Due to software performance improvements, it takes less time to process genotyping data using GenomeStudio software than it does using BeadStudio.

Yes, they are necessary to remove the rRNA.

No specific controls are included with this kit. Illumina does recommend using the PhiX sequencing control (sold separately), which is necessary for calibrating the instrument for each run. For sample prep level positive control, we recommend using the Universal Human Reference RNA from Strategene.

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.

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.

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, dif

This protocol can safely be stopped after the ethanol precipitation or any Qiagen purification step. The samples should be stored at -20°C.

Genotypes generated by iControlDB are in the matrix format, and are forward strand reports.

Yes, BeadStudio projects from v2.1.12 can be opened in v2.3.41. However, changes made in v 2.3.41 may not necessarily be saved if the project is then opened again in v2.1.12.

Yes, v2.1.12 cluster files can be used in v2.3.41.

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.

Yes, the user guide has step-by-step instructions for loading gene expression data from DASL or Illumina genome-wide gene expression experiments.

Yes. GenomeStudio can display data from non-human genomes.

Yes. Select the f() icon from the menu and devise a calculation within an array. The column/calculation will appear in all displayed arrays.

No. If you want to view data from BeadStudio ChIP sequencing projects, you must recreate your ChIP sequencing projects using the GenomeStudio ChIP Sequencing Module.

Yes. The output files from the GoldenGate and Infinium platforms, through BeadStudio software, can be combined in the customer’s database such as Access for genotype analysis. There is a 93% overlap of markers between the GoldenGate and Infinium Linkage panels. Genotypes for samples run separately on the GoldenGate and Infinium platforms can be analyzed in one data set for the overlapping markers.

One OMA can be used per analysis. However, a single BeadStudio project can contain multiple analyses, and users can set up separate analyses using additional OMAs for the same samples.

The Beta value is unique to the Illumina platform and can range from 0 to 1, with 0 indicating no methylation and 1 indicating complete methylation at a given locus. Importantly, the Beta value reports on the intensity ratio between methylated- and nonmethylated-specific assays for each CpG site. Since assay performance can vary between loci, this ratio does not consistently report percent methylation.

No. However, you can export the data table in *.txt, *.csv or *.xml format, and then work with your data files in third-party software applications.

Yes. However, it is very important to design the chips with a sufficiently high density of SNPs to detect the changes of interest. Contact your local Application Scientist or our Technical Support team for more information.

mRNA-Seq libraries are built with paired-end adapters and amplification primers. They can be run on paired-end flow cells, and two paired runs of sequence data will be generated. However, these paired sequences can span unknown splicing junctions, which complicates alignment to a reference transcriptome. For this reason, Illumina does not support this application. However, users with bioinformatics experience can create their own alignment methods to handle these paired reads.

We strongly advise against doing this, as they contain different SNP lists. We recommend only downloading samples from the same array type and version number.

The BeadStudio Methylation module allows users to export data tables for analysis with third-party software applications.

Yes. Click the Export Data icon to export all visible columns of both quantitative and qualitative data.

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

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.

No. GenomeStudio software is intended for use only with Illumina data.

No. BeadStudio software is intended for use only with Illumina data.

The BeadStudio Methylation module has a column import function that allows users to import customized data columns into existing projects. Please refer to the user guide for step-by-step instructions for importing data columns into your project.

Yes. However, you cannot create a GenomeStudio ChIP sequencing project and open it using the BeadStudio ChIP Sequencing Module.

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.

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.

Yes. After initial hybridization store the INT plate at -20°C. For re-hybridization, fully thaw the INT plate at room temperature, pulse centrifuge the plate to 250 xg, and then place the plate in a pre-heated oven at 60°C for 10 minutes. Proceed according to the Experienced User Cards to set up the remainder of the hybridization to a new Sentrix Array Matrix or BeadChip.

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

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

Yes. If you right-click on the bar plot, you can save the image in various formats.

Yes. Right-click on the scatter plot, select Copy Image, and paste the image into another application.

Yes, you can simply click the header of any of the columns and it will sort that column for you.

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

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

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.

No. Only genotype information is contained within the iControlDB at this time.

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.

No. Only the Illumina iControlDB - Client program can be used to download data from the database..

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.

The use of degraded RNA will have a large effect on the performance of the assay. One of the first steps in the process is the purification of poly-A mRNA using a poly-T capture step. If the RNA is degraded, the mRNA that is captured at this step will not be full length, and thus will not be able to give full-length cDNA products after random priming. When we use high quality total RNA, we are able to produce even, end-to-end coverage of each mRNA molecule present in the sample. However, if the RNA is degraded, you will probably observe a noticeable 3'- to 5'-bias in the number of reads observed for most transcripts. If you do not have a choice, and the best you can do from your system is degraded RNA, you can certainly still use this process to create libraries for sequencing on the Genome Analyzer. The more the RNA is degraded, the stronger the 3'-bias you will see in the sequencing. For this reason, when trying to compare expression levels across many samples, it is important to compare counts only from samples that have a similar RIN (RNA integrity number, from the Agilent Bioanalyzer) in order to give the most quantitative results.

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.

Illumina has tried this protocol on FFPE-derived total RNA and it works well. The poly-A purification step is still very efficient for removing the ribosomal RNA in the sample, and the highly degraded transcripts that are present are still purified by binding to the Poly-T beads. However, as described above, the cDNA fragments created from FFPE will be only as long as the length of mRNA that is still attached to the poly-A tail in each molecule. So for instance, if the RNA purified from an FFPE sample is degraded to an average length of 200 bps, then we find that we only get reads in the last 200 nts of most all mRNA molecules in that sample.

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.

No. Only the Illumina iControlDB-Client program can be used to download data from the database.

While this might be possible with as little as 100ng with high quality total RNA, Illumina does not have direct experience with this method.

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.

No, not in the current version of BeadStudio.

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.

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.

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. Up to a 4% decrease in call rate was observed in the limited internal testing performed. The decrease in call rate varies 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.

Yes. Right-click the point and select that probe in the menu (first option). A window with three tabs appears: Data, Manifest, and Ontology.

No. You can only view data from one project at a time. You can, however, have multiple copies of the BeadStudio software open at once. Keep in mind that this may affect performance.

LOH and copy number 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.

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.

Illumina recommends following the protocol as closely as possible. The protocol was optimized with SuperScript II.

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

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

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

Not at this time.

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.

Yes, customers now have access to preliminary scoring using ADT, 24 hours a day, 7 days a week. Web-based access to ADT is available through iCom (http://icom.illumina.com).

Homopolymers do not impact sequencing. The number of uniquely alignable reads is a function of the repeat content, so this will have an impact on productivity. With longer reads and paired-end sequencing, this may be less of an issue.

Yes, you will need to supply an approval letter from your Institutional Review Board (IRB) or equivalent review committee (for international customers) when you begin the upload process. Please see the iControlDB manual and support documents for additiona

No. You can use your current BeadStudio license keys with the corresponding GenomeStudio software modules.

Yes. If you do not already own the module, you need to purchase new license key(s).

Yes, BeadStudio v3.1 or higher is required to upload data to the database.

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.

No. The machines are standardized and will remain stable if left in the original configuration.

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.

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.

During cluster generation cDNA libraries are processed in the same way as genomic DNA. cDNA libraries may be prepared from RNA by two different methods. For directional sequencing of RNA,RNA adapters are ligated serially to RNA fragments followed by conversion to cDNA. For non-directional sequencing of RNA, the RNA fragments are converted to cDNA by hybridization of random primers directly to the RNA fragments followed by reverse transcription to produce cDNA fragments. cDNA may also be prepared from RNA-associated with proteins by immunoprecipitation of the ribonucleoprotein complexes.

Not at this time.

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

Not at this time.

No, not at this time.

Yes, annotation for rsSNPs is available upon request.

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.

This protocol is designed for quantitative shot-gun sequencing of cDNA. Therefore, we end up getting reads from both ends of the cDNA fragments and not from a specific strand. Illumina has determined that this protocol is the best method available for creating even, unbiased coverage of the entire length of the cDNA molecule. We have tested many methods and found that this feature of the protocol provides the most quantitative and reproducible results for counting transcripts. For those who are interested in generating strand-specific information, several publications have demonstrated this capability on the Genome Analyzer (e.g., Lister et al., Highly Integrated Single-Base Resolution Maps of the Epigenome in Arabidopsis, Cell (2008), doi:10.1016/j.cell.2008.03.029)

Yes. Unlike the GoldenGate Assay, the effect of underlying polymorphisms is not critical to overall performance.

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%.

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/

See the GenomeStudio RNA Sequencing Module User Guide, the RNA Counting Methodology Technical Note, and the SNP Counting Methodology Technical Note in the GenomeStudio Portal for information.

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.

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).

Click the Column Chooser icon in the desired table. You can then select the columns of data you would like to view.

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.

p = 10'(DiffScore*sgn(µcond-µref)/10)

To check whether your network performs fast enough for a particular run, do the following: 1. Open the copylog.txt file located in \Data. Each entry has the following format: 7/1/2009,07:01:31.843,0,0,0,Copy D:\Runs\090630_HWUSI-EAS713_97772299_42FRP\Processed\L008\C13.1\s_8_65.cif to \\smb11\smb\sata825\production\090630_HWUSI-EAS713_97772299_42FRP\Data\Intensities\L008\C13.1\s_8_65.cif,406,0,0 2. Look for entries designated Copy (indicated above in blue) after the first series of numbers in the entry. These describe copy events. 3. The third number from the end (indicated above in purple) specifies the time (in ms) it took to copy the particular file to its destination. Collect the file name and copy time. 4. Go to the destination folder and look up the corresponding file size. 5. Divide the file size by the copy time, which will give you the copy speed for that particular file. For a typical image file (7.5Mbyte), in this example the copy speed is 18.5 Mbyte/s. 6. Repeat steps 2-5 for a few more files. The typical copy speed should be at least 10 Mbyte/s, ideally 15 Mbyte/s or higher. 7. If the copy speed is consistently significantly less than 10 Mbyte/s or lower, your network does not perform fast enough. See “What should I do if the copy speed is too slow?” for suggested solutions.

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

If you are running BeadStudio v3.0 or higher and have an active system or software warranty, you can download GenomeStudio software from iCom. Others can request GenomeStudio software from their Regional Account Manager or Regional Sales Manager, or from Illumina Technical Support.

Short fragments tend to create smaller clusters allowing greater data density. The optimal fragment size for single-read is 150 to 300 bp. The optimal fragment size for paired-end is 250 to 500 bp.

See the Sequencing Analysis Software User Guide, available in the GenomeStudio Portal.

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.

To determine the chromosomal location for the potential/putative miRNAs, Illumina performed the following steps: Select 100% hits (of genome BLAST) for each mature miRNA sequence (if we don't get 100% match, a value of "zero" will be shown in the bgx file) For each hit select the upstream and downstream pre-miRNA sequence (i.e. 5'-nnn-miRNA-nnnnnnnnnn-3' and 5'-nnnnnnnnnn-miRNA-nnn-3') Generate mfold structures for both upstream and downstream pre-miRNA sequences Analyze each structure (if the pre-miRNA will fold into the stem-loop structure) Filter out structures that are below the score threshold (estimated from Sanger training set) 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.

Select File | Manage Project Data.

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

Select Analysis | Manage Groupsets. You can make changes to existing groups/groupsets or create a new groupset.

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.

With BeadStudio open, select Help | About.

With GenomeStudio open, select Help | About.

The custom manifests are confidential and will be available through FTP download or iCom.

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.

New software releases are announced via our IllumiNOTES e-newletter. You can request the latest versions by emailing the catalog number and your shipping and billing addresses to orders@illumina.com. Software updates are free for customers with a current service contract (warranty). Hot fixes are available for download from our website. You can also contact your Field Applications Scientist or technical support to find out the most recent software versions available.

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

The Illumina platform works best with relatively intact, high-quality DNA. 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.

The catalog number for the Methylation module is BM-10-101. Contact us for more information.

Please refer to the Designing Custom GoldenGate Assays technical note.

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.

In this version of BeadStudio, you can go about this one of two ways: You can lasso genes of interest by right-clicking, then clicking on a selected region. You can then lasso the points (probes) you are interested in, mark selected rows, return to the table, right-click, and select Show only selected rows. Alternatively, you can filter the table by clicking the Filter icon and then structuring the desired query. The table and scatter plot are linked, and therefore only those genes that you are interested in will be displayed in the scatter plot. You can then export this list of genes by clicking the Export icon. Note that only the columns displayed in the table will be exported.

You can unpack SRF files with the srf2illumina utility supplied with Genome Analyzer Pipeline Analysis Software.

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.

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.

The probe design may be based on either Infinium I or Infinium II, but the Infinium II Assay chemistry is always used.

Illumina’s proprietary method ensures ligation of two different adapters in the required orientation to opposing ends of a DNA fragment. PCR selects for these and finalizes the construct ready for hybridizing onto the flow cell surface. Adapter sequences can be determined by sequencing the ligation fragments, but sequence information alone is not sufficient to uncover the method.

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.

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.

In the GenomeStudio ChIP Sequencing Module, merged tables combine peaks and regions. This allows differences between samples to be compared more easily.

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.

Nebulization is a very reproducible process that produces random fragmentation. Alternative fragmentation methods may be more appropriate depending on sample availability. Other available technologies include sonication, ultrasound, and hydroshearing.

The study number will be the next study number available in sequence.

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.

After amplification, you can store flow cells indefinitely at 4°C in the provided storage buffer. After the blocking step, you can store them for one month at 4°C. After primer hybridization, they should be processed for sequencing within 2 weeks.

A prepared sample will remain stable indefinitely when stored at -20°C.

On the Genome Analyzer IIx, this depends on the number of cycles, which in turn depends on the application. The Genome Analyzer IIx can process approximately 22 cycles in one day.

Image analysis and basecalling happen in real time on the instrument workstation.

It takes approximately four hours on cBot and about five hours on the Cluster Station.

It takes approximately 1:30 to scan a 96-sample SAM.

BeadStudio support is currently planned to end on Dec 31, 2009. BeadStudio users are encouraged to migrate to GenomeStudio software before this time.

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.

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

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

Flow cells are designed for single-use. All eight lanes must be used at the same time. They may be used for the same sample or for dif­ferent samples. You can run eight samples at a time without multiplexing. With multiplexing, you can increase throughput to up to 12 samples per lane or up to 96 samples per flow cell

The number of samples you can load depends on many factors, including but not limited to the following: type of storage selected, amount of physical memory on the PC, whether other programs are running. File-based storage is the default storage configuration for the BeadStudio Genotyping Module. This allows you to load many more samples than memory-based storage. Using memory-based storage can result in better performance if sufficient physical memory is available. For a computer with 2GB of physical memory, approximately 200 samples of SNP data from the HumanHap300 Genotyping BeadChip can be loaded using memory-based storage. For SNP data from the HumanHap550 Genotyping BeadChip, approximately 150 samples can be loaded using memory-based storage.

Illumina recommends > 100 samples including both replicates and trios.

Illumina recommends > 100 samples including both replicates and trios.

The lowest number of SNPs assays available within one GoldenGate OPA or panel is 96 SNPs  (on the BeadXpress platform). Above that, GoldenGate OPAs are available from 384 to 1,536 SNPs in multiples of 96 (on the BeadArray and BeadXpress platroms). Multiple OPAs can be run per sample. One tube of OPA can query 96 samples (one Sentrix Array Matrix).

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

The minimum number of bead types (attempted SNPs) is 7,600. The maximum number of bead types is 60,800.

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

250 ng, 5 ul at 50 ng/ul.

If possible, start the sample prep if possible with 1–5 μg of DNA, although 1–2 μg is enough for many flow cells. For high-density clusters, we add about 100 μl of a 3 pM solution of the prepared sample (i.e., 3 × 10-4 pmol) to each flow cell lane. This amount is enough to visualize around 13 million clusters in one lane.

Updates coincide with dbSNP releases.

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

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

Illumina recommends 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.

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

CASAVA will use splice file that comes with it. You just need to specify which set you want to use (31 or 46), for example: --spliceJunction=splice_sites-31

Illumina recommends using the Agilent 2100 Bioanalyzer to estimate final product concentration, and to check the size of the library. We do not recommend only using the nano drop because its concentration estimate includes primers and other small nucleic acids.

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 were chosen from HapMap phase I validated SNPs. We’ve found that the conversion rate of HapMap-validated SNPs is generally high.

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. TechSupport@illumina.com.

See the GenomeStudio Framework User Guide available in the GenomeStudio Portal for information about how to download a reference sequence from UCSC. Also note that you will need to update Pipeline and CASAVA. For RNA Sequencing experiments, you will also need to create splice sets and exon_coords files. The genome you use for visualization must be the same as the genome used for alignment.

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

One option is to clean all your data and run your command CMD again ˜/ CMD --target=allClean ˜/ CMD

Assuming that you have specify reference genome to be: --refSequences=/data/runs/genomes_human and genome size file: -g conf/human_rna_size.xml you have three options: Use the human reference genome provided by Illumina. This is preferable since the GenomeStudio Software assumes human reference genome provided by Illumina. Change the names of your fasta files in ⁄data⁄runs⁄genomes_human to match the names in human_rna_size.xml. Instead of using conf⁄human_rna_size.xml, use genome_sizes.xml produced by Pipeline. NOTE: For CASAVA for RNA Sequencing all reference sequences and genome_size files are provided by Illumina. If you run into the ERROR: reference sequence ⁄data⁄runs⁄genomes_human⁄c1.fa does not exists problem, you are probably not using files provided by Illumina.

If you have an active Genome Analyzer system warranty, you are eligible to receive a free 1-seat license for each of the GenomeStudio sequencing modules: GenomeStudio ChIP Sequencing Module v1.0 GenomeStudio DNA Sequencing Module v1.0 GenomeStudio RNA Sequencing Module v1.0 Additional licenses for all GenomeStudio modules are available for purchase.

Clicking the 'Download Checked Samples' button only functions to generate the genotype data. The 'Generate Report' button must also be clicked to physically generate the report and save it to your computer.

The Edit Query function only sets up the query to be performed. After the query is set, it is necessary to hit the 'Perform Query' button to have your results updated.

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.

All data that is uploaded is initially deposited in a staging area until it is reviewed by Illumina personnel. Data is not released to the database until it has undergone this review process, which may take up to 10 business days. Other reasons that data

A list of the Illumina barcodes and their corresponding HapMap IDs is available for download.

The FDR is a p-value adjustment. If you select this option, the value in the p-value column reflects the FDR adjustment.

We review the data for many criteria before it is released 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 (i.e. ethnicity, positive phenotype,

No. Only information that is contained in the standard phenotype information columns will be available from iControlDB during the download process.

No, not necessarily. SNPs that do not pass our quality control specifications, and would result in poor data quality, will be removed from the bead pool prior to release. This way, customers receive the highest quality bead pool to initiate Gentraining and subsequent analysis.

With BeadStudio open, select Tools | Options | BeadStudio. In the Settings window, indicate the desired Maximum Project Files.

Double-click a graph point to view data at the resolution of the individual section.

Nothing is wrong with the data. The MIN, AVG, and MAX columns only have different values when the grouping function is used to analyze multiple arrays or BeadChips. If you look at the NARRAYS column, the number 1 appears. This means that only 1 array was reported as a group in this analysis; therefore, the MIN, AVG, and MAX values are the same.

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.

For a targeted approach, there are a number of technologies available to include SureSelect by Agilent, HybSelect by Febit, and RainStorm by Raindance. Additionally, you can use PCR products that are derived from your region of interest as your starting point. The PCR products can be long-range PCR products or as short as 1,500 bp.

Yes. Ethidium bromide or another DNA-visualizing fluorophore can be added to the gel.

Yes. In addition to Windows XP, GenomeStudio can be installed on computers running either the Windows Vista 32-bit or 64-bit operating system.

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

Yes, Illumina data is compatible with Bioconductor, a collection of R packages developed by researchers around the world and distributed for free.

Yes, this is OK. Their presence will not impact the performance of the assay.

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

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

Illumina recommends normalizing mRNA data separately from miRNA data.

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.

No, it is a separate product. The product number is: CT-901-1001 (10 lanes), CT-901-1002 (100 lanes).

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

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

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

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.

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

Illumina cannot provide an optimal number because it depends on experimental design and antibody specificity. For example, specific transcription factors require a smaller number of tags than those that are more general (PolII, Histone, …). The higher throughput of the GAII system gives confidence that data from one or more lanes in a single flow cell may be sufficient for high confidence in the number of peaks found per region.

This process is not currently compatible with automation.

Illumina does not currently offer LIMS support for miRNA.

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.

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.

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.

Yes, you can do this by using a conversion feature available in the GenomeStudio. This feature allows you to convert GenomeStudio projects to BeadStudio projects and vice versa.

You have three options: Download GenomeStudio from iCom. Install GenomeStudio over a network that has a shared DVD drive or a copy of the GenomeStudio image. Purchase a portable DVD drive with a USB port, then install GenomeStudio from the DVD.

This situation is a challenge for any system trying to quantify gene expression levels. Variable concentrations are not nearly as challenging as variable quality for the mRNA-Seq assay. As described in the question about RNA degradation, it is not recommended to try to compare results across samples which have a large range of quality and integrity, because this would have a large effect on the read coverage across the full length of the transcripts. If you notice that your RNA samples are of distinctly different quality, Illumina recommends that you first attempt to re-purify all of the lesser-quality samples with the goal of having the highest quality RNA that you can purify from your particular system. Then try to be sure that all of the samples you would like to compare are of similar quality and quantity if at all possible. We do not yet have analytical methods to normalize for these effects in mRNA-Seq data.

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

Yes. We recently changed vendors for one of the components. The change has been fully validated and you should not experience any difference in performance.

If you have an iCom account and an active system or software warranty, you can download GenomeStudio software from iCom. The installer and associated materials (sample projects, demo data, user guides and release notes) are also distributed on DVD. Customers can place an order for GenomeStudio Module Software & Documentation Sets via their RAM, RSM, or Illumina Technical Support and the order will be filled by Customer Service.

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

We recommend quantitation of amplified RNA by fluorometry using Invitrogen’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.

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.

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.

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.

mRNA-Seq libraries can be sequenced on paired end or single read flow cells.

Illumina has not tested kits designed to label microbial RNA. Reagent vendors such as Ambion® do sell kits for this application. These kits may work, but there is no particular kit that we recommend. It is important that a single source of biotin-16-UTP (i.e., same vendor) is used for all labeling reactions (e.g., Ambion #8452 or #8453).

These SNPs may not be real SNPs, but small indels. A small indel will cause a short run of snp calls (~indel+4) with a concomitant dip in coverage. Check whether the apparent SNP can be explained by a short indel.

During a typical run, SCS real time analysis may occasionally run into copy issues, but it will retry copying the file later. As long as all is fine then, SCS real time analysis will proceed normally. Therefore, seeing a few of these issues in the errorlog is acceptable. However, if you see many of these entries (dozens or hundreds), you may have a problem with your network. You may be using the wrong password, network path, or your network may not be fast enough. Do the following: 1) Check whether the network location is still valid. Your network may be down, or a folder may have been moved. 2) Check whether the proper user name and password are used. 3) Test whether your network is fast enough. See “How can I check whether copying files proceeds fast enough?” for details.

The Features directory contains splice junction/exon sets required for running CASAVA for RNA Sequencing. Please make sure that the Features directory is present in the CASAVA folder.

The file that is downloaded and contains the program also contains 'template' files that can be used instead of a Final Case Report. These do not contain sample genotypes, but rather are the required format to be used as the Final Case Report by iControlD

You can download .NET 3.5 from Microsoft’s web site.

While 200bp is the most ideal size for the GA system, this means the insert will be about 100bp because the combined flanking sequence (including adaptors) will be about 100bp. This will enable 35bp to 50bp single reads. NOTE: If the fragment is less than 150bp, it may be difficult to obtain 50bp reads.

This is a HumanHap550 product.

There are no inherent limits in the software. Illumina scientists have pooled 29 BACs of 130 kb each.

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

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

Yes. Use the Properties tab on the left side to control auto-scaling or to use a fixed scale.

Tag profiling, by concentrating the sequencing power across the relatively small number of tags (~100k or so), is able to produce very large dynamic ranges with very high sensitivity. mRNA-Seq offers broader coverage of the transcriptome, allowing for more discovery applications (e.g., novel transcripts, splice isoforms, cSNPs).

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 table below includes Illumina’s minimum hardware recommendations to run GenomeStudio software.

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.

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.

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 by the customer.

The number of SNPs has been increased to 6056 markers in the Linkage V panel. Importantly, the marker lists for the GoldenGate and Infinium panels are further optimized for genetic spacing with the goal of maximizing information content in your study. Additionally, the linkage panels on the Infinium and GoldenGate platforms are designed to have a high degree of overlapping SNP markers so that samples run on the separate platforms can be included together in a single analysis. We used Genome Build 36.

GoldenGate: 250 ng, 5 µl at 50 ng/µl, Infinium (including HumanHap550 Plus): 750 ng, 15 µl at 50 ng/µl, Custom Infinium (iSelect): 200 ng, 4 µl at 50 ng/µl

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.

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.

To process the data from a single run, the system requires 500 GB to 1 TB (depending on factors such as cycles and cluster density). Images are acquired and stored on the instrument workstation and must then be transferred to an external computer to be analyzed by the Genome Analyzer Pipeline software, which handles image processing, base calling, and sequence alignment. The software runs on all common Unix/Linux variants. A high-end Linux box should be adequate as the analysis computer. However, Illumina software is compatible with Sun Grid Engine and LSF, if you wish to install it on a cluster. The instrument generates ~1 Tb of data during a full 2–3 day run. About 70% of this is TIFF image data that can be sent to tape after a run is finished and you are satisfied a reanalysis is not required.

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.

CPU speed Intel Pentium 2.0 GHz or above, Memory size >2GB, Hard drive >100GB, Video display 1280 x 1024 recommended 1024 x 768 required, Monitor display LCD or CRT 17” recommended Operating system Windows XP - SP2 (32 bit) or Windows XP - SP2 (64 bit), Specific OS requirements Requires Microsoft .NET Framework 1.1 or above

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.

Illumina specifies 40 ng/ul in the assay protocol.

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

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.

The average CV from replicate samples within a SAM was determined to be 0.11. The average CV from replicate samples across multiples SAMs was determined to be 0.14.

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

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. sample from a diabetes patient). In this instance, 'diabetes' may be reflected in the 'Positive Phenotype' for this sample

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.

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).

Please refer to the Assay Design Tool training presentation found here: Illumina Assay Design Tool

In SNP graphs, squares represent samples with replicate errors. Samples with PC (parent-child) or PPC (parent-parent-child) errors are represented with circles and Xs. The parents are represented with circles and the children with Xs.

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.

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

ADT generates probe scores, selects the best probe design, provides assay validation information, populates normalization bins, and allows Infinium II to Infinium I Assay conversion.

BeadScan generates up to five files depending on whether or not you elected to save compressed images. There will be 96 of each of the following file types if you use SAMs, and 12 each if you use BeadChips. NOTE: If you save *.jpgs, you will not get *.locs or *.xml files., *.idat file: size ~22KB, *.xml file: size ~2KB, *.locs file: size ~400KB, *.tif or *.jpg file: size ~450MB/150KB respectively, metrics *.txt file: size ~20KB

These are described in the GenomeStudio RNA Sequencing Module Workflow Technical Note.

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.

The library process can be optimized to reduce the amount of DNA required. For some applications such as ChIP-Seq where DNA amounts may be low, steps such as size selection on a gel may be skipped. Instead, end-repaired DNA fragments may be ligated, purified by a column clean-up step that removes primer-dimers, 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 may be eliminated.

The io_lib package contains a variety of vendor-neutral SRF utilities. Download this utility from http://sourceforge.net/projects/staden/, and compile the code following the instructions provided with this distribution. To unpack the sequences and quality values from SRF file using this package, you can use the utility srf2fastq by entering the command: srf2fastq -c file.srf

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

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.

You can submit the SNP in a SequenceList.

The genome files for non-human species conform to the UCSC format. The GenomeStudio Framework User Guide available in the GenomeStudio Portal describes how to create genome files for non-UCSC genomes. Illumina is currently working on tools that will convert NCBI formats to UCSC format.

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.

The official recommendation is between 1-10 ug. However, Illumina has used as little as 0.1 ug of UHR Total-RNA (highly pure and high quality). NOTE: best results will be obtained with the recommended amounts.

The summary and sorted files output by Gerald are required for use with the GenomeStudio ChIP Sequencing Module. For more information about required input files, see the GenomeStudio ChIP Sequencing Module Workflow Technical Note available in the GenomeStudio Portal.

Between 150bp and 200bp is the ideal size range for single-read libraries. Note that the final product of the sample prep process will be about 100bps longer than the size of the cDNA insert due to the additional length of the Illumina linker and primer regions.

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.

The matrix file accounts for cross talk between dyes. It is used for base calling.

A tile is an image captured by the camera on the Genome Analyzer. The field of view of each image is approximately 760 μm × 720 μm. A flow cell contains 8 lanes with each lane being imaged in two columns. 50+ images or tiles are taken from each column. Each tile or image is a monochrome image, therefore for each base 4 monochrome images are taken for each of the 4 colors.

The ability to distinguish between two or more clusters that are in close proximity to each other.

Illumina is currently working to achieve ISO 13485:2003 certification. We are developing all of the VeraCode products under design control and have design history files for each of our products.

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.

Successfully designed on an Illumina platform, using either the Infinium I Assay or Infinium II Assay.

Paired-end analysis sequences both ends of a DNA fragment. If the fragments are of known size, this method can facilitate de novo sequencing of repetitive elements and help to identify structural variation.

SRF (Single Read Format) is a generic format for DNA sequence data. Some public sequence databases, such as NCBI, require that you post data from published manuscripts and other sequencing projects using SRF. The format is defined at http://srf.sf.net.

Robocopy is a script that copies files from the local drive to a network location while the run is proceeding. This saves a considerable amount of time transferring data after completion of a run.

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.

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.

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.

A lane and a channel are used synonymously. The term channel may also refer to a color channel on the Genome Analyzer (four colors corresponding to the four bases A, C, G, or T).

Tag profiling offers a way of generating a large number of 3'-based sequence tags which can be used for expression profiling. mRNA-Seq, in contrast, generates sequences across the entire length of the transcript through the use of random priming.

The BeadStudio Genotyping module is designed for analyzing GoldenGate and Infinium project data sets. The Genotyping Module allows you to normalize raw data, generate genotype calls, perform clustering, analyze data intensity, and calculate LOH (Loss of Heterozygosity) and CN (Copy Number) scores. Advanced, flexible graphical tools enable the visualization of potential regions of LOH along different chromosomes. The BeadStudio LOH Plus module is designed specifically to study chromosomal aberrations and allelic imbalances. Key features of this module include paired-sample analysis where one sample serves as a reference, auto-detection of chromosomal aberrations, customized bookmarking of genomic regions of interest, and a chromosomal heat map that can be used to examine LOH/CN-related intensity differences along chromosomes.

Cluster generation requires very low amounts of DNA libraries. 1-8 pM final concentration provides the optimum cluster densities. The method of DNA quantitation is important. Calibrating cluster density with DNA concentration may be determined by running a titration flow cell. To maximize data density we recommend a library concentration that delivers approximately 500,000 clusters/mm2

r2 between BeadChips: .99, r2 within BeadChip: .99

The Illumina2srf utility converts analyses from Genome Analyzer Pipeline Analysis Software to SRF. It can create simple SRF archives containing only sequences and quality values. It is also capable of creating SRFs with raw intensity and noise values. If these values are stored, the SRF file is significantly larger (~8X larger), but this allows base calling to be rerun from the archived files. Note: Intensity and noise values are stored in SRF with one less significant digit than is contained in the original pipeline files. illumina2srf runs in Genome Analyzer Pipeline Analysis Software and produces SRF files containing sequences and quality values from the standard pipeline output files. Using Illumina2srf can be specified as part of the GERALD config file: SRF_ARCHIVE_REQUIRED yes Or from the command line: For Genome Analyzer Pipeline Analysis Software v1.3: ${PipelineDir}/bin/illumina2srf -o lane_${lane_no}.srf ${BustardDir}/s_${lane_no}_*_qseq.txt For Genome Analyzer Pipeline Analysis Software v1.0: ${BustardDir}/s_${lane_no}_*_seq.txt where ${PipelineDir} is the root location of the Genome Analyzer pipeline, ${BustardDir} is the bustard directory of the run to be archived, and ${lane_no} is the number of the lane to archive from this run. To include intensity and noise values in the archive, include the “-b” flag: ${PipelineDir}/bin/illumina2srf -b -o lane_${lane_no}.srf ${BustardDir}/s_${lane_no}_*_qseq.txt

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

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

As of January 2009, approximately two billion rows can be loaded, and 128 million rows can be displayed in the Sequence Table.

1.35 fold

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.

Currently ADT retrieves data based on dbSNP126 for sequence, position, and MAF.

The average reproducibility between technical replicates is > 0.98.

=1 in 250,000

The srf2illumina utility unpacks SRF files from Genome Analyzer Pipeline Analysis Software into multiple files. It can unpack these files into a single directory, or it can place files so as to reflect the distribution of files in a pipeline analysis directory tree. To unpack an SRF archive, enter: ${PipelineDir}/bin/srf2illumina file.srf To unpack an SRF archive into a pipeline directory tree, enter: ${PipelineDir}/bin/srf2illumina -b file.srf

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

Since Pipeline release 1.3, the quality scoring scheme is the Phred scoring scheme, usually en-coded as an ASCII character by adding 64 to the Phred value. A Phred score of a base is Qphred =-10 log10(e) where e is the estimated probability of a base being wrong. If a base is estimated to have a 1% chance of being wrong, it gets a Phred score of 20. Phred score=30 corresponds to 0.1% esti-mated error, 40 to 0.01%, and so on. The base scores produced by the base caller use a 4-values-per-base scheme which also en-codes information on the next most likely base call. NOTE: The obsolete prb files (only produced in Pipeline 1.3 and later when explicitly directed) use the old Illumina base scoring scheme: QIllumina_old =10log10(p(X)/(1-p(X)) To convert from an old Illumina score back to a probability value use: p(X) = 1 - 1/(1+10 (QIllumina/10) ) Important point: the highest old Illumina base score and the Phred score are asymptotically identical. This means that for scores of about 15 and above they are so close as to be effectively the same.

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).

“The results were generated using the ELAND module within the Illumina Genome Analyzer Pipeline Software, vx.x (Illumina, Inc., San Diego, CA).”

Please reference the Illumina iControlDB web page and/or Illumina, Inc., San Diego, CA, whichever is the preferred format of the journal to which you are submitting.

Two kits have been tested for use with Illumina’s Gene Expression products: Ambion Total Prep Kit (Catalog No. IL1791) and EpiCenter Target Amp Nano-g Biotin-aRNA Labeling Kit (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.

This depends on the size of the organism you are trying to resequence. For whole-genome resequencing, a 25-fold over-sampling should be adequate. For targeted resequencing involving mixes of many PCR products, 75-fold over-sampling will correct for the inability to mix the PCR products at a 1:1 ratio. Illumina sample prep shows no systematic bias. In sequencing the X chromosome we achieved 16-fold average cover­age, with all sequenceable bases covered at least twice.

If you get this message, you should explicitly install the io_lib. To do this, run the following command from the Pipeline Install directory: /GAPipeline_1.3.2 make WITH_IO_LIB=1 install, then retry the command from Q2, above.

If you are not copying files to the target server fast enough, consider the following: 1. You should use a network connection rated at 1 Gigabit or faster. If not, ask your IT department to upgrade your network. 2. If the server drive is nearly full, copying to it takes much longer. Remove files that are no longer needed from the target server, or copy to a different server. 3. If multiple sequencing instruments all copy to the same server, the server drive may not be fast enough to handle all the traffic. Consider having the instruments copy to different servers. 4. If multiple sequencing instruments share a connection to copy to their target servers, the connection may not be fast enough to handle all the traffic. Consider having the instruments use different routes.

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

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.

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

You can perform gene analysis or differential expression analysis. The data can be visualized as bar plots, scatter plots, line plots, heat maps, and cluster diagrams.

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.

Accepted inputs include: RSList, GeneList, SequenceList, and RegionList. File formats are also available from techsupport@illumina.com and can be found on the Custom OPA Design page.

Illumina has observed that the overall yield and quality of the library may diminish with lower input amounts. One thing to keep in mind is that the PCR cycles used in the enrichment step of the protocol (following the gel) are tuned so that we expect a certain amount of material feeding into the reaction. The protocol calls for exactly 15 cycles of PCR amplification. If the amount of material purified from the gel is significantly less than in a standard 1 to 10 ug prep, then the number of PCR cycles needed to create a clearly visible band may need to be increased beyond 15 cycles. However, it is important to note that we do not recommend "over amplifying" the library, since this can lead to artifacts during the electrophoresis and quantification of the library.

GenomeStudio GT, GX, M, and PT v1.0 include the features you are familiar with from BeadStudio, plus enhancements and bug fixes. A few examples are listed below: In the GenomeStudio v2008.1 Framework, the IGV includes different features for microarray (GT, GX, M, and PT) and sequencing (CS, DS, RS) modules. The GenomeStudio Genotyping Module v1.0 includes support for merging Human1M and Human1M-Duo data in the same project. In the GenomeStudio Gene Expression Module v1.0, heat maps are now automatically generated upon project creation. For more information, see the GenomeStudio v2008.1 Release Notes, available in the GenomeStudio Portal.

Please contact TechSuport@illumina.com and they will forward you files for any recent products.

Admin rights are only available for Illumina personnel who are adminstrators for the iControlDB.

GenomeStudio software is now available to download from iCom.

This template is available with the from iCom or by contacting TechSupport@illumina.com.

The license key for each GenomeStudio software module appears in two places: 1) on a sticker on the outside of each GenomeStudio Software & Documentation Set, and 2) on the inside of the front cover of each GenomeStudio module DVD.

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.

You can find workflow documents for the GenomeStudio ChIP Sequencing, DNA Sequencing, and RNA Sequencing modules in the GenomeStudio Portal or on the Product Documentation web page.

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.

The image viewer is not available in v2.3.41. It will be added to a future version of BeadStudio.

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

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)

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.

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.

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

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.

All customers with an active iScan, BeadXpress, or Genome Analyzer system or software warranty are eligible for a free upgrade from BeadStudio to GenomeStudio software.

Testing indicated that this configuration was optimal for RNA applications.

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.

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.

BeadStudio 2 does not support analysis across different BeadChip types in the same project. However, you can use BeadStudio 1.5 (an older version) for analysis across BeadChip types.

To avoid amplification of adapter dimers.

The data from the control sample are used to generate the matrix file. The analysis tool uses the control to calculate phasing/pre-phasing from this sample, and the relative proportion of the different bases. Without a control lane, the software would assume that the base composition of the sample is strictly balanced. While this is true of a total human genome, it might not be true of non-human genomes or a focused region of the human genome. Therefore, the control is necessary for all expression studies, small RNA studies, and reduced complexity studies.

This allows you to keep track of the samples you have uploaded, and provides an easy reference 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 sa

This fragmentation method takes advantage of the sensitivity of RNA to cleavage by divalent cations and temperature. This is the most robust method for fragmentation of RNA. Fragmentation by this method has been shown to result in more uniform sequencing coverage compared with other methods.

It is perfectly normal to observe both a slight GC bias and a distinctly non-random base composition over the first 12 bases of the data. This is observed when looking, for instance, at the IVC (intensity versus cycle number) plots which are part of the output of the Pipeline. In genomic DNA sequencing, the base composition is usually quite uniform across all bases; but in mRNA-Seq, the base composition is noticeably uneven across the first 10 to 12 bases. Illumina believes this effect is caused by the "not so random" nature of the random priming process used in the protocol. This may explain why there is a slight overall G/C bias in the starting positions of each read. The first 12 bases probably represent the sites that were being primed by the hexamers used in the random priming process. The first twelve bases in the random priming full-length cDNA sequencing protocol (mRNA-seq) always have IVC plots that look like what has been described. This is because the random priming is not truly random and the first twelve bases (the length of two hexamers) are biased towards sequences that prime more efficiently.This is entirely normal and expected.

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.

In general, this message means that the SNP sequence cannot be found in our internal database, which is a filtered version of dbSNP. We filter out any SNPs that are not appropriate for the Illumina platform (e.g., insertion/deletions, multiple nucleotide SNPs, SNPs with ambiguous or multiple localizations, etc.).

It is necessary to run a PhiX control in one of the 8 lanes on the Genome Analyzer in order to insure that the data generated for each run is accurately calibrated during the Image Analysis and Data Analysis pipeline.

It is normal to observe that, unlike other applications, the first two or three bases in the mRNA-Seq reads have slightly elevated error rates compared with what you may be used to when looking at genomic DNA samples. Illumina believes that this is also an effect of the random priming process. The bases at the beginning of each read were likely at the back end of the random primer, away from the extending polymerase, during the priming process. It appears that this observation is a measurement of the mismatch pairing that is tolerated on the other end of the primer during the extension process by the polymerase.

At this time, uploading samples does not include this information. Future releases of GenomeStudio and BeadAccess will incorporate this ability, and at that time you will be able to query on this field.

For the HumanHap550 Plus BeadChip, the HumanHap550 BeadChip Gentrain file will be available for those samples. However, for all custom content (including custom content added to the HumanHap550 Plus) Gentraining must be performed at the customer site.

No. You can install BeadStudio and GenomeStudio on the same computer.

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.

The current BeadStudio Methylation module supports memory-based storage for faster project loading.

Upon release of GenomeStudio, Illumina will no longer release regular software updates for BeadStudio. All new features and updates will be in GenomeStudio software only.