Next Generation Sequencing FAQ’s
Next Generation Sequencing – Frequently Asked Questions
Q: What are Illumina-ready libraries?
Illumina-ready libraries, also called “Client prepared libraries” or “User-made libraries”, are samples that are indexed and contain full adapters on either end. These samples are ready for final qPCR quantitation and loading on a sequencer.
Q: What is my sample type?
Sample type refers to type of nucleic acid you are submitting. In the case of user-made libraries, this refers to the source of your starting material, e.g. RNA, immunoprecipitated DNA, etc.
Q: What is the difference between low-, mid-, and high-output sequencing?
The core offers several flavors of Illumina next generation sequencing. Low-output sequencing is performed on the MiSeq where less than 25 million reads is sufficient for the project. Low-output sequencing is often used to check libraries or for targeted sequencing experiments when high coverage for a few regions is required. Mid-output sequencing is performed on the NextSeq or the smaller (SP and S1) NovaSeq flow cells. Although more expensive than high-output sequencing, you are purchasing an entire flow cell so turnaround time is faster. High-output sequencing is performed on the larger (S2 and S4) NovaSeq flow cells. Here you purchase 5% increments of the flow cell so you will be combining with other projects and will need to wait for a flow cell to fill before running. High-output sequencing is the most cost effective sequencing. Here is a comparison of our three Illumina sequencers in terms of their output, cycles, and reads.
Q: What are the standard read-lengths per kit?
|Number of Cycles in Sequencing Kit||Standard Read-Length||Available On|
|50 cycles||single-end 50bp||MiSeq|
|75 cycles||single-end 75bp||NextSeq|
|100 cycles||paired-end 50bp||NovaSeq|
|150 cycles||paired-end 75bp||MiSeq,NextSeq,NovaSeq|
|200 cycles||paired-end 100bp||NovaSeq|
|300 cycles||paired-end 150bp||MiSeq,NextSeq,NovaSeq|
|500 cycles||paired-end 250bp||MiSeq,NovaSeq|
|600 cycles||paired-end 300bp||MiSeq|
NOTE: deviating from the standard read lengths requires the purchase of a full flow cell
Q: What is the yield per run type?
|Sequencer||Flow Cell||Reads/Flow Cell|
|NextSeq||mid-output (MO)||130 million|
|NextSeq||high-output (HO)||400 million|
Q: Can I share a flow cell?
Absolutely! In fact, the most cost effective way for the core and all of our clients is to get as many high-throughput submissions on to S4 (and S2 for PE-50) flow cells as we can. This is the Core’s standard operating procedure: we strive to fill up S4 flow cells and get them running ASAP.
Q: Do I have to share a flow cell?
No, but it will probably be cheaper if you do share an S2 or S4 flow cell with other submissions. If you feel you really need your own flow cell, you can submit with low- or mid-throughput sequencing options to get 100% of a MiSeq, NextSeq, NovaSeq SP, or NovaSeq S1 flow cell.
Q: How long do I have to wait if I am on a shared flow cell?
The Core strives to fill flow cells as quickly as possible. We will be running two to six flow cells per week on the NovaSeq. The turn-around time for your samples depends on a number of factors, including the quality of your samples (we often get replacements for low-quality samples) and the sample queue length at the time your submission is ready to run. Generally, we should be able to go from “qc passed” to your data in about 4-6 weeks when you share an S2 or S4 flow cell. If you want to try to get your data faster, you can submit with low- or mid-throughput sequencing options to get 100% of a NovaSeq SP or S1 flow cell.
Q: Why do you take so long?
In almost all cases, the issue is a lag in email response time. The Core notifies you at multiple quality control checkpoints in your sample processing. The faster you get back to us, the quicker your samples move to the next step.
Q: What if my submission doesn’t need PE100 or PE150 reads?
Sequencing more bases than needed will not prevent you from doing your experiment. For example, you can trim away uninformative bases. Many users find that the economy of scale makes it cheaper and faster to get longer reads on NovaSeq S4 flow cell than to run the exact required configuration on HISeq 4000. Alternatively, if you can fill a smaller flow cell yourself, we can work with you to configure a NovaSeq run to your specifications.
Q: What is the minimal increment of a high output run I can purchase?
The minimal fraction of a flow cell available for purchase is 5%. For S2 flow cells, this corresponds to ~380 million reads. For S4 flow cells, this corresponds to ~500 million reads.
Q: What is asymmetric sequencing?
Asymmetric sequencing is when the length of read 1 differs from the length of read 2. With the exception of 10X single-cell projects for Gene Expression 3’ and Immune profiling 5’ projects asymmetric sequencing requires the purchase of a full flow cell.
Q: What are the DNA Prep Options supported by the core?
Q: What are the RNA Prep Options supported by the core?
Q: What are the Single Cell Prep Options supported by the core?
Q: Can I see the sample QC reports before library prep starts?
Yes, the DNA Sequencing core will send you the QC metrics (BioA traces, concentrations, RINs etc.) prior to library prep. If the quality and quantity of your samples do not meet the requirements for standard library prep methods, our highly trained staff can provide you with prep alternatives that would accommodate your samples. You will need to make a decision on how to move forward with your samples.
Q: Can I submit replacement samples?
If after initial QC you decide to submit a replacement sample, please create a new submission request and include the original submission ID in the comments section. For each sample in the submission, please reference the corresponding old sample number in the sample notes field.
Q: Does the core run QC on my final libraries?
Yes, the core runs two library QC assays. The Tapestation/Bioanalyzer is used to determine size and general nM concentration of each sample. This QC measurement consumes 1-2 uL of sample. A PDF report of your final library traces will be emailed to you. The Core also runs qPCR (uses 4uL of sample) 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.
Q: What information about my user-made libraries should I provide during submission?
If you are submitting Illumina-ready libraries, please indicate the type of prep or the kit used to make your libraries as well as any changes/alterations to the standard protocol. This information help the core optimize the conditions in which your libraries are sequenced.
Q: Do I need to remove primer or adapter contamination from my user-made sequencing libraries?
Yes, primer or adapter-dimer contamination can seriously impact your sequencing run. They not only reduce the number of clusters available for your samples, but can lead to problems such as index hopping.
Q: Which indexing scheme should I use for Illumina sequencing?
If you prepare your own libraries, we strongly recommend that you dual index your samples to maximize the uniqueness of your sample identifiers. When possible, the core prefers unique dual indices to minimize index hopping. Please contact us if you have specific indexing questions.
Q: For user-made libraries, what volume and concentration should I submit?
When submitting Illumina-ready libraries, please submit them in 1.5 ml tubes with the unique sample numbers (generated during the submission process) on the top of each tube in black ink.
Each instrument has specific requirements for loading. The minimum volume requirements for each instrument are:
- MiSeq: 30 uL at 10 nM
- NextSeq: 30 uL at 5 nM
- NovaSeq: 30 ul at 10 nM
Q: How do I submit custom primers?
Custom primers require the purchase of a full flow cell. You need to verify that the primer sequence is compatible with Illumina platforms prior to submission. In the notes section, please indicate if the custom primer interferes with the PhiX sequencing control (added to all runs by the core). The primer should be submitted in a 1.5 mL tube – the tube must contain 30 uL of the custom primer at a concentration of 100 uM. The tube should be labeled with the following: primer concentration, the PI, the submission ID, and the read that the primer is for.
Q: Does the Core keep my samples?
The core does not store unused. If you wish to retrieve unused sample, please indicate in the notes section at the time of submission. You will be expected to retrieve your unused sample as soon as you receive notification of final library QC. Excess sample will be automatically discarded 7 days after release of data unless previously picked up.
Q: How long does the Core store final libraries?
The core stores final libraries for 3 months. We strongly encourage you to pick up your libraries. If you would like your libraries returned to you, please contact the core to schedule a day/time for pick up at NCRC or delivery to a U-M sample drop off location.
Q: What data will I receive for Illumina sequencing?
You will receive demultiplexed fastq files (gzip compressed), containing only the reads from clusters passing the Illumina quality filter. By default, we do not trim the sequencing data. Unless purchasing a full flow cell, we are no longer able to deliver fastq files associated with the unknown bin or raw bcl files.
Q: 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 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.
Q: What if I need additional sequencing for my samples?
If you would like additional sequencing of samples previously sequenced, please contact us with the sample numbers and submission ID. Include the number of additional reads you would like for your samples. We will contact you with an estimate of additional costs.
Q: What if I need Bioinformatics Help?
An initial NGS project consultation with the UM Bioinformatics Core is free.
Q: What is PhiX?
PhiX is bacteriophage DNA. It contains roughly equal numbers of A, C, T, and G. The Core uses PhiX control V3 on all of our next generation sequencing platforms. This control is used for unbalanced genomes, alignment calculations, quantification efficiency, and as a troubleshooting metric. It is important for the core to know if custom primers will interfere with this control.