Detailed information for our stable of instruments (Illumina, Oxford Nanopore) and library preparation options. Come try our new GridION!
The core offers several flavors of Illumina next-generation sequencing.
- Standalone Runs - 1 million to 10 billion reads.
- Shared Runs - performed on the 10B NovaSeq flow cells. You can purchase 2.5% increments of the 300-cycle flow cell, which means you will be combining with other projects and will need to wait for a flow cell to fill before running. Shared run sequencing is the most cost-effective sequencing.
Run configurations and expected yield are shown in the table below. These specifications are based on a control library, Illumina PhiX. Actual yields will vary depending on the library type, the library complexity, the insert size, and the GC content. The core makes every effort to target the reads listed ± 20% for all runs.
| NovaSeq X Shared Flow Cell | ~Number of Raw Reads | Cycles | Default Run Type |
| 10B | 10 Billion Reads | 300 | PE150 |
| MiSeq Flow Cells | ~Number of Reads | Cycles | Default Run Type |
| Nano | 1 Million Reads |
300 |
PE150 |
| Micro | 4 Million Reads | 300 | PE150 |
| V2 | 13 Million Reads |
50 |
SR50 |
| V3 | 25 Million Reads |
150 |
PE75 |
| NextSeq 2000 Flow Cells | ~Number of Reads | Cycles | Default Run Type |
| P1 | 100 Million Reads |
100 |
PE50 |
| P2 | 400 Million Reads |
100 |
PE50 |
| P3 | 1.2 Billion Reads |
50 |
SR50 |
| NovaSeq 6000 Flow Cells | ~Number of Reads | Cycles | Default Run Type |
| SP | 800 Million Reads |
100 |
PE50 |
| S1 | 1.8 Billion Reads |
100 |
PE50 |
| S2 | 4 Billion Reads |
100 |
PE50 |
| S4 | 8-10 Billion Reads |
200 |
PE100 |
| NovaSeq X Plus Flow Cells | ~Number of Reads | Cycles | Default Run Type |
| 1.5B | 1.6 Billion Reads | 100 200 300 |
PE50 PE100 PE150 |
| 10B | 10 Billion Reads | 100 200 300 |
PE50 |
| 25B | 26 Billion Reads | 300 | PE150 |
For WGS, the core supports different options, including standard DNA and PCR-Free. Standard DNA preps are ideal for large genome sequencing when the amount of available DNA is limited. The PCR-Free option enhances the ability to sequence through more challenging genomic regions but requires at least 1ug of intact genomic DNA.
Kits:
- Standard WGS – NEBNext Ultra II FS DNA Library Prep Kit (NEB, E7805)
- High-throughput WGS – QIAseq FX DNA library kit A-D (384) (Qiagen, 180487)
| Illumina Assay | Input Range | Max Volume | Concentration Minimum |
| Standard Whole Genome | 10 – 500ng | 35 µl | 4.0 ng/µl |
| High Throughput Whole Genome | 1ng – 1µl | 30 µl | 0.1 ng/µl |
Whole-exome sequencing uses biotinylated oligonucleotide probes to capture only the protein-coding regions (gene exons) from the genomic DNA library. Unlike WGS, only the protein-coding genes are sequenced thereby reducing the amount of sequence needed per sample. Our preferred WES option uses the Twist panels, but we do work with other probe sets when requested.
Kits:
- Twist Library Preparation EF Kit 2.0 (Twist, 104207)
- Twist Human Exome 2.0 (Twist, 104134)
- Twist Mouse Exome (Twist, 102036)
| Illumina Assay | Min Recommended Input | Volume | Concentration Minimum |
| Whole Exome | 100 ng | 25 µl | 4.0 ng/µl |
Targeted sequencing is used when deep sequencing depth over specific genomic regions is desired. There are two methodologies for approaching targeted sequencing assays: probe-based capture sequencing or amplicon sequencing. We can help you select a commercially available panel or design a custom solution to interrogate your genes/regions of interest. Please contact the core for more details. Please note input requirements will depend on your custom assay.
Although we do not perform ChIP or 3C/4C/5C/HiC assays in the core, we do prepare the Illumina libraries from DNA obtained from these and other protocols. Please contact the core to inquire about prep options.
Kits:
- NEBNext Ultra II DNA Library Prep Kit (NEB, E7645)
| Illumina Assay | Min Recommended Input | Volume | Quality and Concentration Minimums |
| DNA (low input) Library Prep | 0.5 ng | 25 µl | 0.02 ng/µl |
Messenger RNA accounts for <5% of the RNA in cells. Thus, for transcriptome or gene expression studies, enrichment of samples for mRNAs is preferable. Poly-A selection, in which poly-adenylated transcripts are selected via hybridization to poly-T oligos bound to magnetic beads, is the most commonly used method for enriching mRNA from total RNA samples. For low input (<200ng) samples, the Smart-Seq method starts by hybridizing the RNA to an oligo(dT)-containing primer and then uses a template-switching mechanism to generate full-length cDNA. Both of these polyA-dependent methods generate the highest percentage of reads mapping to protein-encoding genes and thus are the first choice for many investigators. Poly-A enrichment requires high-quality total RNA samples so it is not suitable for FFPE or otherwise degraded samples.
Kits:
- Standard – NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, E7490) and NEBNext UltraExpress RNA Library Prep Kit (NEB, E3330)
- Low input – SMART-Seq v4 PLUS Kit (Takara, R400753)
| Illumina Assay | Min Recommended Input | Volume | Quality and Concentration Minimums |
| Poly-A (standard) Library Prep | 50 ng | 25 µl | RIN > 7 and 1 ng/µl |
| Poly-A (low input) Library Prep | 1 ng | 25 µl | RIN > 7 and 0.095 ng/µl |
Ribosomal RNA depletion is applied when transcripts do not carry polyA (e.g. bacterial RNA), when you want to characterize long non-coding RNA (lncRNA) as well as mRNA, or when your RNA is degraded. There are two methodologies for removing ribosomal RNAs (rRNA) from your total RNA. The more common option is a subtractive hybridization method. Suitable for samples with RINs>5, this method uses oligonucleotide probes and magnetic beads to capture and remove the rRNA from the sample. More appropriate for degraded samples (RINs<5) is the RNaseH method which enzymatically degrades rRNA targeted by complementary oligonucleotides.
Kits:
- Standard H/M/R – NEBNext rRNA Depletion Kit (NEB, E6310) and NEBNext UltraExpress RNA Library Prep Kit (NEB, E3330)
- Low input – SMART-Seq Stranded Kit (Takara, 634444)
- Bacterial – NEBNext rRNA Depletion Kit (NEB, E7850) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, E7760)
| Illumina Assay | Min Recommended Input | Volume | Quality and Concentration Minimums |
| Total RNA (standard) Library Prep | 100-400 ng | 15 µl | DV200 >70% – 6.6 ng/µl (100 ng total) DV200 50-70% – 13.3 ng/µl (200 ng total) DV200 30-50% – 26.7 ng/µl (400 ng total) DV200 < 30% not recommended |
| Total RNA (low input) Library Prep | 1-4 ng | 15 µl | DV200 >70% – 0.06 ng/µl (100 ng total) DV200 50-70% – 0.13 ng/µl (200 ng total) DV200 30-50% – 0.27 ng/µl (400 ng total) DV200 < 30% not recommended |
Due to the size, small RNA is lost during most library prep procedures. For small (micro) RNA, we offer library construction using the SMARTer smRNA-Seq Kit . Small RNA preps can start from either enriched small RNA or directly from total RNA (RIN>7). The minimum amount of total RNA is 100ng, but we recommend starting 500ng-1000ng if possible as the fraction of small RNA in a sample can vary between tissues/organisms.
Kits:
- SMARTer smRNA-Seq Kit (Takara, 635030)
| Illumina Assay | Min Recommended Input | Volume | Quality and Concentration Minimums |
| small RNA Library Prep | 10 ng | 15 µl | if submitting total RNA RIN > 7 |
Absolutely! In fact, the most efficient way for the core operate is to get as many projects on S4 flow cells as we can. This is the Core’s standard operating procedure: we strive to fill up 300 cycle S4 flow cells and get them running ASAP.
The Core strives to fill flow cells as quickly as possible. We run 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 a 300-cycle S4 flow cell. If you want to try to get your data faster, you can submit with low- or mid-output sequencing options to get 100% of a flow cell.
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 as a previous experiment. Alternatively, if you can fill an entire flow cell yourself, we can work with you to configure a NovaSeq run to your specifications.
The minimal fraction of a 300 cycle S4 flow cell available for purchase is 2.5%, which corresponds to ~200-250 million reads.
Yes, the Advanced Genomics core will post your QC metrics (BioA traces, concentrations, RINs etc.) in the attachment section of your MiCores request 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.
If after initial QC you decide to submit a replacement sample, please contact the core via your MiCores request so additional samples can be added to the request.
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.
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 helps the core optimize the conditions in which your libraries are sequenced.
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.
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 service request ID, and the read that the primer is for.
| RNA Sequencing Method | polyA-enrichment | ribo-depletion | small |
| Starting Material | DNase-treated Total RNA | DNase-treated Total RNA | DNase-treated Total RNA |
| RNA Quality | RIN >7 | DV200 > 30% | RIN >7 |
| Preferred Input | 200 - 1000ng | 200 - 1000ng | 100 - 1000ng |
| Preferred Volume | 25ul | 25ul | 25ul |
| Minimum Input | 1ng | 1ng | 10ng |
| Recommended Minimum Reads/Sample | 30 million | 45 million | 15 million |
| Recommended Sequencing Platform | NovaSeq | NovaSeq | NextSeq or NovaSeq |
| Recommended Run Configuration | PE150 | PE150 | SR50 (can be longer) |
| Deliverables | fastq files, count matrix | fastq files, count matrix | fastq files |
| Estimated per sample cost (assumes 12 sample submission) | $274 | $389 | $287 |
The Advanced Genomics Core offers long-read DNA and RNA sequencing on the Oxford Nanopore Technology PromethION platform. While ONT technology allows sequencing of nucleic acid molecules with lengths greater than 100,000 bp, typical samples produce read lengths of ~ 40,000 bp. The length of an ONT read is dependent on the quality of the sample and the preparation method. There are a variety of protocols available enabling sequencing of genomic DNA, PCR products, methylated DNA, cDNA, and RNA. ONT reports good results with high molecular weight gDNA when the average fragment size is >30kb.
Oxford Nanopore library preparation is sensitive to contaminants that can be revealed by Nanodrop readings. The following is quoted from ONT’s QC protocol:
- We recommend that sample DNA has a 260/280 ~1.80, and a 260/230 ~2.0-2.2
- A 260/280 which is higher than ~1.8 indicates the presence of RNA
- A 260/280 which is lower than ~1.8 can indicate the presence of protein or phenol
- A 260/230 significantly lower than 2.0-2.2 indicates the presence of contaminants, and the DNA may need additional purification
The following extraction methods are recommended:
- NEB Monarch HMW DNA Extraction Kit
- Circulomics
- Qiagen Genomic-Tip
The following extraction methods are NOT recommended:
- Phenol-chloroform
- Spin-column based methods (will reduce fragment length)
Other species- and sample-specific extraction methods developed for ONT sequencing can be found on the ONT community website (which does require you to log in with a free account) here: https://community.nanoporetech.com/extraction_methods
| MinION | PromethION | |
| Guaranteed minimum of available pores | 800 pores | 2675 pores |
| Sequencing life under optimal conditions | ~48 hours | ~72 hours |
| Flow cell can be washed, allowing for multiple runs | Yes | Yes |
We highly recommend you join us at one of our Monthly Workshops to discuss your project and experiment design.
Nanopore sequencing at the Advanced Genomics Core begins with the purchase of a ONT flow cell. Each flow cell can be expected to have a minimum of pores and a sequencing lifespan of approximately 48-72 hours when run with high-quality, chemically pure samples. Often, we can get more sequencing time out of the flow cell, so long as no contaminants were present in the original samples. Contaminants in samples can damage the pores irreversibly, significantly decreasing the sequencing life. With the 48+ hours of sequencing lifespan, you have several options, discussed in detail in the following sections.
Ligation Sequencing
Ligation sequencing is the most popular ONT library prep we offer. It can sequence gDNA, cDNA, or amplicons of any size. This PCR-free method generates 1D reads equal to the fragment length. For clients who are seeking long or full-length reads, this method is ideal. ONT currently rates this library prep method for one of their highest throughputs, so you will get the most amount of data using this kit; however, it is important to realize that long reads are negatively correlated with throughput due to physical complications with sequencing very high molecular weight DNA.
For the longest reads, we recommend starting with 1-3 ug of gDNA, with fragment sizes >30 kbp. With 1 ug, we are usually able to produce enough library for a single run on a GridION flow cell, but with 3 ug we are usually able to produce enough for 2-3 runs from a single prep. Long fragments tend to clog the pores on the flow cell quickly, so having enough library generated to wash and reload improves output. Sometimes, we may use up the library generated from the first prep, but there will still be pores available for sequencing left on the flow cell. At this point, we will reach out and see if you would like us to re-prep more library, if the sample remains, and continue sequencing.
For cDNA or amplicons, we recommend starting with 100-200 fmol, which typically produces plenty of library for 2+ runs on a GridION flow cell. Since shorter fragments do not clog flow cells as quickly as long fragments, we do not need to wash and reload as often, and therefore a single prep of cDNA or amplicons is usually enough to use up a flow cell’s sequencing life.
Rapid Sequencing
Rapid sequencing is the second most popular ONT library prep we offer. It is recommended for only gDNA with fragments >30 kbp. Unlike with ligation sequencing, this PCR-free kit generates libraries using a transposase, which cleaves the gDNA at random loci to attach adapters. Due to the difference in chemistry, you can expect 1D reads of slightly shorter length than the input. Some protocols do use this kit to generate ultra-long reads by starting with many micrograms of extremely long fragments; please reach out to the AGC prior to submission if you are interested in this. ONT rates this kit for intermediate throughput, so the expectation is lower than the ligation sequencing kit.
For this kit, we recommend starting with 400 ng gDNA, which produces enough library for a single run on a GridION flow cell. The limiting factor in this kit is the transposase adapter, so we are not able to produce more than one run’s worth of library from a single prep. This does mean that read length is correlated to input amount; if we start with a large amount of long DNA, the fragments produced will be longer.
Direct RNA Ligation (SQK-RNA002)
The Direct RNA library preparation is the only RNA sequencing protocol that we offer currently. This library prep enables sequencing of RNA molecules directly. This sequencing method has two advantages – 1) end-to-end transcript sequencing allows quantification of long-range modifications like isoforms and fusion transcripts with ease, and 2) direct RNA sequencing allows estimation of base modifications, like pseudouridine, 5mC and m6A. This prep is PCR-free and targets only 3’ poly-A transcripts preferably >200 bp. The library prep method involves an optional reverse transcription that makes an RNA-cDNA hybrid for better throughput, however, only the RNA bases are read during the sequencing. We suggest ONT’s recommended RIN score of above 7 for total RNA, as higher RIN number directly correlates with longer read distribution.
ONT recommends input of 500 ng poly-A enriched RNA in 9 ul for this library prep. However, we will also need to estimate quality with a RIN score for a good sequencing run. RIN score can only be estimated with whole RNA. Due to this reason, we recommend submitting total RNA that contains 500 ng poly-A enriched RNA. According to ONT, 1-5% of total RNA can be poly-A tailed mRNA. With that, we arrive at an estimation of 10-50 ug of total RNA as starting input to obtain 500 ng of poly-A enriched transcripts. We recognize that this is a huge amount to extract, so we ask that you submit as much total RNA as you can for us to ribo-deplete it to ONT’s recommendation. We will reach out to you about your RNA sample quality before sequencing.
For DNA sequencing, both the ligation and rapid sequencing methods allow for multiplexing of samples. You have two options for multiplexing: we can run all samples at once, or we can run them in a series of runs with a nuclease wash between them. The first option is most likely to get you an even number of reads between all samples, while the second option is good if your samples are significantly different from each other (different lengths, different species, etc), since they will not compete with each other for pores.
For direct RNA sequencing, ONT at present does not offer any multiplexing solutions. So all samples will need to be sequenced either on multiple flow cells or one at a time on a single flow cell with a nuclease wash step between each sample.
Our technicians will monitor your flow cell during the run and adjust as necessary to improve output and performance. Below are a few common situations that arise during ONT sequencing:
- When sequencing long fragments, we will see an accumulation of saturated pores that are “clogged” and unable to sequence. The only way we can “unclog” the flow cell is to perform a nuclease wash and reload with more library. If we do not have more library to reload with and there is more of the original sample remaining, we will reach out to you and ask if you would like us to prepare more library and continue sequencing. If we do not have more of the original sample remaining, we will reach out to you and ask if you would like to submit more sample to be sequenced.
- When sequencing shorter fragments, the flow cell will run out of fuel and the translocation speed drops. When this happens, we will pause the experiment, refuel, and resume without needing to reload with any additional library.
- For any size fragments, eventually the number of available pores will decline. Once the number of pores drop below 10-20%, we assume that the flow cell is exhausted. At that point, we will do a nuclease wash and see how many pores are recovered for sequencing. While this is handled case-by-case, generally if the pores stay below 200, we will declare the flow cell and run as finished. Alternatively, if the recovery was higher, we will reload more library and continue sequencing. If we do not have more library, we will reach out to you and let you know how many pores are left so you can decide how to proceed.
Yes, the Advanced Genomics core will post your QC metrics (BioA traces, concentrations, RINs etc.) in the attachment section of your MiCores request 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.
If after initial QC you decide to submit a replacement sample, please contact the core via your MiCores request so additional samples can be added to the request.
University of Michigan
2800 Plymouth Rd.
Ann Arbor, MI 48109-2800
