Otogenetics Frequently Asked Questions

Capabilities

Is the NGS provider trained by major companies that supply the equipment and reagents?

Otogenetics has been trained by Illumina for NGS services using the HiSeq2500 and by Agilent for providing exome services for using their capture kits. Our sample processing methods and NGS data quality meet the high industrial standards set by these companies.

Is your NGS work guaranteed?

Yes. Otogenetics works closely with its customers to ensure we achieve the best possible data given the samples they provide.

How are the samples tracked?

Otogenetics uses Genologics Clarity LIMS for sample and project tracking. We also provide LabLink for customers to track their own project in real-time.

Sample Processing

Will the samples be shipped overseas for processing?

Otogenetics processes all the samples at our facility in Atlanta.

What are the advantages of paired-end vs single-end sequencing?

Paired-end sequencing provides extra positional information that is useful in reliable mapping of reads to a reference genome based on the library fragment size. Given the same number of fragments, sequencing of both ends dramatically increases the power in detecting alternative splicing and chimeric transcripts relative to single-end sequencing.

What types of samples to you accept?

Otogenetics accepts all types of DNA and RNA samples, as well as cells and tissues, except those which are infectious or have been treated with radioisotope.

For DNA, blood, saliva and extracted gDNA may be submitted at room temperature, or solid tissues may be submitted flash frozen.

For RNA, cells and tissues may be submitted flash frozen, submerged in RNAlater or homogenized in Qiazol. All samples undergo QC testing upon receipt. You will receive a QC report of your RNA once testing is complete, along with any concerns we may have regarding your sample.

If your samples do not meet our QC standards, we will give you the option of submitting a new sample or eliminating the sample. Meanwhile we can proceed with your other samples or hold them until your new sample is received, per your direction.

What does the standard bioinformatic analysis offer?

For DNA, standard analysis includes mapping & alignment, coverage, sequence variation (SNPs/Indel report (.VCF file), vcf annotations, functional prediction, and population frequency.

For RNA, standard analysis includes mapping &alignment, annotations and expression levels. Expression level comparisons also available.

We offer custom bioinformatic analysis packages, so please let us know if you have special requirements.

Sequencing from DNA (Exomes, Disease Panels)

How do we prepare our DNA sample?

Compatible genomic DNA purification kits include Qiagen QIAampDNA Blood Mini Kit (Cat# 51104) for body fluid and OmegaBiotek Tissue DNA Kit (Cat# D3396-01) for frozen or fresh tissues; Qiagen QIAamp DNA FFPE Tissue Kit (Cat# 56404). Otogenetics recommends RNase-treatment step during genomic DNA preparation for exome and epigenetic modification profiling. If you have limited amounts of DNA, a minimum of 200 ng of gDNA may be submitted. For mtDNA samples, we recommend extracting genomic DNA containing mtDNA (whole DNA) with Qiagen QIAamp DNA Blood Mini Kit (Cat#: 51104), which can also be used for general genomic DNA preparation as well.

What is meant by a qualified DNA sample?

We look at several parameters when assessing DNA quality. We will test the concentration and OD values and perform agarose gel electorphoresis to determine that the DNA is not degraded. Some degradation is expected with FFPE samples, which can still be successfully sequenced; however, data quality will vary from higher quality DNA samples.

Are the targets covered uniformly?

Otogenetics provides graphs representing across-target coverage available upon request.

RNA Sequencing

What are the advantages of RNA-seq verses microarray?

RNA-seq enables rapid profiling and deep investigation of the transcriptome. Unlike microarray analysis, RNA-seq is unbiased with respect to potential content, so it can detect previously unidentified genes or transcripts which may be involved in the phenotype of interest. One of the biggest advantages of RNA-seq over microarray analysis is the ability to detect single nucleotide variants, small insertions and deletions (indels) and transcript isoforms.

RNA-seq also offers improved specificity than microarrays, so it can detect different isoforms of a transcript, and offers increased dynamic range of expression over microarray analysis. Reproducibility of data set results is also improved in mRNA-Seq versus microarray analysis, where the expression signal tends to be compressed. Studies have shown that for human RNA-seq, 2 million mapped reads provides data comparable to a single microarray, and 25 million reads are necessary for repeatability equivalent or better than microarray platforms.

How do we prepare our RNA sample?

If you extract your own RNA, most commercial kits are acceptable as are Trizol-based extraction methods. We request you do not use DEPC-treated water to elute or re-suspend your RNA however, as it can inhibit the polymerases used at several steps in NGS sample preparation. If you plan to do small- or microRNA-seq, you will need to confirm that your chosen extraction method retains these small molecules, as most column based kits do not. More details can be found in the Otogenetics sample preparation & QC instruction.

How many reads do I need?

For smaller genomes (roughly 100Mb or smaller) or for small- and microRNA-sequencing projects, 8 million reads will be sufficient. For larger genomes, we recommend 20 million reads for gene expression studies. If you are interested in structural variations or low frequency transcripts, higher reads depths are recommended. Otogenetics can help you decide based on the specifics of your project.

How do I ship my RNA?

RNA should be shipped on dry ice, next day delivery if possible. If the RNA is not frozen on arrival, it may impact the quality of your RNA. Alternatively, you may ship RNA at ambient temperature, including internationally, if it has been treated with RNAstable according to the vendor instructions.

What is meant by a qualified sample for RNA?

We look at several parameters when assessing RNA quality. If sufficient RNA is provided, we will test the concentration and OD260/280 and OD260/230 ratios using Nanodrop. We also test all samples using the Agilent Bioanalyzer to determine the integrity (RIN) as well as rRNA ratio when applicable (28S/18S). Ideally, the OD ratios should be greater than 1.8, RIN should be 8 or higher, and rRNA ratio should be 1.5 or higher.

What are my options if my RNA sample is less than ideal?

For some samples, Otogenetics may be able to perform a column clean up of your sample (low OD260/230, organic contaminant, samples in DEPC water). Only those samples which have been submitted at 1ug or more will be treated for clean-up to account for inevitable loss on-column.

What is the lowest amount of RNA you can deal with?

As little as 10 cells can be used to generate high fidelity gene expression profiles using direct RNA-Seq assays optimized by Otogenetics.

Otogenetics now offers direct RNA-seq with polyA cDNA synthesis from low input samples. Please contact Otogenetics ([email protected]) for instructions of sample handling, preparation, and shipping specific to your sample type and volume.

Please see our page for Direct Input RNA-Seq for Low Sample Quantity.

What is rRNA depletion and why would I want to have this process performed on my samples?

For many species, rRNA constitutes 70-80% of total RNA. Most of these are not poly-adenylated, and will not significantly impact polyA-based cDNA preparation methods. However, these molecules will be reverse transcribed when a random-primed cDNA preparation method is used. As a result, if these molecules are not depleted from a sample the vast majority of your data would be rRNA. If you are not interested in rRNA you will obtain very little useful information, and the data you would have would be on only the most abundant transcripts. We use a hybridization method to deplete rRNA from total RNA samples, specific to eukaryotic or bacterial species, and depletion is reported to be greater than 95% using these methods.

Does Otogenetics offer globin RNA depletion from whole blood total RNA samples?

Otogenetics recommends globin RNA depletion for projects using whole blood total RNA, regardless of the cDNA preparation method used. We use a hybridization method to deplete globin RNA from total RNA samples and depletion is reported to be greater than 95% using these methods.

What makes small- and microRNA sequencing distinct?

All the approaches to isolate small and microRNA purification are based on size-selection. Otogenetics offers this service at $1,498 per sample. The protocol used by Otogenetics for small- and microRNA-seq uses adaptors ligated directly to the RNA in your sample. This allows us to amplify the RNA with universal primers, followed by subsequent size selection of the cDNA to purify the small- and micro-cDNAs from tRNA, rRNA and larger transcripts. Because of this size selection, you will not obtain sequence data on the other populations.

Can I get data on micro- and smallRNAs in the same experiment as larger RNA?

No, the process involved in preparing samples for standard RNA-seq does not retain small cDNAs due to size exclusion. A distinct protocol is used for micro- and smallRNA-seq that selects only for the desired size range of these populations after linkers have been added. This excludes the large RNAs from analysis. If you desire information on both populations, your sample will need to be split for separate processing and each will be treated as a distinct sample for your project.

Payment and Turnaround Time

What is your turn-around time?

For most services, Otogenetics turnaround time for NGS services is 4-6 weeks, but many services are faster. We also offer expedited services to provide the fastest turnaround in the industry if needed.

What forms of payment are accepted and what are the terms?

Otogenetics accepts institutional purchase orders, credit cards and wire transfers. Payment is required prior to data release.

Other

Can you provide me with a list of former customers that recommend your service?

For references please contact us at [email protected]

Can you provide me with a list of publications that reference your service?

Please see our publications page.