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.
Will the core send QC results before they start my library prep?
Yes, the DNA Sequencing core will send you RIN scores of your RNA samples when applicable along with concentration. If the quality and quantity of your samples do not meet the requirements for the library prep that has been selected the core will let you know, as well as give you some possible options that you will need to make a decision on how to move forward with your samples.
What are the input RNA requirements for library prep?
TruSeq Stranded, mRNA or Ribo Depleted: 500ng-1ug total RNA in 20ul water
SMARTer Stranded RNA: 100ng-1ug total RNA in 10-20ul water
Small RNA: 100ng-1ug total RNA in 10-20ul water
*These are just general input requirements for the current kits offered by the core. A specific kit may have different requirements, please consult the core or the kits website/tech support for those requirements including, input and quality of RNA.
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 should also email the core to let them know of any changes or oddities to the protocol.
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 that you do not wish to move forward with. In the new submission please reference the old sample for each old one being replaced.
When creating a submission for replacement samples you can submit the number of lanes to be zero (0).
MiSeq and NextSeq run parameters
Since the MiSeq and NextSeq sequencing options are based on the number of cycles in the kits available, please put in your submission notes the length and type of read (Single End or Paired End) you would like for your samples.
For example: 75 High Output kit, you would put in the notes 75 Single Read, or 38 Paired End.
How long does the core keep my samples for?
Due to limited space in the DNA Sequencing cores freezer we only keep your samples for 6 months. If you would like your samples returned to you 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.
Custom Primer requirements for samples.
When samples have a custom primer, please include this information in the submission notes, along with what read the primer is for.
Make sure the primer sequence is compatible with Illumina platforms (contact Illumina Tech support or the primer companies tech support), also check if the primer will interfere with PhiX (a control added by the DNA Sequencing core).
Pleas submit the primer in a 1.5 mL tube.
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.
Volume and concentration requirements for client prepared libraries.
When submitting client prepared libraries, please submit them in a 1.5 ml tube with the unique sample numbers generated after the submission is complete on the tope of the tube in black ink.
Also please submit at least 20-30uL at 10nM for the HiSeq 2500 (V4) and the MiSeq. Submit at least 20-30 uL at 5 nM for the HiSeq 4000 and the NextSeq.
We ask that we have these minimum volumes for the automation that has been created for sample quality control steps.
Does the core run QC on my samples?
Yes. For client prepared libraries and core prepared final libraries the DNA Sequencing core runs two QC measures.
We run the Tapestation/Bioanalyzer for size and general nM concentration of your sample. You will get a PDF report of your final sequencing library from this QC measure, if there is anything amiss the core will point it out before proceeding to qPCR. This QC measure uses 1-2 uL of sample.
The Core will then run qPCR on the sequencing library to get an accurate nM concentration. If there is anything that stands out on this QC measure the core will email the client to alert them to the issue before proceeding with sequencing. This QC measure uses 4uL of sample.
PhiX Control V3
The DNA Sequencing Core uses PhiX control V3 on all of our Next Generation Sequencing platforms (HiSeq 2500, HiSeq 4000, MiSeq, NextSeq and NovasSeq).
This control is used for unbalanced genomes, alignment calculations, quantification efficiency, and a troubleshooting measure for clustering problems.
It is important for the core to know if custom primers will interfere with this control.
What if I want more sequencing?
If you would like/need additional sequencing of samples that have previously sequenced please contact the DNA Sequencing core with the sample numbers and submission ID that you would like additional sequencing for. Please also include the number of additional or total reads you would like for your samples. Please also consult your bioinformatician.
What are the limitations of the sequencers?
HiSeq 2500: A 10% adapter peak threshold, a minimum final sample/pool concentration of 5nM with a total volum of 30uL.
HiSeq 4000: A 2% adapter peak threshold, no free primers peak (50bp), average final library length of 580bp, minimum final sample/pool concentration of 2 nM with a total of 30 uL.
NextSeq 550: A 10% adapter peak threshold, a minimum final sample/pool concentration of 0.5 nM with a total of 40 uL.
MiSeq: A 10% adapter peak threshold
How do I get my Data?
- The Core offers data copy to external drives.
- We bill the cost of the drive to your shortcode, there is no charge for the copy service.
- If you do not chose the external drive option, your data will be posted to a Download Server for ** 2 WEEKS ** before being deleted.
- The core will not archive your data – it will be PERMANENTLY DELETED as necessary to make space for new clients.
If you have problems downloading your data, please contact the core immediately.
Please also make proper backups of your data as we cannot keep data for more than 6 months
What is the cores shipping address?
If you need to ship samples, library prep kit, or sequencing kit to the DNA Sequencing Core please ship it to the below address.
Also if possible please put the name of the PI and the contact at the core that you have been corresponding with. Please email a copy of the shipping notification if available to the core.
Attn: UofM DNA Sequencing Core (Illumina Sequencing)
NCRC Bldg 14 Rm 122
2800 Plymouth Rd
Ann Arbor, MI 48109-2800
How do I provide feed back to the core?
The DNA Sequencing Core appreciates feedback from our clients on the services that we have provided. It helps the core to validate kits, pick run concentrations, pooling strategies, and quality control measures.
Any feedback after analysis would help the core with optimizing protocols for future experiments.
Also if you would like to give a lunch and learn type of presentation to the core about your project(s), please contact us to set up a date and time.
Please send your feed back to firstname.lastname@example.org
Important Miscellaneous information.
The DNA Sequencing core uses a control library called PhiX in all sequencing runs.
If your species of your samples have a know unbalanced genome, for example 30% GC content please let the DNA Sequencing core know in the submission notes.
When labeling your sample tubes please put the unique sample numbers on the tops of the tubes in black marker.