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NextSeq 500 sequencing at Oklahoma State Genomics center



Frequently Asked Questions (FAQS)

 

 

 

How much does sequencing cost?
A: It depends. You need to determine how much sequencing you need for us to answer that question. We suggest you visit Illumina's support pages for a "coverage calculator" (http://support.illumina.com/downloads/sequencing_coverage_calculator.html) and their Sequencing method explorer (https://tinyurl.com/lka854b) and their custom protocol selector (https://support.illumina.com/downloads/custom-protocol-selector-link.html). The NextSeq500 produces up to 120Gbp of sequence data from up to 400,000,000 reads. Current pricing for kits is located on our Pricing Page.

How do I submit a sample for sequencing?
A: Please use our online request/submission form at: http://bioinfo.biochem.okstate.edu/index.php?option=com_content&view=article&id=84:submission&catid=1:latest&Itemid=47 (https://tinyurl.com/kb4h8e5) then arrange for your samples to be brought to the Genomics center at a mutually acceptable time.

Can I multiplex samples?
A: Yes and we expect lots of multiplexed samples using indices. Please remember that multiplexed samples should all use the same (single or dual) number of indices, and that they should all have approximately the same total length. We can multiplex, and then demultiplex samples with either 6bp, 8bp or 12bp indices. It is important that we know exactly which index you use for each sample. However, we can only multiplex up to 24 samples simultaneously using Illumina kits. If you make your own libraries with third party kits we can sequence and demultiplex them but can't guarantee results.

What is cross-talk?
A: Cross-talk is when sequencing errors cause reads to placed in the wrong index set after demultiplexing. Some core facilities report up to 0.1% of reads can be incorrectly demultiplexed. Illumina reports that this issue will be dramatically reduced in future releases (2017-2018) of indexing kits. Dual indexing or longer index sequences reduce cross-talk.

How much sample do I need?
A: It depends. Determine which library sequencing kits you need.  After gaining experience, it is clear that you should have at least 150ng of high quality nucleic acid and a minimum concentration of 10ng/ul.  Use Illumina's support pages to help you decide what is best for you.  A local description of the sample amounts needed for different projects is under development.

How can I tell if my sample is good enough to sequence?
A: We suggest you submit gel-images of your samples as requested in the submission request, then pay for the Genomics center to do quality-control on your samples using fluorometric determinations ($4 to $35), Bioanalyzer analyses ($4-$60)*, and qPCR ($20-$40).
*There are multiple options for Bioanalyzer analyses before and after library production.

What happens if my samples are not good?
A: They will be returned to you with an explanation of the QC-results and you will need to pay for the quality control. Samples will have to be resubmitted.

What happens if my samples are good?
A: If you prepared your own libraries, they will enter the sequencing queue. If we are preparing your libraries they will enter the sequencing queue at the library-preparation stage. An invoice for all charges will be sent which is due at the conclusion of sequencing.

Where will my data be stored?
Most data will be streamed to the OSU High Performance Computing Center resources, where it can be copied to your system or backup drive after we send you a direct link for downloading. All data will be immediately compressed and archived in redundant tape archives using the Oklahoma Petastore at OU. 

How long will the Genomics center store my data?
A: We have arranged to store your data locally on the HPCC for one month. After that it will only be available as archived tape storage at the Oklahoma Petastore. If you wish to retrieve your data from the Petastore there may be a $35 charge. If you do not wish for your data to be archived, please let us know and it will simply be deleted from local storage.

Will the Genomics center analyze my data for me?
A: Some analyses can be done at the Genomics Center under collaborative arrangements, or using a fee-for-service of $35 per hour. Unfortunately, our availability is sporadic, and can be limited. Alternatively, analyses can be done by the Bioinformatics specialist in the HPCC. To learn how to analyze your data, we suggest attending bioinformatics workshops, Software/Data Carpentry workshops, or taking online bioinformatics modules as they are developed.

How long will it take to get my sequencing data?
A: It depends. The longest run on the instrument is 28 hours. The longest library preps take 2.5 days. If you want your data rapidly, assemble enough samples to use an entire sequencing run. You may want to mix samples with another researcher and split the costs. Our goal is to eventually run the instrument 3 times a week. Our new robotic sample preparation robot, funded through the OSU VPR office, will greatly facilitate multiplexing samples. However, custom protocols must be written in Javascript. For more information please contact us.


What types of sequencing can you do?

A: Almost any type offered by Illumina up to the limits of the instrument and we will do everything possible to support your project!


Can you perform 16S RNA analyses?

A: Not optimally... Most 16S analyses rely on longer individual sequences than the NextSeq can produce. 16S analyses, are best done with a smaller MiSeq instrument. Contact the Genomics center before starting your experiment and we can discuss options or find you a sequencer elsewhere to use.


I'm confused about....

A: Contact the Genomics center and arrange for a meeting with This e-mail address is being protected from spambots. You need JavaScript enabled to view it or This e-mail address is being protected from spambots. You need JavaScript enabled to view it .

 

"Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation."