General Submission Advice
- We will assess the quantity and quality of the DNA/RNA you submit upon receipt and inform you of the results. For this, we use the most sensitive/specific techniques available (Qubit for concentration measurements, TapeStation or Femto Pulse analysis for size distribution and checking for degradation). DNA and RNA samples for short and long read library preparation should be free of degradation.
- Standard gel electrophoresis can be used to check the quality of your sample prior to submission, however, use of a Nanodrop to assess concentration can produce highly unreliable results so we would advise against this where possible.
- If in doubt please contact us regarding your quality analyses.
- For libraries prepared yourselves (‘ready-prepared libraries’), see bottom of is this page.
- Please contact us if you would like to discuss using any protocol other than those listed below. Similarly, if you are wondering which sequencing protocol would be most suitable for experimental goals please don’t hesitate to get in touch for advice.
PacBio Service Requirements
- There are five main PacBio services offered by the facility, with different sample requirements (see table below). These are ‘ideal’ criteria – don’t worry too much if you can’t meet the minimum amounts, concentrations or quality threshold*.
- We recommend you follow this guide when extracting High-Molecular Weight (HMW) DNA for PacBio whole genome sequencing applications: https://www.pacb.com/wp-content/uploads/Technical-Note-Preparing-DNA-for-PacBio-HiFi-Sequencing-Extraction-and-Quality-Control.pdf . Use of alternative methods can have a detrimental effect on library preparation results.
| Method | Concentration req. | Suggested volume | Amount req. per sample | Key QC requirement(s) |
| HiFi (Genomic DNA) | ≥50 ng/µl | ~250 µl | ≥12500 ng (12.5 µg) | DNA Integrity Score (DIN) > 8.0 |
| Ultra-low (Genomic DNA) | ≥5.0 ng/µl | ~15 µl | ≥50 ng | DNA Integrity Score (DIN) > 6.0 |
| Iso-Seq (Total RNA) | ≥50 ng/µl | ~15 µl | ≥750 ng | RNA Integrity Score (RIN) > 7.0 |
| Amplicon (amplicons or linearised plasmid) | ~20 ng/µl | ~50 µl | ≥1000 ng (1 µg) per pool or sample | Appropriate size distribution (PCR products should be similar sizes) |
| Iso-Seq Kinnex (Total RNA) | ≥100 ng/µl | ~10 µl | ≥1000 ng | RNA Integrity Score (RIN) > 7.0 |
| 16s Kinnex (Microbiome/environmental DNA) | ~10 ng/µl | ~20 µl | ≥200 ng | DNA Integrity Score (DIN) > 6.0 |
*NB. If you can’t meet these requirements, please contact us in advance of sending samples.
Illumina Service Requirements
There are several Illumina library preparation services offered by the facility, with different requirements:
Method | Sample type | Concentration req. | Suggested volume | Amount req. per sample | Quality recommendation(s) |
TruSeq DNA PCR Free | Genomic DNA | >30 ng/µl | 60 µl | 1.8 µg | DIN ≥5 |
TruSeq DNA Nano | Genomic DNA | >7.5 ng/µl | 65 µl | 488 ng | DIN ≥5 |
Illumina DNA prep (tagmentation) | Genomic DNA | >12 ng/µl | 35 µl | 420 ng | DIN ≥5 |
Illumina DNA PCR Free (tagmentation) | Genomic DNA | >50 ng/µl | 30 µl | 1.5 µg | DIN ≥5 |
Nextera DNA XT | Genomic DNA | >5.0 ng/µl | 10 µl | 50 ng | DIN ≥5 |
TruSeq Stranded mRNA | Total RNA | >30 ng/µl | 55 µl | 1.65 µg | RIN ≥7 |
Illumina Stranded mRNA – ligation | Total RNA | >20 ng/µl | 30 µl | 600 ng | RIN ≥7 |
Illumina Stranded Total RNA – ligation | Total RNA | >85 ng/µl | 20 µl | 1.7 µg | RIN ≥7 |
Illumina Stranded Bacterial RNAseq – ligation | Total RNA | >85 ng/µl | 20 µl | 1.7 µg | RIN ≥7 |
Oxford Nanopore (ONT) Service Requirements
- There are four main Oxford Nanopore library preparation services offered by the facility, with different requirements.
- The standard DNA ligation protocol can be performed with shearing and size-selection or without. Typically, shearing and size-selection produces more data but requires more input DNA.
- We recommend you follow the guides provided by ONT when extracting samples for submission. Guides for several different species and tissue types are provided at the following link (you may have to register): https://community.nanoporetech.com/docs/prepare/extraction_protocols
Method | Sample Type | Concentration req. | Suggested volume | Amount req. per sample | Key QC requirement(s) |
Ligation | Genomic DNA | ≥40 ng/µl | ~50 µl | ≥2.0 µg | DNA Integrity Score (DIN) > 7.0 |
Ligation with Shearing and Size selection | Genomic DNA | ≥25 ng/µl | ~400 µl | ≥10 µg | DNA Integrity Score (DIN) > 8.0 |
Rapid | Genomic DNA | ≥50 ng/µl | ~20 µl | ≥1.0 µg | <30kb fragment size only |
Ultra-long | Genomic DNA | ≥40 ng/µl | ~1.0 ml | ≥40 µg | Most species require ≥50% over 50kb fragment size – can be discussed |
Direct RNA | Total RNA | ≥50 ng/µl | ~20 µl | ≥1.0 µg | RNA Integrity Score (RIN) > 7.0 |
Ready-prepared Libraries for Illumina Sequencing
- We can sequence Illumina libraries you have prepared yourself. Please pool and prepare an aliquot appropriate for the intended flowcell, as specified in the table below
- Please let us know if the libraries require the addition of custom sequencing or index primers for the sequencing run. Please submit 30µl of each primer at 100 µM.
- At present, we only accept user prepared libraries for Illumina platforms and flowcells. This may change in the future. If you would like to submit a library you have prepared yourself for any other platform please contact us to discuss your requirements.
| Flowcell | Concentration req. | Minimum volume | Key QC requirement(s) |
| MiSeq | 5nM | 50 µl | <1% Adapter dimers |
| NovaSeq SP/S1/S2 | 5nM | 100 µl | <1% Adapter dimers |
| NovaSeq S4 | 5nM | 200 µl | <1% Adapter dimers |
NB. for multiple flowcells, please submit multiples of minimum volume.