o Copy Number o Allele Ratio o Exon o Gene Expression o Tiling e Next Generation Sequencing o Import Criteria o ChIP seq o RNA Seq e Visualization e Computer Specifications e Statistics e Project Partek Frequently Asked Questions Import Question I am having problems getting my data into Partek what steps should I take Answer Choose File gt Import choose the appropriate format of your data and follow the import wizard Please see Help gt Online Help gt Chapter 4 for detailed information Question How do I import Affymetrix library files Answer Partek automatically downloads the appropriate library files and annotation files if you are importing Affymetrix CEL files Question How do I import annotation files Answer Partek automatically downloads the appropriate an
30. sors Answer Partek is a single threaded process it cannot take advantage of multi core CPUs While there may be a slight boost to performance in a quad core processor the benefits might not outweigh the expense Question How much RAM can Partek handle in 32 bit Windows XP Vista Answer 3 4 gigs of RAM Partek is a 32 bit application though we are planning to make it 64 bit in the future As of right now somewhere around 3 5 gigs is the most Partek and 32bit windows can use by default Question Do you have any recommendations for video cards Answer Partek recommends Nvidia video cards Partek is not a graphics intensive application a powerful video card is not necessary NVidia has the best support for OpenGL and right now has been the most stable with Partek Question Any thoughts on hard drive specifications Answer Import and certain other parts of analysis can become very hard drive bound as the large files are being read To increase speed at these steps Partek recommends that you get either a fast 10k RPM hard drive like the Western Digital Raptor or a similarly fast SCSI drive which you use for only your current project Or you can setup a RAID O0 array which will give you access to large amounts of fast hard drive space but at the cost of reliability Partek will run without issue on current standard issue hard drives These recommendations are only for researchers looking for high performance computing Statistics Q
31. t rma data bgadj background FALSE 4 the values in exprs data expr should be the same as Partek s There are other possible causes for example 1 The order of the CEL files are different E g 1 CEL 2 CEL and 3 CEL will produce different numbers than 3 CEL 2 CEL and 1 CEL 2 Sys setlocale LC_COLLATE C was not set in R 3 Bioconductor and Partek are using different library files E g if the CDF file or the chip sequence file are different the result will be different Interplatform Comparison Question How do I generate a list of genes that are common to two different platforms which share probe set names e g Affymetrix HG U133A and HG U133_Plus_2 Answer create a list of common probeset ids from both platforms import just one cel file of each platform choose Transform gt Create Transposed Spreadsheet to put probesets on rows on each spreadsheet choose View gt Venn Diagram to select the two transposed spreadsheets use the common region to create a list delete all the extra columns only keep the probeset ID column and save it as a text file Note this simply creates a list of common IDs between the arrays DETE d l File Edit View Window Help ke k a x t list4 HG U133A 20590 TistS HG U133_Plus_2 21973 Partek Frequently Asked Questions 4 Question How do I import data from two different platforms e g Affymetrix HG U133A and HG U133_Plus_2 using t
32. tion How do I adjust the view add labels or standardize my clustering data Answer In the cluster viewer choose Edit gt Plot Properties On the Style page you can configure the viewer on the Rows amp Columns page change the Axis Labels from off to any other options to add labels for row or column dendrograms To standardize the clustering only on the viewer you can choose Edit gt Standardize Intensity Plot from the menu of the viewer to make the mean as 0 and standard deviation as 1 for all the columns Question Why do I run out of memory on gt 30k elements Answer To do hierarchical clustering on 30K elements requires at least 3G of RAM just for itself if you don t have enough RAM you can choose to do 2 pass option for the Clustering method on the dialog Genomics Copy Number Question Why is quantile normalization not selected as a default option during import CEL files on copy number workflow Answer Quantile normalization does not work well when the assumption of identical distributions is violated We do not recommend using quantile normalization on any samples that are expected to have any significant amount of variation from true biological signal cancer samples for example In addition we have not seen large improvements in performance when using quantile normalization Question Should I smooth my copy number data Answer Smoothing is not necessary for many analysis algorithms Visual smoothing is
33. uestion Why did we get question marks for the p values of treatment combination and so did for the contrasts in the ANOVA result spreadsheet Answer There are two reasons could result in the question marks in the ANOVA result One is that there are missing treatment combinations when designing the experiment The other is there is no replicate for each treatment combination when conducting the experiment Question What does the question mark mean for LSMean Answer It means the LSMean is not estimable Let L as LSMean matrix If ABS L LH gt C x10 then L is declared nonestimable H is the X X X X matrix and C is ABS L Question When doing a statistical test like ANOVA why doesn t my factor show up in the candidate list Answer Make sure the sample information column property has the attribute status defined as factor Right click on the column header choose Properties change the Type to categorical or it can be numeric such as scaling factor or age etc the attribute is factor click OK Partek Frequently Asked Questions 19 Question How do I get the mean of groups e g Tumor vs Normal Answer The column of the groups should be categorical choose Stat gt Descriptive gt Column Statistics select the Group By check button on the dialog choose the groups column and move Mean to the Selected Measures panel and select OK Question What is the difference between ratio and fold change Answer When comp
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