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Frequently Asked Questions

» SNP genotyping and cytogenetic/CNV analysis
1. Using DNA extracted from saliva on HUMAN660W-QUAD chips.
2. Gene annotation files for standard Infinium panels
3. OPA design: recommended SNPs
4. Failure codes and validation bins in ADT results file
5. Custom genotyping: SNP not found in the database

» Gene expression analysis
1. RNA extraction from plasma or serum for direct-hybridization assays on Human HT-12 BeadChips.
2. RNA quality: the recommended values for rRNA ratio, RIN and % total area 18s/28s

» Next generation sequencing
1. Sequencing microRNAs from paraffin-embedded samples with GAII technology
2. Release of the multiplexed sequencing kit for microRNAs
3. Concentration of purified small-RNA required for sequencing
4. The amount of material needed for a mRNA-Seq experiment
5. Choosing a target enrichment method
6. Target enrichment: "bait", "bait group" and "library" definitions
7. Target enrichment: creating a library by bait tiling


Questions and Answers:

» SNP genotyping and cytogenetic/CNV analysis

1. Using DNA extracted from saliva on HUMAN660W-QUAD chips.

Keywords: DNA;extraction;saliva;genotyping;HUMAN660W-QUAD

Question: DNA extracted from human saliva can contain 10 to 90% of DNA of microbial origin. Could give us some recommendations for the optimal amount of such DNA to be used on HUMAN660W-QUAD chip?

Answer: Answer from Illumina:
We do not have any specific recommendations for protocol modification for DNA extracted from saliva. This is the information which I have found on our website in FAQs (http://www.illumina.com/support/faqs.ilmn):
While we do not have experience using DNA extracted from saliva, 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.
Here are a couple of publications which quote using Infinium assay with DNA isolated from saliva:

Links: 2. Gene annotation files for standard Infinium panels

Keywords: gene annotation; standard panel

Question: Where can I find gene annotation files for standard Infinium panels?

Answer: They can be found on Illumina's public ftp site. To access the site, please follow the link displayed below (using your Internet browser).
You will need to enter the following data:
User name: guest
Password: illumina
Folder name: Whole Genome Genotyping Files

Links: 3. OPA design: recommended SNPs

Keywords: genotyping; custom; OPA; GoldenGate

Question: Which SNPs should I use to design my OPA?

Answer: Illumina:
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 affect adversely the performance of the entire OPA.

4. Failure codes and validation bins in ADT results file

Keywords: genotyping; custom; ADT; GoldenGate

Question: What are the failure codes and validation bins in my ADT results file?

Answer: Please refer to the Assay Design Tool training presentation to be found in Illumina Assay Design Tool document.

5. Custom genotyping: SNP not found in the database

Keywords: genotyping; custom; ADT; SequenceList

Question: What can I do if my SNP is not in your database?

Answer: You can submit your SNP in a SequenceList - for more information, please see Illumina Assay Design Tool document.

» Gene expression analysis

1. RNA extraction from plasma or serum for direct-hybridization assays on Human HT-12 BeadChips.

Keywords: RNA;extraction;plasma;serum;direct hybridization

Question: Do you have any suggestions on how to use RNA extracted from plasma or serum in direct-hybridization assays on Human HT-12 BeadChips?

Answer: Answer from Illumina:
As our gene expression array protocols begin at the hybridization step, we do not have specific recommendations for RNA extraction. For specific recommendations on RNA extraction and quality, I would refer to the protocols for one of the labeling kits we recommend:

• Ambion Illumina TotalPrep RNA Amplification Kit (Ambion Catalog # IL1791).
• Ambion Illumina TotalPrep-96 RNA Amplification Kit.
• Epicentre TargetAmp™ Nano-g™ Biotin-aRNA Labeling Kit for the Illumina System (Epicentre Catalog # TAN07924).
• The NuGEN Ovation® amplification kits have been shown to work with Illumina Gene Expression BeadArray technology.

In general, RNA extracted from plasma or serum tend to produce low yields. Based on the Ambion TotalPrep protocol, the recommended amount of input RNA is: recommended amount of total RNA 50 - 500 ng, minimum amount of total RNA 25 ng, maximum volume of RNA 11 µl.
After extracting the RNA from serum or plasma, I would suggest quantifying the RNA with the Molecular Probes Invitrogen Quant-iT RiboGreen assay as recommended in the DirectHyb assay guide. The RiboGreen assay can quantitate small RNA volumes. Unlike OD260 measurements, it is relatively insensitive to silica fines that can contaminate the sample.

2. RNA quality: the recommended values for rRNA ratio, RIN and % total area 18s/28s

Keywords: gene expression;RNA;quality;BeadChips

Question: What are the recommended parameter values for RNA quality for expression analysis on BeadChips (rRNA ratio, RIN, % total area 18s/28s)?

Answer: Illumina answers:
For RNA quality, we do not have any set criteria for rRNA ratio and % total area 18s/28s. For RIN, again we do not have an official recommendation but we have found that using RNA of RIN 7-9 achieves good results. We recommend that the RNA is of general good quality, i.e. minimal degradation and high purity (as judged by the OD260/280 ratio).

» Next generation sequencing

1. Sequencing microRNAs from paraffin-embedded samples with GAII technology

Keywords: small RNA;micro RNA;paraffin embedded;sequencing;Genome Analyser

Question: Is it possible to sequence microRNAs isolated from paraffin-embedded samples with GAII technology?

Answer: Answer (from Illumina): We have not specifically validated the Small RNA Sample Prep kit for sequencing small RNA from archived samples such as FFPE but, there is no reason to think that the method would not be appropriate. The small RNA in FFPE samples is more resistant to degradation and, our latest v1.5 small RNA sample prep kit uses a method that specifically targets small RNA molecules within the total RNA pool. This allows us to start directly from total RNA at low levels without the upfront enrichment of miRNA species. The important thing to note for using FFPE samples for small RNA sequencing is to use a kit that extracts total RNA including the small RNAs. I would then suggest you do not use a column based extraction method, as they tend to lose all of the small RNAs. Instead, organic extraction methods are preferred.

Links: 2. Release of the multiplexed sequencing kit for microRNAs

Keywords: microRNA;multiplexed;sequencing;sRNA

Question: When is Illumina going to release the multiplexed sequencing kit for microRNAs? Is there any pre-release?

Answer: Answer (from Illumina): The multiplex sRNA kit is in development and due for release this year but, we currently do not have any information on when this will be available. There is no pre-release protocol because the sRNA kit uses RNA adapters instead of DNA adapters, so the multiplexing kit needs to be redeveloped.

3. Concentration of purified small-RNA required for sequencing

Keywords: small RNA; sRNA; sequencing; concentration

Question: We would like to run a small-RNA sequencing on GAII but not with total RNA, as we have already isolated small-RNA. What is the concentration of small-RNA required?

Answer: Answer from Illumina:

None of our internal research teams have experience with running this protocol with sRNA as the starting material but I can pass on the following recommendations:

1) It is known experimentally that the ratio of sRNA in human samples can vary between 0.5 - 9.2 % of total RNA. ç These differences may be due to the quality of the total RNA samples, the better quality the higher the amount recovered; and also the way the samples were prepared can have an impact in the final amount of sRNA recovered. The ratio has also been reported to be tissue- specific.

2) As we recommend a starting input of 1-10ug of total RNA for the sRNA v1.5 protocol it would be a fair assumption to say that you should attempt to start with a minimum of ~50ng.

4. The amount of material needed for a mRNA-Seq experiment

Keywords: mRNA;sequencing;RNA amount;sample

Question: How much starting material do I need for a mRNA-Seq experiment?

Answer: You will need 1-10ug of total RNA or a minimum of 100ng of mRNA.

5. Choosing a target enrichment method

Keywords: DNA;sequencing;target enrichment

Question: How do I know which DNA target enrichment method is the best for my study?

Answer: There are several considerations one should take into account. These are summarized in agilent_web.pdf file, please see the link below.

Links: 6. Target enrichment: "bait", "bait group" and "library" definitions

Keywords: DNA;sequencing;target enrichment

Question: What are "bait", "bait group" and "library" ?

Answer: Bait: a single oligo sequence of pre-determined length (120bp) that complements a targeted region of the genome.
Bait group: consists of a number of baits designed to complement a single or a set of targeted intervals. Can be formed by baits generated within eArray, baits uploaded by the customer into eArray, or bait search results within eArray.
Library: consists of one or more bait groups and represents the sets of oligos that will be produced for the kit.
For more information, please see the Agilent link below.

Links: 7. Target enrichment: creating a library by bait tiling

Keywords: DNA;sequencing;target enrichment

Question: What steps do I have to follow to create a library by bait tiling for target enrichment system (in solution)?

Answer: Register and login into Agilent's eArray (see this link)

1. Enter a name for the design job.
2. Design strategy:
1. Use parameters already optimized for bait tiling (recommended)
2. Change parameters
   • Design strategy (centered or justified)
   • Bait length (bp)
   • Bait tiling frequency (see file)
   • Allow overlap between avoid region (bp)
3. Genomic target intervals
1. Type in the genomic intervals to be targeted.
2. Upload a file that includes the genomic intervals to be targeted (format of entries for both cases: chrX:1000003816-1000003948).

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