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NextSeq 500 sequencing FAQS





Frequently Asked Questions (FAQS)


1. How much does sequencing cost?

A: It depends. You need to determine how much sequencing you need for us to answer that question. We can 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 reaads.

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 mutally acceptable time.

Can I multiplex samples?

A: Yes and we expect lots of multiplexed samples with indecies. Please remember that multiplexed samples should all use the same (single or dual) number of indecies, and that they should all have approximately the same total length. We can multiplex, and then demultiplex samples with either 6bp, 8bp or 12bp indecies. It is important that we know exactly which index you use for each sample.

What is cross-talk?

A: Cross-talk is when sequencing errors cause reads to placed in the wrong index poll after demultiplexing. The NextSeq has the lowest measured cross-talk of all Illumina instruments and cross-talk is estimated to be  

How much sample do you need?

A: It depends. Determine which library kit, and sequencing kit you need.  TruSeq no-PCR kits require at least 2 micrograms of sample. Nextera kits can use as little as 50 nanograms or even 1 nanogram of sample. Use Illumina's support pages to help you decide what is best for you.

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 $10), 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 QC-results, and you will need to pay for the quality control.

What happens if my samples are good?

A: They will be entered into the sequencing queue starting at the library prep stage, or the sequencing stage (if you have prepared your own libraries). An Invoice for all charges will be produced which is due at the conclusion of sequencing.

Where will my data be stored?

It depends. it can be put directly onto your USB drive if you have a 1-TB USB flash or SSD drive. Most data will be streamed to the OSU-TIGER cloud (part of the Cowboy supercomputer), where it can be copied to your system or backup drive after we send you a direct link for downloading. We hope to be able to transfer your data directly to your home or scratch folder on Cowboy upon request, but this has not been validated yet.

How long will the Genomics center store my data?

A: We will store your data locally on the cloud for one month. After that it will be moved to the archive storage at the Oklahoma Petastore for long-term storage. If you wish to retreive your data from the Petastore we can do it for a $35 charge. If you do not wish for your data to be archived, it will simply be deleted from local storage.

Will the Genomics Center analyze my data for me?

A: It depends. 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 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. Turnaround times will likely be longer as we first start taking samples. For concerns please contact us.

Can you perfom 16S RNA analyses?

A: It depends. Some 16S analyses use longer sequences than we can produce, however, there are shorter 16S regions that are often used, or multiple regions can be used.  Because of the complexity of these projects it is best if you contact the Genomics center before starting your experiment.

I'm confused about....

A: Contact the Genomics center and arrange for a meeting with Dr. Hoyt or Dr. Hwang.