Next Generation Sequencing FAQs
What is NextGen sequencing?
Next Generation sequencing, a.k.a. “NextGen”, is the use of instruments designed to sequence thousands or even millions of DNA fragments in a highly parallel fashion. This contrasts with the older automated sequencing, based on Sanger sequencing and computer-interpreted electrophoresis systems, which handle only one or perhaps a few dozen DNA fragments simultaneously.
What NextGen sequencers are available in the UM DNA Sequencing Core?
As of early January, 2018, we have six Illumina HiSeqs (a mixture of Models 2500 “V4” and a 4000). The core has a MiSeq, a NextSeq and the new NovaSeq. Plus a Pacific Biosciences RS II+, an Ion Torrent Personal Genome Machine and an Ion Proton sequencer.
How do the sequencers differ?
The Illumina sequencers, our primary workhorses, as you can imagine based on the number of those we own. They specialize in sequencing hundreds of millions of DNA fragments and returning relatively short sequence data from each (ca 50-600 nt). They generally produce the most sequence data for the least price (but not always!).
The Ion sequencers are also niche specialists. They give read lengths of 100-400 nt t a moderate cost. The company provides some nice support services (experimental design, custom reagents, specialty data handling) that give their system an important edge in certain applications.
Which of the NextGen sequencers should I use for my project?
Each of the sequencers have strengths and weaknesses, and it is important to choose the correct system for your own project. We can help. Please contact the Core (see contact info at the bottom of the page) for consultation. Note, however, that very often our first step will be to send you to a qualified Bioinformaticist, who can field some of your questions that we cannot answer.
How do I submit samples for NextGen sequencing?
Before you can submit samples, it is important that you get help from a qualified and experienced Bioinformaticist with experimental design. In order to fill out our Sample Submission forms, you will need to answer a number of questions that you probably can’t answer without help. You will also need to provide us with the contact information of your Bioinformaticist anyway!
The first time you attempt to submit samples for NextGen sequencing, we will generally question you very closely to make sure that you understand (1) the possible cost of the experiment, (2) the method by which your data will be analyzed and (3) the experimental design parameters. NextGen sequencing can be a huge waste of money if you are not prepared appropriately.
We strongly suggest that you prepare the actual samples before you enter the sequencing request into our website. Please look in the pulldown menus (“Platforms’ or ‘Services” – either will get you to the same places) to find the sequencing method that you and your Bioinformaticist and I have chosen for your project. Those menu pages give important sample preparation details.
Once you are ready to fill out the Sample Submission forms, look for the ‘Submit Samples’ menu pulldown at the top of our home page. Choose the appropriate submission type, and fill out the form. You will be give a tracking number to write on each of your tubes. Please make sure it is written legibly! You will also be give a “Submission ID”, which is a short number from which we can look up (i) who you are, (ii) what type of sequencing you need, and (iii) what sample numbers to expect on your tubes.
What if my sample type is not listed in the submission?
If the prep method for your samples is not listed in the pull down menu under DNA/RNA on the submission form, please write the prep method in the submission notes section. You can also email the core to let them know any changes or oddities in your samples.
How do I submit replacement samples?
If after initial QC you need to submit more of your sample to the core please create a new submission when the new sample is replacing an old one tat you do not wish to move forward with. In the new submission please reference the old sample for each old one being replaced.
How long does the core keep my samples for?
|Due to limited space in the cores freezer’s we can only keep your samples for 6 months. If you would like your samples returned to you after sequencing please contact the core to schedule a day and time to come and pick them up.|
How do I pick up samples after my sequencing is finished?
Due to limited space in the core’s freezer’s we can only keep your libraries/samples for 6 months, after that they will be disposed of. If you would like any part of your samples back please contact the core to schedule a time to come pick your samples. If you have RNA you must come to NRCR to pick them up or we can send them back to MSRB II if they are not RNA.
Volume and Primer requirements for samples.
When your samples have a custom primer, please include this information in the submission notes, along with what read the primer is for.
On the tube with the custom primer in it please submit 30 uL at 100 uM concentration. Please write the concentration on the tube, the PI name, the submission ID number, and the read that the primer is for.
When submitting samples that you have prepared yourself, please submit them in a 1.5 mL tube with a minimum of 20uL.