Questions & Answers

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  • How long does it take to scan an Infinium MethylationEPIC array on the iScan?

    ~18-37 minutes per BeadChip

  • Can I upload data from my custom GoldenGate OPA or my custom iSelect product?
    The iControlDB is intended only for standard content Illumina products. BeadStudio will allow you to upload your data from a custom project, but this data will not pass the validation stage and will not be released to the database.
  • Can I use GenomeStudio to download data from the iControlDB database?
    No. Only the Illumina iControlDB-Client program can be used to download data from the database.
  • Do you consider underlying polymorphisms in the Design Score calculation?

    The Design Score reflects underlying polymorphisms based on the extent that the polymorphisms are annotated in dbSNP. If you have additional annotation information regarding polymorphisms around SNPs of interest, you should consider submitting these SNPs in a sequence list to be sure this information is taken into account.

  • What RNA Amplification kit does Illumina recommend?

    Two kits have been tested for use with Illumina’s Gene Expression products: Ambion Total Prep Kit (Catalog No. IL1791) and Epicenter TargetAmp-Nano Labeling Kit for Illumina Expression BeadChip(Catalog No. TAN07924). In addition, the Ovation Amplification Kits from NuGen have been shown to produce acceptable results with the use of a modified protocol. Please note that Illumina does not provide technical support for these kits.

  • What is the lowest detectable fold change?

    During product development experiments, the lowest detectable statistically significant fold change was 1.2 fold.

  • What are the requirements to run Infinium MethylationEPIC arrays?

    Illumina Automation Control v5.3.0, Illumina LIMS v4.8.1, Tecan Tip Guide-E, Standard Teflow glass back plates and spacers, iScan Control Software v3.3.29, GenomeStudio 2011.1, BeadArray Controls Reporter.

  • Is the controls dashboard different for the Infinium HumanMethylation27 BeadChip versus the HumanMethylation450 BeadChip?

    Yes, there are several differences in the control dashboard due to the inclusion of Infinium II Assay designs for the Infinium HumanMethylation450 BeadChip. Please see the Infinium HumanMethylation450 BeadChip User Guide for more details.

  • What are the computing requirements for the PC Module?

    The PC Module has the same computing requirements as other GenomeStudio Microarray modules listed here.

  • What do ‘TOP/BOT’ strand and ‘A/B’ alleles mean in the Illumina manifests and reports?

    Please refer to our technical note entitled “TOP/BOT” Strand and “A/B” Allele (PDF).

  • I clicked the 'Download Checked Samples' option, and it completed the download. Why can't I find the genotyping results?

    When you click the Download Checked Samples button only functions to generate the genotype data. You must also click the Generate Report button to generate the report and save it to your computer.

  • What if my SNP is not in your database?

    You can submit the SNP in a SequenceList.

  • Can I use whole-genome amplified (WGA) DNA for GoldenGate genotyping?

    Please refer to our technical note entitled Using Whole-Genome Amplified (WGA) DNA Samples in the GoldenGate Genotyping Assay (PDF) before conducting your genotyping experiment. You can also find a link to the publication referenced in the technical note here: Two Methods of Whole-Genome Amplification Enable Accurate Genotyping Across a 2320-SNP Linkage Panel.

  • Can I quantify my cRNA by A260 instead of Ribogreen?

    We have found quantification by RiboGreen to be more accurate than spectrophotometer readings. Contaminants from purification columns can give artificially high readings. If you must use A260, sample measurements should be > 0.5 to minimize the impact of contaminants on amplification.

  • How should I send DNA samples to Illumina for genotyping?

    We will provide to you barcoded 96-well plates for you to ship samples to us along with a step-by-step preparation protocol.

  • There are samples from the HapMap Project in the database. What HapMap sample IDs do these Illumina barcodes correspond to?

     A list of the Illumina barcodes and their corresponding HapMap IDs is available upon request from techsupport@illumina.com.

  • How many components can I expect with my order?

    Orders will receive five items: four reagent boxes including the MAP, and one package containing arrays.

  • Does Illumina recommend background subtraction for analysis?

    Background subtraction is required when you compare data run on different types of scanners because of technical disparities; for example, the iScan and HiScan have different offsets. Background subtraction has a much smaller effect when you scan chips on the same scanner, and might not be necessary. Analyze a subset of data with and without subtraction, and choose the subset of data you prefer based on your results.

  • Is the Infinium MethylationEPIC BeadChip controls dashboard similar to the Infinium HumanMethylation450 BeadChip?

    Yes, the same controls types are present.

  • How are the VeraCode Universal Beads used?

    Each uniquely coded VeraCode Universal Bead has a unique oligonucleotide capture sequence attached and can be used to design nucleic-acid based assays. For example, to develop a 3-plex reaction using a single-color detection assay, such as Allele Specific Primer Extension (ASPE), pool together six different tubes of unique VeraCode Universal Oligo Beads (one bead type per allele).

  • How often is the annotation updated?

    This depends on updates to the Sanger miRNA database, http://microrna.sanger.ac.uk/sequences/. We plan to update the annotation as needed.

  • Can I run FFPE samples on the Infinium Methylation Assay?

    FFPE samples are NOT recommended for the standard protocol. FFPE samples are already highly degraded, with a high level of crosslinking, so conversion does not occur effectively. However, you can run FFPE samples on the Infinium HumanMethylation450 BeadChip using the FFPE automated or FFPE manual protocol along with the Infinium FFPE DNA Restoration Solution kit.

  • How can I take data from samples that have been run in the past and see if those same found regions appear in samples that are run in the future?

    You can adjust the information contained within the known regions report. Refer to the user guide, which describe how to customize the known regions table so you can track your favorite regions in the additional samples.

  • Can I compare data from experiments with MCS3 to experiments with MCS4 plus RTE?

    We have observed high concordance between detected probes using MCS3 versus MCS4 plus RTE and have found the variability in raw signal intensity correlations between MCS3 and MCS4 plus RTE to be comparable to the variability between freshly made MCS3 and MCS3 nearing the end of its shelf life. As is to be expected, signal intensity correlations and detected probe concordance decrease with input RNA quality.

  • What is the throughput of the BeadXpress Reader?

    Throughput is dependent upon the level of multiplexing and whether you are running a single-color or dual-color detection scanner. Typical throughputs are:

    Multiplex

    Single-Color Detection

    Dual-Color Detection

    10

    140 samples/hr

    120 samples/hr

    96

    90 samples/hr

    68 samples/hr

    384

    44 samples/hr

    30 samples/hr

  • How does Illumina select SNPs and develop the SNP genotyping assays?

    For custom genotyping projects, we will validate and develop the SNP genotyping assays for you. Prior to development, we will work with you to select SNPs, screening those SNPs informatically. Assay development success depends upon the source of the SNP, its frequency in the population, and the assay system. For instance, many SNPs derived from databases are sequencing errors, or exist in too low a frequency to be useful in most genotyping studies.

  • Can I transfer called SNPs from CASAVA to use in custom BeadChip design?

    Called SNPs from CASAVA can be parsed to ADT via two reports: a dbSNP Report for the start and stop positions of the region flanking the SNP, and a Sequence Report which provides the actual sequence of the region flanking the SNP.

  • Can I transfer called SNPs from CASAVA to use in custom BeadChip design?

    Called SNPs from CASAVA can be parsed to ADT via two reports: a dbSNP Report for the start and stop positions of the region flanking the SNP, and a Sequence Report which provides the actual sequence of the region flanking the SNP.

  • Does the install of the PC Module require a license key?
    No, the PC module is a standalone software which does not require a license key.
  • In what format will Illumina provide the final data at the end of the project?

    We will provide you with compact discs containing the final data in standard comma-delineated text. For large genotyping studies, we will be happy to discuss custom formats with you.

  • Does custom Infinium support Tri/Tetra-allelic SNP assays?

    Not at this time.

  • Can I quantify my cRNA by A260 instead of Ribogreen®?

    Illumina has found that quantification by RiboGreen is more accurate than spectrophotometer readings. Contaminants from purification columns can give artificially high readings. If you must use A260, sample measurements should be >0.5 to minimize the impact of contaminants on amplification.

  • How was the default known region list generated?

    The known region list covers 244 known regions and was generated by compiling information from multiple sources including publications, databases, and public information available on which regions are typically examined in cytogenetics labs.

  • What is the Decode File (dmap) Download Utility? How does it work?

    It is a software application that runs from your PC desktop and enables you to download multiple DMAP (decode) files for Illumina BeadChips more easily than manually copying files from CDs one by one.

  • What are the system requirements for the Decode File Download Utility?

    The software will work with any PC that has internet access (port 80) and for which you have rights to install new programs. You may need help from your IT department if you do not have sufficient rights to install new software on your computer. There are no other firewall or security restrictions.

  • What types of inputs are supported by ADT?

    Accepted inputs include RSList, GeneList, SequenceList, and RegionList.

  • Which Illumina-supplied consumables are necessary to run the GoldenGate Assay for Methylation?

    GM-17-211 GoldenGate Methylation Cancer Panel I (standard panel oligo for methylation assay), GM-12-109 Sentrix Universal-96 Array Matrix (1,536-plex) , GM-95-201 Single-Use Activation Kit (576 Samples) or GM-95-202 Multiple-Use Activation Kit (576 Samples), GM-95-203 GoldenGate Assay Kit (96 Samples) or GM-95-204 GoldenGate Assay Kit (576 Samples), or GM-95-205 GoldenGate Assay Kit with UDG (96 Samples), or GM-95-206 GoldenGate Assay Kit with UDG (576 Samples)

  • I have uploaded my data, but it is not visible using the iControlDB download software. Was there a problem with the upload?

    All uploaded data is initially deposited in a staging area until it is reviewed by Illumina personnel. Data is not released to iControlDB until it has undergone this review process, which may take up to 10 business days. Additionally, data will not be released if they do not pass our call rate threshold of 98%. If you still do not see your data after 10 business days, please contact techsupport@illumina.com for further investigation.

  • Can I calculate PC and PPC errors in the PC Module?

    No, this is not an option in the PC Module.

  • To which genome build are the current probe coordinates mapped?

    The probe coordinates of v1 and v2 MAPS are mapped to Genome build 36.2.

  • What is a cluster file?

    The cluster file contains the mean (R) and standard deviation (theta) of the cluster positions, in normalized coordinates, for every genotype, for every SNP. The cluster file also includes cluster score information, as well as the allele frequencies from the training set used to generate the cluster file. A cluster file is required for KaryoStudio. Illumina provides a standard cluster file for each product. Alternatively, customers may generate their own cluster file.

  • What total-RNA quantification method do you recommend?

    Any routine method is fine. Illumina recommends using RiboGreen to quantify total-RNA concentration.

  • How well do SNP genotype calls from the Infinium Assays compare to those called from the GoldenGate Assay?

    Although we have not tested this specifically, the expectation is that the conversion efficiency will be high.

  • Can I use the GoldenGate Linkage panel with samples from many ethnic groups?

    Yes. We provide a list of the markers in the Linkage V Panel and include minor allele frequency information for samples from the three major ethnic groups represented by the HapMap samples. The panels were constructed to optimize information content across Caucasian samples, and it has been augmented to increase information content for the African population.

  • How many samples can I load into KaryoStudio?

    There currently is no limit on the number of samples that can be loaded into KaryoStudio.

    Refer to Appendix A of the KaryoStudio Software User Guide or see the KaryoStudio Benchmark Performance Technical Note for more information about KaryoStudio software performance.

  • Can you identify tag SNPs for me?

    Not at this time.

  • Can I use cRNA prepared according to Affymetrix's protocol on BeadChips?

    Illumina has had poor results when using fragmented sample on our arrays. The Affymetrix protocol requires fragmentation during sample preparation due to the short probes used on their arrays as it reduces the secondary structure of RNA. Illumina's longer probes allow for more stringent hybridization conditions which preclude the need for RNA fragmentation. If you have labeled sample which has NOT been fragmented, you may obtain satisfactory results, depending on the age and quality of the sample.

  • Have Illumina’s gene probes been mapped to any public database?

    Yes. Our probes (human and mouse) have been mapped to Ensembl. The Illumina probe track can be turned on by checking the box next to Illumina Probes on the DAS Sources dropdown menu in Contigview. When the page refreshes, blue features indicate the location of probes. Clicking on a probe opens up a floating menu with probe information and a link to a page giving more information about the probe. The GFF text files giving the mapping information for probes to mouse and human can be downloaded from the Sanger Institute website ( http://www.sanger.ac.uk/Software/ formats/GFF/): - Human GFF file (right-click and select Save As) - Mouse GFF file (right-click and select Save As) More information about the GFF data format with an explanation of the fields can be found at: http://www.sanger.ac.uk/Software/formats/GFF/

  • I have downloaded specific samples before, but now when I open the iControlDB - Client, it says “FALSE” in the Downloaded column for these samples. Why does this happen?
    The TRUE and FALSE entries in the Downloaded field are only applicable to the current session. If you have downloaded samples in previous sessions, this will not be reflected in the current iControlDB - Client session.
  • Can I upload copy number variation data?
    No. Only genotype information is contained within the iControlDB at this time.
  • Can I use the modified cycling incubation protocol for the bisulfite conversion recommended for Infinium Methylation analysis on samples to be analyzed on the GoldenGate Methylation protocol?

    Yes, the modified cycling incubation can be used on the GoldenGate Methylation Assay. It is not required, however, because that assay is more tolerant towards incomplete conversion.

  • Why is a study number used for uploaded samples?

     A study number allows you to keep track of the samples you have uploaded, and provides an easy reference to identify your control samples for any publications you are preparing. It also allows others who are referring to your publications and studies to replicate the analysis by downloading the same samples.

  • How do I prevent the magnetic beads from drying out during GoldenGate processing?

    To avoid this, decrease the number of columns processed at one time during each of the wash and buffer exchange steps. To resuspend beads that have partially dried, re-vortex the ASE plate containing the UB2 buffer in the Add Mel step and/or the IP1 during the Inoc PCR step.

  • Is the Illumina-recommended bisulfite conversion kit a plate- or column-based kit?

    Zymo Research provides EZ DNA Methylation Kits for columns (#D5001 and #D5002) or 96-well plates (#D5004).

  • For custom Infinium content, what is the expected overall conversion to functional assays?

    The conversion rate depends on many factors including our stringent QC criteria during manufacturing, the sequence nature of the chosen SNPs, and criteria used for Gentraining. To maximize chances of success, we recommend selecting validated SNPs and/or SNPs with high design scores. In general, we expect the final Design Conversion Rate to average 80%.

  • What are some guidelines for improving data quality?

    With Infinium products, the two main parameters for copy number are the B Allele Frequency (based on genotypes) and the Log R Ratio (based on intensities). The Log R Ratio is the log (base two) of the "observed intensity" divided by the "expected intensity". The "expected" intensity is generated from the cluster file.

    Because of this direct comparison, accurately measuring the sample input amount is vital. Essentially, your input amount should match the recommended value (400ng for Infinium HD Duo; 200ng for Infinium HD Quad and Infinium HD 12-sample products). If this is this case, your Log R Ratio signal will tend to have low noise.

    When the DNA samples are inaccurately quantified, you may see an "undulation" pattern (this looks like waves) in the log R ratio. This tends to be in GC-rich regions of the genome. This wavy pattern makes it difficult to do CNV analysis as the waves themselves look like copy number changes. This tends to confuse algorithms and confound analysis. On the other hand, call rates tend to be only slightly affected by this change (but this varies).

  • How much DNA is required for bisulfite conversion?

    Single use, manual process: > 500 ng, Single use, automated process: > 1,000 ng, Multi use, manual process: 2,000 ng, Multi use, automated process: 2,000 ng

  • Is it possible to know who submitted the individual samples?

    Samples are listed as being submitted either by Illumina, or by Other. The User Agreement that is necessary to comply with the Health Insurance Portability and Accountability Act (HIPAA) does not allow the identification of the source of the data from the download tool. However, it is projected that many submitters will reference their study number in their publications.

  • Over what range of intensities can I detect the median significant detectable fold change?

    The intensity range over which a fold change of ~1.35 is significantly distinguishable is greater than 3 logs.

  • What information is captured during my dmap download session?

    Illumina servers record the login ID and the date, time, and serial numbers of any downloads. This is necessary to allow you to filter your selections during future uses of the software. No cookies or information other than the *.sdf and *.dmap files are written to your computer.

  • How do I place an order for a custom OPA?

    Please refer to the Designing Custom GoldenGate Assays technical note.

  • How do I determine which version of GenomeStudio software I am using?

    With GenomeStudio software open, go to the Help menu and select About. The About screen includes GenomeStudio software version information.

  • What concentration does my RNA need to be?

    Illumina specifies 40 ng/ul in the assay protocol.

  • Is Illumina data compatible with Bioconductor?
    Yes, Illumina data is compatible with Bioconductor, a collection of R packages developed by researchers around the world and distributed for free.
  • Under what conditions and for how long can DNA be stored after bisulfite conversion?

    Bisulfite-converted DNA can be stored at -20°C for up to one month.

  • Can I run FFPE samples on the Infinium Methylation Assay?

    FFPE samples are already highly degraded, with a high level of crosslinking, so conversion does not occur effectively. However, you can run FFPE samples on the Infinium MethylationEPIC Array using an FFPE Methylation Protocol (available in 2016) along with the Infinium FFPE DNA Restoration Solution kit. 

    To run FFPE samples on the InfiniumMethylationEPIC Array, perform the following procedures:

    1. QC extract FFPE samples with the Infinium FFPE QC Kit.
    2. Perform bisulfite conversion on only samples that pass QC with 1 of the approved kits.
    3. Restore bisulfite converted samples with the Infinium FFPE DNA Restoration kit.
    4. Input restored FFPE samples into the Infinium Methylation Assay.
  • BeadStudio allows for the customization of genotype reports (i.e. forward strand report, TOP strand report, design strand report, etc). What format is generated by iControlDB?
    Genotypes generated by iControlDB are in the matrix format, and are forward strand reports.
  • Is there a maximum distance from the 3' end of the RNA transcript to which probes hybridize?

    No, there is no set maximum distance. However, strong preference is given to probes closer to the 3' end.

  • Which metrics can be used to evaluate data in the SNP Table to identify SNPs that require further editing and poorly performing SNPs?

    SNPs can be evaluated and sorted by Call Freq, # no calls, Poly 10%, and Poly 50%.

  • What is the general efficiency of bisulfite conversion using the Illumina-recommended bisulfite conversion kit?

    Conversion efficiency generally exceeds 99% using the EZ DNA Methylation Kit.

  • What is the sensitivity for Illumina Gene Expression BeadChips?

    =1 in 250,000

  • Are there restrictions to the Minimum Number of Points in Cluster?
    This number can be any number different from 0, however we recommend that the Minimum Number of Points in Cluster is set to match the biology of your samples and size of your dataset.  A general guideline is to set this number to 1-4% of the number of samples that are performing well in the data set.
  • Can I submit custom designs for species other than human or mouse?

    Illumina does not currently have custom design support for this product.

  • Do I need to have a copy of BeadStudio to upload data to the Illumina iControlDB?
    Yes, BeadStudio v3.1 or higher is required to upload data to the database.
  • Are there duplicate samples in the Illumina iControlDB?

    Samples are checked for duplicate barcode numbers, and duplicates are not allowed. This does not prevent the same sample from being run on different barcodes. There is not a check performed at the genotype level to ensure unique samples. Additionally, different sources may have run the same commercially available sources of DNA and submitted the data. Therefore, we recommend performing genotype-level comparison of the data that you are downloading prior to analysis to ensure the most accurate frequency information for your study.

  • Do I need to begin clustering with one of the two default algorithms, OPTICS or DBSCAN?
    No, you can also cluster by defining #clusters, if known based on the biology of your samples.
  • I cannot find a Report Wizard in the PC Module. How can I create reports from my data?

    Data can be exported directly from the Samples Table, SNP Table, and Full Data Table for downstream analysis. Mark the columns and rows you wish to export and click the icon for "Export displayed data to file" to save selected table content in *.txt or *.csv format.

  • When did the new MCS4 plus RTE formulation ship?

    The new MCS4 plus RTE formulation started shipping in late April, 2012. All new orders were automatically shipped with the new material. There is little or no inventory left of MCS3.

  • How do you ensure you have enough markers for linkage mapping?

    This is an optimal set of markers for linkage mapping. By simulation studies, it has been suggested that a 1 to 2 cM bi-allelic map of polymorphic markers (minor allele frequency 20–50%) will extract most of the inheritance information and that for common linkage study designs, adding more markers provides diminishing returns (Kruglyak, 1999). In a study of 188 meioses, the average information content over all chromosomes was over 97% and never dropped below 83%. This high information content throughout the genome can be attributed to both the appropriate level of marker density and high heterozygosity of SNPs used in the panel and will ensure maximal power for detecting linkage to a disease or trait and defining the linkage interval.

  • How can I calculate a p-value from the DiffScore?

    p = 10'(DiffScore*sgn(µcond-µref)/10)

  • Is it possible to get the data for each feature on the chip?

    Yes, this is known as bead-level data. Contact your Field Application Scientist (FAS) or Technical Support Representative for assistance.

  • When changing parameters in the Clustering Options dialog box, will this impact or change the existing clustered data?
    No, the new settings will only be applied to any SNPs clustered after applying changes.
  • What kind of data can I export from KaryoStudio?

    You can export data displayed in the Found Regions table as either a single row or the entire table.

    • To export a single row: right click in the Found Regions table and select Copy Row to Clipboard.
    • To export the whole table: right-click in the Found Regions table and select Copy All to Clipboard
    • Paste the data into an Excel file or import it into another downstream, third-party application. In addition, you can export data in the form of a cytogenetics report.

    There are no other reporting functions available in KaryoStudio.

  • Do you support an automation option for custom Infinium?

    Yes, both manual and automated product offerings are available.

  • I don't have an iCom account. Can I still access the Illumina iControlDB to download genotypes?
    No. Access to downloading the genotypes is only available with an iCom username and password. Illumina customers can sign up for an iCom account by visiting http://icom.illumina.com
  • What is the reproducibility of the GoldenGate for Methylation Assay?

    The average reproducibility between technical replicates is > 0.98.

  • How do I obtain genotypes from my data with KaryoStudio?

    Genotype information is not provided by KaryoStudio. To obtain the SNP genotypes from your data, you must load your data into the GenomeStudio Genotyping Module. For more information, refer to the GenomeStudio Genotyping Module V1 .0 User Guide.

  • What has Illumina done to ensure a high level of genotyping accuracy?

    Many factors play roles in determining genotyping accuracy including the quality of the DNA, the quality of the genotyping assay, and the quality of the system. At Illumina we optimize genotyping accuracy by testing samples of DNA before initiating a genotyping project and then by performing quality control procedures on each DNA that we receive. In addition, we have developed a robust genotyping system that is fully automated with built-in internal controls that are run with every genotype. For every SNP assay, our informatics system calls each genotype and assigns a GenCall confidence score. The GenCall score statistically measures the clustering of a particular result against the standard clusters for each genotyping assay.

  • Why can't I successfully log in to the Decode File Download Utility?

    You need a valid user ID and password for the Illumina iCom site and a working internet connection. Verify that your username and password are correct by logging in to your iCom account. If you do not have a login ID for iCom, complete and submit the form on the website for new users. If you believe you have a valid login ID and password for iCom, launch your browser and load any external page to verify you have a working internet connection. If you are still unable to log in, contact Technical Support.

  • If I have added additional information in auxiliary columns in my BeadStudio project, will this be included in the released data?
    No. Only information that is contained in the standard phenotype information columns will be available from iControlDB during the download process.
  • What is the limit of detection for the miRNA assay?

    The limit of detection is 100,000 molecules when spiked in the background of 200 ng total RNA.

  • How do I view genoplots with SNP information?

    You must load your data into the GenomeStudio Genotyping Module.

  • What is the expected correlation for hybridization replicates between BeadChips? Between arrays within a BeadChip?

    r2 between BeadChips: .99

    r2 within BeadChip: .99

  • Why can't I find the serial number of my BeadChips when I open the Decode File Download Utility?

    Click Find to initiate a search. If there are still no chips visible, ensure that you requested data for a BeadChip that shipped at least 24 hours previously, and has not been on the server more than 30 days.

  • What is the minimum aberration size I should use to filter my data?

    This varies for each user depending on the type of cytogenetics lab in which the samples are analyzed. For example, some cytogenetics labs tend to look at all aberrations greater than 250kb in size. Others may look at aberrations 100kb or greater in size. Therefore, it is up to the user to decide.

    You may want to set a threshold for the minimum number of SNPs per region, such as 50. This information can be found in the # SNPs column in the Found Regions table.

    Remember that more aberrations appear in the Found Regions table when you allow for regions of a smaller size. In this case, many of the regions may not yet be linked to a specific phenotype. Therefore, users may want to try a "top-down" approach, beginning with the largest regions that may be most likely already linked to a phenotype. Depending on the product in use, you may find aberrations as small as 1kb in size.

  • Which software should I use for downstream analysis of clusters and for generating genotype calls?

    Illumina does not provide recommendations for downstream analysis outside of GenomeStudio.

  • What amplicon decontamination protocol does Illumina recommend?

    Illumina recommends using a combination of UDG and routine lab cleaning with a 10% bleach solution. Also, physical separation between pre- and post-PCR areas is ideal.

  • The iControlDB tool requires a Final Case Report (.txt) file. What if I don't have that yet because I am just in the design phase of my experiment?

    When you downloaded iControlDB, you received template files that can be used in place of a Final Case Report. These template files do not contain sample genotypes, but are in the format required by iControlDB.

  • Is there a protocol for dual-color hybridization and detection?

    No. The low variability and high inter- and intra-slide reproducibility of Illumina's arrays make it unnecessary to perform dual-color experiments.

  • How much DNA is required for the GoldenGate Assay for Methylation?

    250 ng, 5 ul at 50 ng/ul.

  • What if my hard disk fills up during a download using the Decode File Download Utility?

    The program automatically checks the disk space available on the directory you select before the download begins, and prompts you if the available space is insufficient. HumanHap300 and HumanHap240S Genotyping BeadChip files are approximately 3.7MB each, and HumanHap550 Genotyping BeadChip files are approximately 6.5MB each. The software checks the available disk space after every *.dmap file, in case another user is filling up the disk space with another process after the initial check (for example, writing BeadScan files to the same disk). The utility alerts you when free disk space approaches 50 MB and stops if the available disk space drops below 50MB.

  • Can I use whole-genome amplified (WGA) samples with the Infinium Assay?

    Illumina does not recommend using WGA samples as input for the Infinium Assay as the first step in the protocol is a WGA step. Up to a 4% decrease in call rate was observed in the limited internal testing performed. The decrease in call rate will vary depending on the specific sample and WGA method used. Additionally, any allelic bias present in the original WGA sample may be compounded by another WGA reaction.

  • Can I design my custom Infinium BeadChip to detect chromosomal aberrations including Loss of Heterozygosity (LOH) and Copy Number (CN) changes?

    Yes. However, it is important to design the chips with a sufficiently high density of SNPs to detect the changes of interest.

  • What are the recommended system requirements to run KaryoStudio?

    For systems information, refer to the KaryoStudio Benchmark Performance Technical Note  or to Appendix A of the KaryoStudio User Guide.

  • What are the input requirements for the Direct Hyb Assay?

    50–500 ng of Total RNA, 11 µl at 50–500 ng/µl, according to Ambion Total Prep Kit #IL1791.

    cRNA to load on chips: 750ng in 5 µl (150 ng/µl) for 8- and 12-sample, 1.5 µg in 10 µl (150 ng/µl) for 6-sample.

  • How do I analyze the Infinium MethylationEPIC array data?

    The GenomeStudio Methylation Module extracts beta values and provides clustering and normalization for Infinium MethylationEPIC array data. In addition, there are many freeware applications in Bioconductor, such as chAMP and RnBeads, which provide enhanced normalization and visualization options. Illumina does not support these freeware solutions directly.

  • What is the minimum recommend total-RNA starting amount?

    Illumina recommends starting with 100 ng of total-RNA. However, highly reproducible (R2 0.94) miRNA expression profiles have been generated with as little as 2 ng total RNA input.

  • My OPA tubes arrived at room temperature but the label indicates that they should be stored at -20ºC. Are they still usable?

    Yes. OPAs are shipped at ambient temperature; stability testing indicates that OPAs are quite stable for short term storage (3-4 weeks) under these conditions. For long-term storage (> 1 month), we recommend storage at -20ºC.

  • My OPA tubes arrived at room temperature but the label indicates that they should be stored at -20ºC. Are they still usable?

    Yes. OPAs are shipped at ambient temperature; stability testing indicates that OPAs are quite stable for short term storage (3-4 weeks) under these conditions. For long-term storage (> 1 month), we recommend storage at -20ºC.

  • Does Illumina recommend a specific kit for bisulfite conversion?

    EZ DNA Methylation Kit for 50 DNA reactions (supplied with capped columns) (Zymo Research, catalog # D5001), EZ DNA Methylation Kit for 200 DNA reactions (supplied with capped columns) (Zymo Research, catalog # D5002), EZ-96 DNA Methylation Kit for 2x96 DNA conversion reactions (deep-well Zymo-Spin I-96 Filter Plate) (Zymo Research, catalog # D5004) Any other methylation kits are not recommended for use with this assay.

  • Why are SNPs included in the manifest of the methylation BeadChip?

    SNPs were included on the BeadChip so investigators could generate a DNA "fingerprint" of their samples as an added level of quality control. Please find further information in the Infinium HD Methylation Assay Protocol Guide. SNP assays on the BeadChip are not mentioned in the assay guide and only briefly described in the GenomeStudio Methylation Module. Follow this method to confirm the identity of samples from the same individual:

    1. Highlight the SNP assays in the sample methylation profile, right-click, and "show only selected rows."
    2. For any given pair of samples that are supposed to be from the same individual, plot the beta values in a scatter plot from the sample methylation profile.

      Samples from the same individual:

      Infinium HumanMethylation450 samples from one individual.

      Samples from different persons:

      Infinium HumanMethylation450 samples from different people.

      The scatterplots differ is because the beta values calculated from SNP assays will cluster, much as they do in a standard genotyping theta graph. Samples from the same individual have the SNP results fall along the identity line in a scatter plot, whereas samples from different persons scatter into the 9 different possible spots, based on their genotypes.

  • What are the call rates for GoldenGate genotyping?

    The call rates for GoldenGate genotyping for our standard products are generally > 99%. However, for custom OPA design the locus conversion rate is highly dependent on the quality of genotyping assays that are chosen.

  • Can I design for species other than human, mouse and rat with NuPCR?

    DesignStudio allows user to submit sequences for design. Select Add New Target - Sequence. When submitting for species other than human, mouse, rat it is important to turn off "Specificity" checking against the source genome.

  • Is there a manual way to include or exclude samples from clusters?

    Yes, in the SNP graph, use the curser to draw a box around the samples you wish to manually edit, right-click and choose the cluster samples should be assigned to, or NC (no call) if you wish to remove samples from any clusters.

  • When I right click the mouse on the samples table, I see an option that says “Request to be Admin”. When I select this, it says I do not have permission. What is this function?
    Admin rights are only available for Illumina personnel who are adminstrators for the iControlDB.
  • I do not have GenomeStudio installed on my computer, but would like to work with the PC Module. Do I need to install GenomeStudio prior to installing the PC Module?

    Yes, please download the GenomeStudio 2011.1 installer from the Illumina website. It is sufficient to install the GenomeStudio Framework by clicking the respective box in the install wizard, which does not require a license key. It is not required to install the GenomeStudio Genotyping Module on the same computer on which the PC Module is installed. However, the polyploid workflow does require generating a genotyping project in the GenomeStudio Genotyping Module prior to taking the data to the PC module for polyploidy clustering. 

  • My SUD pellets will not resuspend. What can I do?

    Complete resuspension of the DNA is required for optimal assay performance. Repeat shaking several times to resuspend. If the pellet does not resuspend, store the plate overnight at 4 °C followed by repeat shaking prior to proceeding.

  • Should I save *.tif images on my iScan or HiScan for my bead chips?

    Save *.tif images if you are interested in re-extracting the intensity information with third-party software. While image re-extraction is not supported by Illumina, some users may want to do this for various academic purposes. The 'beadarray' software package can be used and is available at www.bioconductor.org. NOTE: Illumina cannot provide support for this software.

  • How long will it take to download the files using the Decode File Download Utility?

    This is highly dependent on the speed of your internet connection. Times can vary from less than 30 seconds to five minutes per chip for most high-speed connections. In general, it will take less than one minute to set up a download session for an unlimited number of files, unlike the frequent interventions needed to manually copy files from CDs. In addition, you can have extended download sessions for large numbers of chips going on in the background constantly with almost no impact to your computer's performance. If there is any interruption in your internet connection during a download you will not lose files that were transferred before the interruption.

  • Can I run other applications while the Decode File Download Utility is running?

    Yes. The application places practically no load on CPU or RAM, and has minimal impact on write speed to your hard disk.

  • Can you tell me about the VeraCode Microbeads?

    The VeraCode technology is based on cylindrical glass microbeads measuring 240 μm in length × 28 μm in diameter. Illumina uses a proprietary technology to inscribe digital holographic elements within each bead. When a laser beam shines through the bead, the holographic elements diffract the light, creating a code image. Each different bead type contains a unique code that can be used to represent information such as the target of interest in multiplex assays. It can also be used to track critical information, including sample ID, laboratory ID, reagent lots, etc. The high-density codes (24 bit) offer a virtually unlimited number of unique bead types.

  • Can samples processed with the MCS3 reagent and the MCS4 plus RTE reagent be analyzed within the same GenomeStudio project?

    Yes, samples can be analyzed in a single experiment. We have observed high concordance between detected probes using MCS3 versus MCS4 plus RTE and have found the variability in raw signal intensity correlations between MCS3 and MCS4 plus RTE to be comparable to the variability between freshly made MCS3 and MCS3 nearing the end of its shelf life.  As is to be expected, signal intensity correlations and detected probe concordance decrease with input RNA quality.  

  • Did the catalog numbers of the kits that contained MCS3 change when the new MCS4 plus RTE formulation was released?

    No, all catalog numbers remained the same.

  • I have noticed double peaks in methylation value that seem to be related to Infinium I versus Infinium II probe designs. What is causing these double peaks?

    What you are seeing are histograms of the beta values in bins of 0.02 steps and categorized by Infinium design type. The difference in beta value ranges between the Infinium I and Infinium II assay design types cause the double peaks. In general,  the beta peaks at the extremes of Infinium I probes tend to be further out than the beta peaks for Infinium II.

    These differences do not affect the final analysis of the project. Individual CpG assays are not intended to be compared directly with other CpG assays, as each probe (or probe set for Infinium I designs) has different binding characteristics and behaves differently than any other probe or probe set. Rather, each assay is compared between 2 samples or sample groups (ie, in determining a relative rather than an absolute methylation value). For the Infinium HumanMethylation450 and Infinium MethylationEPIC array, we expect technical replicates to show > 98% correlation, and often observe > 99% correlation.

  • How do I determine which genes are accurately detected?
    Filter the genes using the Detection p-value. Setting detection at .99 (p value <0.01) means that there is a 1% false positive rate.
  • What is the minimum amount of total RNA I can use with the Ambion TotalPrep™ Kit?

    Illumina recommends a minimum of 50 ng total RNA. This generally produces 5-10 ug of amplified RNA, which should be sufficient for 10-20 arrays.

  • Can I still order individual components?

    Current kit configurations include all components, and are orderable via a single catalog number. See this web page for more information: http://www.illumina.com/pagesnrn.ilmn?ID=70

  • Are the data from GoldenGate Linkage V and/or Infinium HumanLinkage-12 projects compatible with linkage analysis software products on the market?

    The output genotyping files from the GoldenGate and Infinium Linkage Panels are compatible with most linkage analysis software on the market. Details about Illumina’s software compatibility with downstream linkage analysis software can be found at Illumina’s website on the illumina.Connect program page.

  • Does Illumina recommend any the commercially available methylation standard DNA: methylated, hemimethylated, or unmethylated?

    Illumina has not extensively tested any of the commercially available standards, and does not currently recommend any of them. In-house, Illumina uses standards generated in our own laboratory. Our methods for generating the standards are described in the following article:

    Bibikova M, Le J, Barnes B, Saedinia-Melnyk S, Zhou L, et al. (2009) Genome-wide DNA methylation profiling using Infinium assay, Epigenomics 1:177-200

  • What is the recommended operating system requirements for analyzing Infinium MethylationEPIC array data in GenomeStudio?

    Infinium HumanMethylation450 samples from different people.

  • Can FFPE DNA samples be used for the GoldenGate Assay for Methylation?

    While not specifically validated by Illumina Scientists, our commercial test sites observed good results with FFPE-derived DNA samples. Technical reproducibility is generally lower, with the r2 for Beta values ranging from 0.90 to 0.96.

  • Does DNA require quantitation after bisulfite conversion?

    No. However, the user guide provides directions for quantifying the DNA before bisulfite conversion.

  • What size DNA is recommended for the Infinium Methylation Assay?

    Similar to the Infinium genotyping assay, we recommend DNA that is sized ≥ 2kb, which can be assessed on an agarose gel.

  • Do I need to cluster all SNPs in the data set by the same algorithm and clustering options?

    No, you can use different algorithms and clustering options for different SNPs. The goal is to find optimal parameters for each SNP matching the biology of your samples.

  • Which metrics can be used to evaluate data in the Samples Table to identify poorly performing samples that should be excluded from the data?

    Samples can be evaluated and sorted by Poly Call Rate, Poly 10%, and Poly 50%. We recommend using the scatterplot function within the Samples Table to plot Poly 50% against Poly Call Rate to graphically visualize sample outliers.

  • How often is the content updated?

    We update content as often as warranted by Sanger miRBase. Our current content covers Sanger v12.

  • What is the major difference between the DBSCAN and OPTICS algorithm, and how do I choose between them?

    OPTICS is an acronym for "Ordering Points to Identify Clustering Structure". It is a sub-algorithm of DBSCAN, developed to be more robust to changes in input parameters. This trait makes OPTICS more suited for initial clustering. DBSCAN is an acronym for "Density-based Spatial Clustering of Applications with Noise". This algorithm is more sensitive to initial input parameters such as cluster distance. DBSCAN is more suited for differentiating clusters that are very close together, and should typically applied to SNPs for which OPTICS does not yield satisfactory results. 

  • When I downloaded the iControlDB program, I got the Final Case Report template files but the product I am interested in is not contained in the files. How can I get this file?
    Please contact TechSupport@illumina.com and they will forward you files for any recent products.
  • How does changing Cluster Distance in the Clustering Options dialog box affect my results?

    Cluster distance specifies the maximum distance that samples can be away from each other and still considered part of the same cluster. Increasing cluster distance will result in fewer clusters that are larger in size, while decreasing cluster distance will result in more clusters which are smaller in size. A cluster distance of 0.06 is typically a good starting point for initial clustering.

  • What do 'Positive Phenotype' and 'Negative Phenotype' mean?

    These are supplied by the submitter of the data. Samples may have been used as part of the case group in an experimental design (e.g., a sample from a diabetes patient). In this instance, "diabetes" may be reflected in the "Positive Phenotype" for this sample. These samples are still valuable to other studies that are not looking at this Positive Phenotype, and in fact it may be very valuable to obtain accurate population representation in control sets. Similarly,  "Negative Phenotype" is supplied by the submitter and is intended to indicate which phenotypes the sample has been screened for and are negative. Please note that some submitters have chosen also to use the Phenotype field to note the sources of the samples (e.g., blood, lymphocytes). In addition, the entry "HapMap" indicates HapMap samples from Illumina.

  • How do I know if the quality of my DNA is sufficient for Illumina microarray assays?

    For the Infinium Assay, we recommend fragment sizes of at least 2 kb. The GoldenGate Assay can tolerate shorter fragments (> 200 bp) and is more forgiving of degraded samples.

  • Is the GoldenGate Assay for Methylation protocol LIMS-supported?

    No, there are no current plans to incorporate LIMS support for this product.

  • How do you achieve multiplexing with the VeraCode Beads?

    Multiplexing is achieved by pooling together beads with unique codes. Illumina offers standard products to enable users to develop anything from a single-plex to several hundred-plex reactions per sample in a single well.

  • Is this product compatible with automation?

    This process is not currently compatible with automation.

  • Which SNPs would you recommend I use to design my GoldenGate OPA?

    We strongly recommend using GoldenGate validated SNPs or two-hit validated SNPs with SNP scores (design scores) of 0.60 and above.

  • Why is overall intensity low?

    This can be caused by a number of factors: Poor total-RNA quality. Check using an Agilent Bioanalyzer or Gel electrophoresis. Low factor setting. Increase the scanner factor setting. Expired or degraded reagents (e.g., Biotinylated UTP, Cy3, cleanup columns). Check the expiration dates on all reagents. Did not add the specified mass of cRNA to the array. Sample specific effects. Relatively few expressed genes (necrotic tissue). Stringency was too great in hybridization solution. Review the protocol for making up the hybridization solution. Temperature not set properly during hot-wash step. Review the protocol for making up the wash

  • How can I get GenomeStudio software?

    The software is provided without charge and can be downloaded from the Downloads page. 

  • Can genotyping projects that were opened and modified in the PC Module be imported back into the GenomeStudio Genotyping Module?

    No, a polyploid project (.pcm) can only be opened using the PC Module.

  • How do I use the Decode File Download Utility to download files for specific chips that are already in my lab without seeing such a long list?

    Click BeadChips by Barcode on the Main tab to open a text box. You may use the barcode scanner from any Illumina workstation to barcode-scan the chips into the system, or simply type them into the box. This feature only retrieves the files for the barcodes you specify.

  • How do I add more data (samples or lanes) to my GenomeStudio project?

    Create a new GenomeStudio project with all of the samples and lanes you want to include.

  • How is specificity achieved for mature miRNA over pre-miRNA?

    Specificity is achieved in the Extension step (in the Add MEL step) and cycling. The Extension step favors mature miRNAs because longer sequences will not achieve complete extension to the PCR primer portion of the Make CSP poly-dT primer to the same degree as mature miRNAs.

  • How often do you update your internal database?

    Updates coincide with dbSNP releases.

  • How do I find the region containing the greatest number of SNPs?

    In the Found Regions table, click the # SNPs column header to sort the number of SNPs found in each region.

  • What is the compelling reason for me to use this methylation tool?

    The Illumina GoldenGate Assay for Methylation is the first array-based platform capable of high-throughput and multiplexed measurement of DNA methylation status with single CpG resolution. 96 samples per plate with 1,536 separate reactions each = 147,456 quantitative DNA methylation measurements per plate. Flexibility in feature content: oligo sets can be customized and optimized. Entire process complete in less than one week from bisulfite conversion to data analysis.

  • What are the input requirements for the VeraCode GoldenGate Methylation Assay?

    > 500 ng for two single-use activation reactions, 45* µl at 50–400 ng/µl.

    *According to Zymo Bisulfite Conversion Kit D5001, D5002, D5004. Kit D5003 is not recommended because the elution volume is too large for this assay.

  • What if Illumina does not use all of the DNA?

    We destroy or return the remaining DNA to you at any end of the project at your option.

  • Can I use whole-genome amplified (WGA) samples with the Infinium HD Assay?

    Illumina does not recommend using WGA samples as input for the Infinium HD Assay because the first step in the protocol is a WGA step. Our scientists observed up to a 4% decrease in call rate during limited internal testing. The decrease in call rate varied depending on the specific sample and WGA method used. Additionally, any allelic bias present in the original WGA sample might be compounded by another WGA reaction.

  • Which genomic DNA storage buffer does Illumina recommend?

    TE buffer with a composition of 10 mM Tris, pH7.5; 1 mM EDTA.

  • Can the PC Module call genotypes?

    No, the PC Module performs cluster assignment, but does not call genotypes. This is because the assignment of genotypes polyploid species is highly dependent on the population and biology of the organism. Any downstream genotype assignment should be done with the biology and evolutionary history of the population taken into consideration.

  • Is automation available for the GoldenGate Assay for Methylation protocol?

    Yes, see the user guide for instructions on the automated protocol, robot use, and liquid-handling automation.

  • Which SNPs would you recommend I use to design my OPA?

    We strongly recommend using SNPs from these two categories: GoldenGate validated SNPs and two-hit validated SNPs with SNP scores (design scores) of 0.60 and above. We discourage use of SNPs scoring < 0.40 as they have a low likelihood of converting into successful assays and they have the potential to adversely affect the performance of the entire OPA.

  • Can I hybridize Cy3-cDNA to my BeadChip?

    The probe design and hybridization conditions were optimized for use with amplified cRNA. Varying the sample to cDNA would require re-optimization of every step of the protocol’s hybridization and wash conditions. Our protocol does not include a pre-block step of the microarray before adding sample. Adding Cy3-labeled sample would produce high background and poor results.

  • How are methylation levels measured by the BeadChip?

    A beta value (β) is calculated for each CpG locus.

  • Can I use DNA extracted from saliva?

    While we do not have experience using DNA extracted from saliva using the GoldenGate Assay, internal testing was performed using the Infinium I Assay with DNA extracted from saliva with a kit from DNA Genotek. The call rates and reproducibility from this test were > 99%. We have not tested kits for DNA extraction from saliva from any other source.

  • What are typical performance metrics of the SNP-Based Linkage IV Panel when my samples are evaluated by Illumina Genotyping Services?

    Key metrics of Linkage IVb mapping panel: 6000 validated SNP markers, 43% average heterozygosity in Caucasians, 38% in African Americans, 36% Asians, 99.6% reproducibility, 0.006% Mendelian inconsistencies, 99.8% genotype call rate, < 45-day turnaround from receipt of DNA. Performance metrics: Call rate, reproducibility, Mendelian inheritance rate, and heterozygosity are based on studies defined in Illumina's SNP-Based Linkage IV Panel: Design and Validation Technical Bulletin. Performance depends on the quality of the DNA samples.

  • Is there a change in price with the new MCS4 plus RTE formulation?

    No, the pricing remains the same.

  • Can I load a project that contains multiple manifests?

    No, KaryoStudio only accepts data from a single Infinium product at a time.

  • Does Illumina recommend specific bisulfite conversion kits?

    Yes. Illumina recommends the following of-the-shelf bisulfite conversion kits from Zymo Research.

    Catalog Number

    D5001    50 reactions (single-column format)

     

    D5002    200 reactions (single-column format)

     

    D5004    2×96 reactions EZ-96 DNA methylation kit (deep-well format)

  • Does iControlDB generate different report types other than the matrix format, forward strand report, because my Final Case Report is not in the forward strand orientation?
    No, not at this time.
  • Is there LIMS support for Infinium Methylation BeadChips?

    LIMS support is available for the Infinium HumanMethyation450 BeadChip and Infinium MethylationEPIC BeadChip.

  • Is requantification of DNA samples after bisulfite conversion recommended?

    If you use at least 250-1000 ng DNA for the bisulfite conversion, requantification is not necessary. It is critical to quantify the input DNA concentration with PicoGreen to make sure that you add sufficient DNA to the bisulfite conversion reaction. Bisulfite conversion renders DNA less complementary, so much of the DNA are denatured and more difficult to quantitate accurately.

  • Can I view data from more than one sample at a time in the ICB?

    Yes, data from a second or third sample can be plotted concurrently by selecting Settings | Trio View from the menu.

  • How are the VeraCode Carboxyl Beads used?

    Carboxyl beads have a carboxylated surface, enabling development of protein-based assays. To achieve multiplexing, pool together different tubes of uniquely coded carboxyl beads after immobilizing with proteins of interest.

  • How can I combine two regions in close proximity into one larger region?

    When you create a merged table, you can specify how close two regions must be in number of base pairs.

  • What is the difference between Call Rate and Poly Call Rate in the Samples Table of the PC Module?

    Call Rate is carried over from the Samples Table in the GenomeStudio Genotyping Module in which the original genotyping project (.bsc) was created. Entries in the Call Rate column do not change when SNPs are clustered in the PC Module. In contrast, the Poly Call Rate is calculated from clustering SNPs in the PC Module and represents the percentage of SNPs for which a given sample was assigned to a cluster.

  • What differences can I expect if I am analyzing a tumor sample versus a typical congenital sample?

    Depending upon the number of division cycles, a tumor sample typically has a large number of aberrations across the length of the whole genome. You will see many regions of loss and gain in copy number. cnvPartition will identify many found regions in these samples. Genotyping call rates in these samples may be as low as 60%. The percentage of defects in these samples can be as high as 50% (or even higher). Congenital samples tend to have fewer aberrations and may only have <10 large deletions and duplications. Genotyping call rates in these samples should be above 95% more likely to be 98% or greater. The percentage of defects in these samples should be less than 1%.

  • Does Illumina supply automated protocols for use with their gene expression products?

    Automation of the DASL® Assay is available. However, Illumina currently does not offer automation for the Direct Hyb protocol. Contact Ambion for information about automation of the TotalPrep protocol.

  • What is the MCS4 formulation change and reason?

    Illumina found that separating the enzyme from the buffer provides a minimum of a nine month shelf life from date of manufacture. The MCS3 had a four month shelf life. As a result, the MCS3 has been split into two tubes containing MCS4 (buffer) and RTE (Reverse Transcriptase Enzyme).

  • Can I sort the data displayed in iControlDB?
    Yes, you can simply click the header of any of the columns and it will sort that column for you.
  • Do you recommend an RNA purification protocol?

    Illumina does not recommend a specific RNA purification product. However, any product that yields pure, intact RNA of good quality that retains (at least) most of the small RNAs should work well with our miRNA assay. We have generated good data with RNAs extracted with the Ambion kit, Qiagen kit, Trizol, etc. NOTE: For any given study, it is ideal to isolate the RNAs using a single method.

  • Which parameters can be configured to manipulate the default clustering algorithms?

    Minimum Number of Points in a Cluster, Cluster Distance, and Maximum Number of Clusters in the SNP Table, which can be found in the Clustering Options dialog box.

  • What is the Diff Score and how does it relate to the p-value?

    The Diff Score is a transformation of the p-value that provides directionality to the p-value based on the difference between the average signal in the reference group vs. the comparison group. The formula is: DiffScore = 10*sgn(µcond-µref)*log10p; For a p-value of 0.05, DiffScore = ± 13; For a p-value of 0.01, DiffScore = ± 22; For a p-value of 0.001, DiffScore = ± 33 The p-value column is hidden by default. To display this column, use the Column Chooser.

  • Which assays and platforms are compatible with the PC Module?

    The PC Module is compatible with GenomeStudio Genotyping projects created from Infinium and GoldenGate genotyping assays run on the iScan/HiScan, BeadXpress and BeadArray Reader.

  • What is the median statistically significant, detectable fold change of the GEX BeadChips?

    1.35 fold

  • Can we use total-RNA extracted from blood?

    Yes, total-RNA extracted from blood has been successfully used with this product.

  • How should I quantitate my DNA before GoldenGate genotyping?

    We recommend using a method of direct quantitation specific to double-stranded DNA (dsDNA), such as with the PicoGreen reagent. Alternative indirect methods such as UV spectrometry/NanoDrop can incorrectly report the concentration of dsDNA in your sample when single-stranded DNA, oligonucleotides, RNA, and/or proteins carried over from DNA extraction are present in solution.

  • What if I don't have enough template DNA?
    The library process can be optimized to reduce the amount of DNA required. For some applications such as ChIP-Seq where DNA amounts might be low, steps such as size selection on a gel can be skipped. Instead, end-repaired DNA fragments can be ligated, purified by a column clean-up step that removes primer-dimers, and then quantitated and directly introduced to a flow cell for cluster amplification. For genomic DNA from small samples, fragmentation can be optimized to increase the number of DNA fragments that are within the target size range. If these DNA fragments are within a tight size range, then the agarose gel step can be eliminated.
  • Where can I find a marker list of all the CpG sites on the array?

    The manifest for the array can be found in the Downloads section of the BeadChip support page.

  • Which normalization method does Illumina recommend for miRNA data?

    If technical replicates are used within a SAM or across multiple SAMs, Illumina recommends our newest Sample Scaling normalization, followed by Quantile normalization. If there are no technical replicates, we recommend Quantile normalization.

  • Where can I find information about integrating probes on a methylation BeadChip with probes on the HumanHT-12 Expression BeadChip?
  • Why doesn't the Infinium HumanMethylation450 BeadChip contain specific distances from the CpG site to the transcriptional start site (TSS) of the listed genes?

    The array density is much higher in the Infinium HumanMethylation450 BeadChip. There are a large number of CpGs for which multiple transcripts are listed and for which the CpG site can fall into different annotation categories. Consequently, the task of calculating TSS distances would be a large bioinformatic undertaking, and the numbers would have to be modified every time the genome was updated. Additionally, the column "UCSC_RefGene_Group" has content about the location of the CpG relative to specific regions and features of the associated genes, which is in many ways richer than the simple distance relative to transcriptional start site.

  • How large is the output file for an Infinium MethylationEPIC array?

    On average, ~2.75 Gb with JPEG files, which is the default setting.

  • Should QC be performed after the in vitro transcription reaction (IVT)?

    Illumina recommends quantitation of amplified RNA by fluorometry using Molecular Probe's RiboGreen® Reagent. We also recommend additional qualitative analysis with the Agilent Bioanalyzer or by electrophoresis through agarose gel. A less precise RNA quantitation method is the measurement of A260 absorbance with a spectrophotometer.

  • How well do SNP genotypes called using the GoldenGate Assay compare to those called using the Infinium Assay?

    Over 65% of the SNPs genotyped by the HapMap project phase I were done using the GoldenGate Assay. The SNP content on the HumanHap300 Genotyping BeadChip was chosen from HapMap phase I validated SNPs. Our scientists found a high conversion rate of HapMap-validated SNPs.

  • Can I import raw intensity files (idats) into the PC Module?

    No, it is not possible to generate a new project in the PC Module directly from idats. The polyploid workflow requires to first generate a genotyping project in the GenomeStudio Genotyping Module from the idats. The genotyping project (.bsc) can then be opened in the PC Module for polyploidy clustering.

  • Is this product robust against degraded RNA?

    Yes. Excellent data has been generated using RNA extracted from FFPE tissue samples. Technical reproducibility is highly similar to intact RNA. In addition, Illumina has profiled artificially-degraded RNA samples (95°C heat for 30 minutes). Excellent reproducibility was obtained with these samples. Further, the profiles generated with these samples are comparable to those generated with corresponding intact RNA samples.

  • Where can I find the probe coordinates for the probes in the Human and Mouse MAPs?

    Illumina has the probe coordinates (i.e., chromosomal location information) for most of the miRNA, including those not in the Sanger miRBase. You can find this information in the *.bgx file. See http://www.switchtoi.com/annotationfiles.ilmn for more information.

  • For custom Infinium can I assay SNPs that are next to each other (i.e., < 60 bp apart)?

    Yes. In the Infinium Assay, the effect of underlying polymorphisms is not critical to overall performance.

  • What do the column headers GeneSymbol, GID, and Accession reference on the gene list for Illumina's standard BeadChips, and where do the numbers come from?

    Descriptions for all of the column headers can be found in the document "Bead Manifest Field Descriptors" located on the documentation CD included in the startup kit.

  • Can I use Formalin-Fixed Paraffin-Embedded (FFPE) samples in the GoldenGate Assay?

    The GoldenGate Assay can tolerate relatively short stretches of target DNA (> 200 bp) and can be quite tolerant of degraded FFPE samples. Internal experience with FFPE samples used with the GoldenGate Assay indicates that high-quality genotyping data can be obtained. Decreased call rates from FFPE samples compared to genomic DNA samples may be observed, but the decrease in call rate depends on the level of sample degradation.

  • Must the Ethnicity field comply to any specific requirements?

    No, this field is optional, or can be completed with any user-defined categories. Our intent is to provide the most comprehensive and varied dataset, therefore we do not limit ethnicities at this time to certain entries.

  • What does OPA stand for?

    Oligo Pool All. An OPA contains all of the assay-specific or SNP-specific primers.

  • What is Illumina's current regulatory status?

    For information on Illumina's regulatory, quality, and instrument safety certifications, including ISO certifications, go to the Regulatory and Quality Information page.

  • What technologies does Illumina use in its Genotyping Services business?

    We have created a highly multiplexed SNP genotyping system using our proprietary BeadArray technology. In addition, we have developed a fail-safe sample-tracking LIMS system to ensure error-free processing.

  • What criteria should I use for LOH/CN analysis?
    You should consider density, physical spacing, and Minor Allele Frequency (MAF). Loci with high MAF (~ 0.5) may be more informative than rare polymorphisms for this type of analysis. The control set must match the test sample population (MAF). You must have control samples with minimal amounts of chromosomal aberrations.
  • How many SNPs can be added to the HumanHap550 Plus Genotyping BeadChip?

    A maximum of 121,600 bead types (attempted SNPs) can be added to the HumanHap550 Genotyping BeadChip.

  • Is it recommended to normalize mRNA and miRNA together in the same project?
    Illumina recommends normalizing mRNA data separately from miRNA data.
  • What correlation is expected from technical replicates?

    At 100- 200 ng total RNA input, R2 = 0.97 is expected.

  • Can I use the Zymo EZ DNA Gold kit for bisulfite conversion?

    Yes, you can, but you must use this kit for your entire project. That means you cannot use a mixture of samples that have been converted with different kits on the same arrays, or within the same project.

  • Is two-round amplification an option?

    Illumina has not tried any two-round amplification kits. However, some customers have reported successful use of two-round IVT kits with Illumina's gene expression arrays.

  • Does Illumina provide annotation (gene, distance) for the rsSNPs?

    Yes, annotation for rsSNPs is available upon request.

  • Why are both Infinium I and Infinium II probes used for this BeadChip? Does the design affect the data output / quality?

    For more information, see the Infinium Methylation Coverage Technical Note.

  • What are the advantages of the Infinium Linkage-12 panel and GoldenGate Linkage V Panels?

    Infinium Linkage-12 uses the powerful PCR-free Infinium Assay chemistry and protocol at a very attractive price point (the most cost-effective on the market for linkage analysis). GoldenGate Linkage V uses the proven GoldenGate Assay technology and is the linkage product of choice for degraded samples such as FFPE and WGA samples.

  • Can I study copy-neutral LOH with KaryoStudio? Can cnvPartition do this?

    Yes, it is possible to study copy-neutral LOH with KaryoStudio and cnvPartition. To ensure that you have the latest version of the cnvPartition algorithm, contact Illumina Technical Support (technicalsupport@illumina.com). Note that the amount of copy-neutral LOH present across a typical genome can be quite large. Illumina recommends setting the filter to a large size in order to limit the number of regions found by the algorithm.

  • Can samples be stored after DNA activation?

    Once precipitated and resuspended as per the user guide, DNA can be stored at 4°C overnight or -20°C for up to six months.

  • Can the Infinium HumanMethylation450K BeadChip or the Infinium MethylationEPIC BeadChip distinguish 5-hydroxymethylcytosine from 5-methylcytosine?

    Illumina has not validated the array for 5-hMc. However, publications have used the Infinium HumanMethylation450K array for 5-hMc analysis and it is possible that this protocol will works on the Infinium MethylationEPIC array.

    For more information, see Nazor, Kristopher L., et al. "Application of a low cost array-based technique—TAB-Array—for quantifying and mapping both 5mC and 5hmC at single base resolution in human pluripotent stem cells." Genomics 104.5 (2014): 358-367.

  • Should I use miRNA-enriched samples?

    Illumina recommends using total-RNA. The enrichment process seems to reduce the precision of the assay, increases the noise due to technical variation, and requires more starting material.

  • Can I use BeadStudio to download data from the database?
    No. Only the Illumina iControlDB - Client program can be used to download data from the database.
  • What’s new in the GenomeStudio microarray modules (GT, GX, M, and PT v1.0)?

    Please see the GenomeStudio Software 2009.2 Release Notes, available in iCom and the GenomeStudio Portal, for details.

  • How can I obtain KaryoStudio?

    Contact your account manager for information about getting new versions of KaryoStudio software.

  • Is performance impacted based on the RNA isolation technique used?

    Illumina has tested only RNA isolated using RNEasy® from Qiagen®, one of the most frequently used methods. We do not anticipate a major impact on performance with other appropriate, well-established isolation techniques.

  • What is a beadpool manifest?

    Also referred to as a SNP Manifest, this is a file containing the SNP-to-beadtype mapping, as well as all SNP annotations. For the Infinium assay, this is a *.bpm file in binary format.

  • How many samples should I run for clustering on custom Infinium content?

    Illumina recommends that you run at least 100 samples including both replicates and trios.

  • Can I assay all SNP classes ([A/T], [C/G], [A/C], [A/G], [T/C], [T/G]) with custom Infinium content?

    Yes, all biallelic SNPs can be assayed using a combination of Infinium I and II probe designs.

  • How do I find the largest aberration in my samples?

    Click the Size column header to sort the data in the Found Regions table.

  • I am getting an error when running the Upload Test Application. What should I do now?

    Please save a screenshot of the error, along with any logs that were created from the Test Application. If the Test Application has completed, these files are saved in the same folder on your computer that the program is in. Contact TechSupport@illumina.com.

  • What are the advantages of using the Decode File Download Utility instead of the CDs shipped with my BeadChips?

    This software allows you to download an unlimited number of files at one time automatically without physically handling the CDs. This can be done overnight, over a weekend, or one BeadChip at a time, and you can easily select only those files that have not previously been downloaded. You can also select BeadChips based on purchase order (PO) numbers, or you can barcode-scan one or many BeadChips as you receive them. Using this software will likely save you many hours of hands-on time moving and copying files from CDs.

  • Do I need to remove the ribosomal RNA prior to labeling?

    It should not be necessary to remove rRNA before labeling. The ribosomal RNA does not get amplified in the protocol and should represent a very small percentage of the final product.

  • How do I get the Decode File Download Utility?

    You can download the software free of charge from the Illumina iCom site. After logging into the system, click Downloads | Software. If you cannot access iCom or see the software in your list of downloads, please contact Customer Service for assistance.

  • Can I get bead-level data?

    Yes, bead-level data is available. Please contact Technical Support for assistance with this feature.

  • What are .idats?

    An *.idat file is an intensity data file. It contains statistics for every bead type on your BeadChip. The statistics in an *.idat file include the number of beads, the mean, and the standard deviation for each color sample. There is one *.idat file per sample per channel.

  • What steps are automated in the GoldenGate Assay for Methylation protocol?

    The automated processes specific to GoldenGate Assay for Methylation are:

    • Make BCS/BCM
    • Precipitate BCS/BCM
    • Resuspend BCS/BCN
    • Make ASE

    The remaining steps are identical to genotyping using the GoldenGate Assay.

  • For the bisulfite conversion, do I need specific hardware?

    Perform this reaction in a thermal cycler to keep the temperature stable. Any PCR machine can be used for the bisulfite conversion reactions, but this reaction must be performed in the pre-PCR room.

  • Do I need to order the robot upgrade to automate the GoldenGate Assay for Methylation?

    Automation for the GoldenGate Assay for Methylation is processed using a new robot control application bundled into the GTS robot control software. Please contact Technical Support at techsupport@illumina.com for the latest version of robot control software.

  • Can I import data from non-Illumina platforms into GenomeStudio software?
    No. GenomeStudio software is intended for use only with Illumina data.
  • Is there a standard panel available for Methylation analysis on the Illumina platform?

    Yes, our first standard methylation panel is called "GoldenGate Methylation Cancer Panel I". The catalog number for this panel is GM-17-211. It allows the researcher to interrogate 1,505 CpG sites carefully selected from 807 genes where 230 genes contain one CpG site per gene, 462 genes contain two CpG sites and 115 genes have three or more sites. Please refer to the gene annotation text file on our product web page for more information.

  • Will my data be kept confidential?

    Yes. All data generated by us will be kept strictly confidential. The data belongs solely to the submitting investigator or company. Illumina will only accept anonymized samples with no patient association.

  • Is there a demo data set for the Infinium MethylationEPIC array?
  • Why is Illumina's Gene Expression assay called Direct Hyb?

    Direct Hybridization (Direct Hyb) refers to the direct hybridization of probes on the array to the complementary cRNA molecule in your sample. This is in contrast to Illumina's flagship GoldenGate® Genotyping Assay, which incorporates a unique oligonucleotide onto the sample sequence. This unique oligonucleotide then hybridizes to the complementary probe on the array.

  • How long are decode files available via the Decode File Download Utility?

    Files appear on the server approximately 24 hours after they are shipped. We guarantee that files remain on the server for at least 30 days from the time they are shipped. Downloading the files changes their status in the database so you can filter them, but you can download files as many times as you like as long as they remain on the server.

  • There is a product listed in iControlDB called “11218540_WGGTManifest.csv”. What product is this?

    This is the HumanHap550v1 product.

  • How much DNA is required for the Infinium Methylation Assay?

    Going into the bisulfite conversion, at least 250 ng is required for the manual protocol, and at least 1000 ng is required for the automated protocol. Using less than the recommended amount does not guarantee R2 performance to meet specifications.

  • How do I know if my network will allow me to upload data to the Illumina iControlDB?
    We have created an application that we strongly recommend running before you attempt an upload. This will allow your network security settings to be examined, and for you to know if the security and firewalls will allow for uploading. This Upload Test App is available for download from the Illumina website.
  • Where can I get the OMA manifest file for the GoldenGate Methylation Cancer Panel I?

    Log onto iCom, our eCommerce site on Illumina's website, and click Downloads. All of the product support files, including the OMA manifest, can be downloaded from this location.

  • Is there an effect on the data from the MCS4 formulation change?

    Our goal is to provide as consistent performance as possible and as with any molecular biology technique, however there may be some additional variation. See the Tech Note that describes the results.

  • What are the input DNA requirements for the GoldenGate Genotyping Assay (standard and VeraCode)?

    250 ng, 5 µl at 50 ng/µl.

  • I uploaded my data, and it has been reviewed and released by Illumina. Why are some of my samples missing?

    We review the data for many criteria before releasing to the database. Any samples that do not pass our call rate criteria are not included in the released data. Additionally, there may be user-entered information (e.g., ethnicity or positive phenotype) that is unclear or not compliant with the Health Insurance Portability and Accountability Act (HIPAA). These samples will not be included in the released data.

  • How many bases constitute a region under a peak?

    See the GenomeStudio ChIP Sequencing Module User Guide available in the GenomeStudio Portal for information.

  • Is miRNA compatible with LIMS?

    Illumina does not currently offer LIMS support for miRNA.

  • Can I download data from different sample types such as the HumanHap550 and the HumanHap300 in the same report?

    Illumina strongly advises against this, as each product contains a different SNP list. Instead, download samples from a single array type and version number in each session.

  • I tried to cluster a set of SNPs, but no clusters are visible in the SNP Graph. What went wrong?

    SNPs are only clustered for samples selected in the Samples Table (marked in blue). If no samples are selected in the Samples Table, SNPs are clustered for all samples in the Samples Table (except non-excluded samples). Thus, if you wish to cluster SNPs for all your non-excluded samples please make sure that no samples are selected in the Samples Table at the time of clustering (e.g. by clicking onto an area in the SNP graph).

  • Are custom panels available for ordering?

    Custom designed assays for Methylation profiling are now available to provide the ultimate flexibility of content! Please contact your Regional Account Manager to discuss your plans for custom designs.

  • What does "design score" mean? What is the relationship between design score and assay conversion?

    A proprietary algorithm gives a score range from 0 to 1. It is intended as a metric to assist you in selecting SNPs. The higher the score, the more likelihood an assay will convert. This does not guarantee an individual locus with a high score will convert, nor that a low-scoring locus will fail.

  • I created a query using the 'Edit Query' button, and then clicked 'OK'. Why didn't my list of samples change?

    The Edit Query function only sets up the query to be performed. After the query is set, click Perform Query to update your results.

  • Can I run GenomeStudio software on a VMware virtual machine?

    Yes. However, this is not officially supported by Illumina due to very slow performance.

  • How can I zoom into a specific region of interest?

    You can zoom into a specific region of interest by clicking on the specific aberration that is shown within the Found Regions table. After this is done, a closer view of the aberration of interest is shown in the chromosome viewer.

    Alternately, use the zoom functions available in the toolbar or drag and stretch the red box on the ideogram to zoom in for a closer view of your data.

  • cnvPartition did not find the exact breakpoint of my found region. Is there a way to adjust this?

    Yes, you can adjust the start and stop positions of a found region by right-clicking the found region and adjusting the genomic positions. You may need to use the zooming and panning functions to determine the exact position to which you would like to adjust the found region.

  • How is the beta value calculated?

    The beta value (β) uses the ratio of intensities between methylated and unmethylated alleles to estimate the methylation level of the CpG locus.

  • What cnvPartition confidence values cutoffs are recommended?

    Users should set confidence value thresholds based on the type of analysis they are doing, and their individual goals for the results. In general, confidence values of 35 or higher are typically found to represent the most reliable results from this algorithm. Values lower than 35 may represent false-positives and should be validated by eye in the Chromosome Browser. If you would like to be a bit more conservative in your found regions, you should begin by looking at regions with confidence scores of 50-100, or even greater.

  • What is Illumina's Genotyping capacity?

    Our genotyping facility was designed as an ultra-high throughput, scaleable service business. Currently, we have capacity to perform well over 1,000,000 genotypes per day.

  • What if I have further questions on the MCS4 plus RTE formulation change?

    Documentation pertaining to this change is available on MyIllumina, particularly the Bulletins section on your MyIllumina home page.  You can also contact Illumina Technical Support.

  • What DNA quantification method does Illumina recommend for Infinium arrays?

    For Infinium arrays, Illumina recommends use of PicoGreen reagent for DNA quantification. UV-based methods may can overestimate DNA concentration by 2–10 fold.

  • What do the designators "A", "S," "I," and "M" in the manifest files mean?

    A = All isoforms. The probe is designed to hit all splice isoforms of a gene.

    I = Isoform specific. The probe is designed to hit a specific splice isoform of a gene, for which multiple isoforms are known to exist.

    S = Single isoform. The gene has only one known splice isoform and our probe hits it.

    M = Multiple isoforms. This gene has multiple isoforms. The probe targets more than one and fewer than all of them.

  • Is there LIMS support for FFPE samples on MethylationEPIC BeadChips?

    LIMS support is not currently available for FFPE samples on the Infinium MethylationEPIC BeadChip.

  • What quality control measures are in place before, during, and after the genotyping services?

    Throughout our process, we have developed quality control procedures to ensure you with the highest quality of data possible. These include (1) quality control and quantification of incoming DNAs, (2) multiple internal controls built into each genotyping assay, (3) barcoded labeling of sample plates, (4) error-free sample tracking under active database control to provide error-free handling of samples, assays and data (PosiTrack), and (5) statistical measures of success for assay development and genotyping confidence scores (GenCall).

  • How do I find the smallest aberration in my samples?

    In the Found Regions table, click the Clear filters button to allow aberrations as small as 1 kb to appear in your data. Then sort the data in the by clicking the Size column header.

  • Can LOH (Loss of Heterozygosity) / CN (Copy Number) analysis be performed with GoldenGate genotyping?

    LOH and CN changes have only been validated using the Infinium Assay. We do not support interpretation of LOH/CN data on the GoldenGate Genotyping Assay. Custom assay pools (OPAs) must provide high density and, therefore, high resolution to generate meaningful data for LOH and CN analysis.

  • For publication purposes, how should I cite that I have uploaded or downloaded samples from iControlDB?

    Please reference the Illumina iControlDB web page http://www.illumina.com/science/icontroldb.ilmn and/or Illumina, Inc., San Diego, CA, whichever is the preferred format of the journal to which you are submitting.

  • Which products are available for online downloads using the dmap Download Utiltiy?

    Decode files for all BeadChip products except the HumanHap300 v.1.0 Genotyping BeadChip, Expression BeadChips ordered in 1- or 2-packs, iSelect Custom Genotyping BeadChips, and Sentrix Array Matrices (SAMs) are available using this software.

  • Do I need any special approval to upload samples to the Illumina iControlDB?

    Yes, you need to supply an approval letter from your Institutional Review Board (IRB) or equivalent review committee (for international customers) before you begin the upload process. Please see the iControlDB support documents for additional information about the IRB documentation required.

  • What are the key differences between the protocols for genotyping using the GoldenGate Assay and the GoldenGate Assay for Methylation?

    DNA samples require bisulfite conversion prior to use in the GoldenGate Assay for Methylation but not for genotyping using the GoldenGate Assay. There are four oligos, two allele-specific oligos (ASOs), and two locus-specific oligos (LSOs) required for each assay locus in the Oligo Pool for Methylation Assay (OMA): one probe pair for the methylated state of the CpG site and a corresponding pair for the unmethylated state. The GoldenGate Assay for Methylation is not LIMS-supported. Analysis of methylation data requires the BeadStudio Methylation module.

  • What types of assays can be developed with the VeraCode Beads?

    The glass surface of the VeraCode beads make them ideal for a number of bioassays, including genotyping, gene expression, methylation, and protein-based assays. Solution-based assays, in conjunction with microarrays, can also be developed.

  • Is the Infinium Methylation Assay a one-color or two-color assay?

    This is a two-color assay.

    For the Infinium HumanMethyation27 BeadChip assay, which is based on Infinium I Assay designs, the color incorporated depends upon the base preceding the CpG locus being queried. This can be either green or red.

    The Infinium HumanMethylation450 BeadChip assay includes Infinium I and Infinium II study designs.  In the latter case, a single base extension from the 3' end of the probe sequence (which is one base upstream of the query base) will result in either a red or green signal depending on whether the query site was unmethylated or methylated.

  • How does KaryoStudio determine which aberrations go into a report?

    After you set how many aberrations should go into the report, KaryoStudio shows the aberrations based upon descending size. Only found regions with a checkmark in the Found Regions Table are displayed in the report.

  • Can I adjust the settings for cnvPartition?

    Some settings for cnvPartition can be adjusted with a supplied configuration file. For information on adjusthing the configuration file, refer to the algorithm release notes. For more information on this algorithm, see the CNV Algorithms Technical Note.

  • Is Globin reduction recommended?

    There is no need to perform Globin reduction. It is likely that doing so could introduce additional experimental variation.

  • Is the miRNA product a one-color or two-color assay?

    Illumina's miRNA product is a single-color assay.

  • What is "information content" and why is it important for a linkage study?

    Information content is a measure of how informative a marker or map of markers is in a collection of pedigrees in order to extract the maximum amount of inheritance information for a linkage analysis. Information content is a function of marker heterozygosity and the number of meioses in the genetic study. For multi-point linkage analysis, information content is also a function of marker density and spacing. It is important to have high information content throughout the genome for genome-wide searches for disease susceptibility loci or other traits so that regions of no linkage can be excluded, regions of significant linkage can be detected, and the linkage interval can be accurately defined.

  • How is the study number determined for the samples I have uploaded?

    The study number assigned to your samples is the next study number available in sequence.

  • Is there a GenTrain and/or cluster file for Infinium Methylation Arrays?

    Since Infinium Methylation arrays are designed to compare relative methylation levels between two samples or sample groups (such as normal versus tumor, or pancreas cells versus liver cells), there is no GenTrain and/or cluster file for this product. It is similar to doing a paired-sample analysis.

  • What are the major differences between the miRNA and DASL protocols?

    There are four new reagents, Polyadenylation Single (PAS), cDNA Synthesis Single (CSS), miRNA Assay Pool (MAP) and Single-Color Master Mix (SCM). There are also two new steps in the protocol: "Make PAP" for the polyadenylation and "Make CSP" for cDNA synthesis. In addition, the "Make ASE" and "Cycle PCR" steps are modified.

  • How can I visualize my results and compare them to GEX data?

    miRNA data can be combined with mRNA gene expression BeadStudio projects (Whole genome or DASL) in BeadStudio Gene Expression v3.2 or later. One tool to help visualize positive and negative correlations is hierarchical clustering w/ absolute correlation.

  • How can I obtain new versions of cnvPartition?

    Updated versions can be obtained through the GenomeStudio Portal or by visiting http://www.illumina.com/iconnect.

  • Can I adjust what is in the Known Regions list?

    Yes, this can be adjusted using Microsoft Excel. Information in any of the rows can be edited or deleted, or new rows can be added; however, no columns can be deleted. You can also create an entirely new known regions file and load it using the Filter Table interface. For more information about adjusting or creating new Known Regions lists, see the User Guide.

  • Can I generate a call report?

    A call report can only be generated from the GenomeStudio Genotyping Module.

  • What is the Polyploidy Clustering (PC) Module?

    The GenomeStudio Polyploidy Clustering Module (PC Module) can identify clusters for samples where the standard diploid clustering algorithm is inappropriate or not useful, such as for polyploidy organisms like wheat and potato.

  • What standard panels are available for GoldenGate genotyping?

    LinkageIVb, Mouse LD Linkage, Mouse MD Linkage, MHC Panel Set, Cancer Panel, DNA Test Panel

  • What is the best way to view the genes in a specific region of interest?

    Gene information is preloaded into KaryoStudio and displayed in the IGV or in the Genes column of the Found Regions table. In addition, you can use the link to the UCSC Genome Browser, the Database of Genomic Variants, and DECIPHER; all of which provide additional gene information.

  • How can I be sure we are targeting only miRNA in the total-RNA?

    This is achieved by two-step discrimination (1) sequence hybridization - the specificity of the miRNA specific probe which targets the pre and mature miRNA species, and (2) enzymatic primer extension - to enhance the discrimination between members of miRNA families and between miRNA and other similar sequences in the total-RNA (e.g., mRNA targets). Illumina has obtained very similar expression profiles with total-RNA and enriched small RNA species, suggesting that cross-hybridization (if any) from the total-RNA is minimal.

  • What do the failure codes indicate in my ADT results file?

    Critical Failures (undesignable):

    • 101 = Flanking sequence is too short.
    • 102 = Polymorphism or sequence formatting error. Possible causes:
      • Check polymorphism format: SNP => [X/Y], INDEL => [-/XYZ], CpG => [CG]
      • More than one set of brackets in sequence
      • Missing brackets around polymorphism
      • SNP alleles not separated by a "/"
      • Spaces found in submitted sequence
    • 103 = Top/Bot strand cannot be determined.
      • Low sequence complexity
    • 104 = Polymorphism is not appropriate for Illumina platform. Possible causes:
      • Tri- or quad-allelic SNP
      • Contains characters other than A, G, C, or T
    • 105 = Polymorphism is located on the mitochondrial genome. Mitochondrial polymorphisms are not recommended for Golden Gate oligo pool.
    • 106 = Degenerate nucleotides in assay design region.
      • W, R, S, N, etc.
    • 107 = SNP sequence not found.
    • 108 = Final score falls below assay limit.

    Warnings:

    • 301 = Polymorphism in duplicated/repetitive region.
    • 302 = Tm outside assay limits.
    • 304 = There are known SNPs within the probe region. See Underlying_SNP column for details.
    • 340 = Another polymorphism in this list is equal to or less than 60 nucleotides away.
    • 360 = Low score warning.
    • 399 = Multiple contributing issues.
    • 601 = Potentially non-specific against the genome.



  • How did Illumina determine the chromosomal location for the potential/putative miRNAs?

    To determine the chromosomal location for the potential/putative miRNAs, Illumina used the following procedure:

    1. Select 100% hits (of genome BLAST) for each mature miRNA sequence (if we don't get 100% match, a value of "zero" is shown in the bgx file)
    2. For each hit select the upstream and downstream pre-miRNA sequence (i.e., 5'-nnn-miRNA-nnnnnnnnnn-3' and 5'-nnnnnnnnnn-miRNA-nnn-3')
    3. Generate mfold structures for both upstream and downstream pre-miRNA sequences
    4. Analyze each structure (if the pre-miRNA will fold into the stem-loop structure)
    5. Filter out structures that are below the score threshold (estimated from Sanger training set)
    6. Select the structure with the best score for each blast hit site

    When multiple 100% hits are found in the genome for one particular mature miRNA sequence, the chromosomal coordinates are listed by the score, i.e., the location with the best score is listed first.

  • What is the best way to ensure that my samples have been processed and analyzed without issues?

    KaryoStudio provides several items for you to examine when you are QCing data. The LogRDev metric provides a measure of noise in the intensity data, and is essentially a measure of standard deviation of Log R values across the autosomes. The "percent aberration" metric is a sum of all of the found regions in a sample divided by the entire length of the genome. In a blood sample, where you expect to have little to no aberrations, you will see a very small (<1%) measure for % aberration. In cases where you have a higher number, it may indicate a sample processing issue. Both of these metrics can be impacted by real biological variation in samples, so they should be examined holistically while taking into account the data viewed in the IGV.

    For specific troubleshooting issues and access to the controls dashboard, load your data into GenomeStudio, if you have access to that software. Otherwise, contact technical support with any additional questions on troubleshooting your data.