Next Generation Sequencing FAQ’s

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ALL Illumina NGS projects require acknowledgment of Bioinformatic support BEFORE WE CAN ACCEPT YOUR SAMPLES. Support can be provided by

The initial NGS project consultation with the Bioinformatics Core is free. Contact them to help choose the best sequencing options before requesting sequencing cost estimates.

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 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. ALL Illumina NGS projects require acknowledgment of Bioinformatic support BEFORE WE CAN ACCEPT YOUR SAMPLES. Support can be provided by

The initial NGS project consultation with the Bioinformatics Core is free. Contact them to help choose the best sequencing options before requesting sequencing cost estimates.

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.

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 3 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 3 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.

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

NovaSeq: TBD

How do I get my Data?

You can choose to download your data from our FTP server or have it copied to an external drive.
If you choose the FTP download option, your data will be posted for 2 weeks before being deleted from the server. To access your data on the server, you will need to download an FTP client. We recommend FileZilla (https://filezilla-project.org/). After installing, open the program and enter the information we provide in the data email into the fields at the top of the window and click ‘Quick Connect’. You will see your data on the right hand side and your local file system on the left.
If you choose the external drive option, we offer new 1TB drives for purchase or your lab can submit a drive to one of our locations (MSRBII or NCRC). New drive purchases will be billed to the shortcode of the sequencing run. If submitting your own drive, make sure the drive is in secure packaging and labeled with the PI name and file format if known. We cannot support mac formats (NTFS is fine). There is no charge for the copy service.
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. After 6 months it will be PERMANENTLY DELETED to make space for new clients.
We can also transfer data directly to the bioinformatics core and MBNI servers.

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 illumina-manager@umich.edu

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.

What if sequencing fails?

An analysis performed by the UMDSC can fail for many reasons, including failures on the Core’s part (e.g. instrument malfunction, Core technician error) and failures on the Customer’s part (e.g. misquantitated sample, misdesigned experiment, impure sample). We will, to the best of our ability, assess whether a failed analysis is due to a failure on the part of the Core, and we may in such cases repeat an analysis at our cost. When, in our sole judgment, a failure is not due to error on the part of the Core, we reserve the right to refuse repeating the analysis. We will not be held liable for analytical failures arising due to errors or problems in the Client’s laboratory. We will not bear the cost of repeat runs unless we explicitly state that we are doing so of our own accord. If, on completion of any repeat, the results prove that the Core was at fault in the original failure, we may at that time choose to absorb the cost of the repeat analysis. Determination of the cause of failure is based solely on the judgment of the Core staff members, and ultimate decision making in such cases resides with the Director of the UM DNA Sequencing Core.