Illumina Input Recommendations
The Advanced Genomics Core prefers >200 ng for most assays. The higher input yields better performing assays and more robust data.
|Illumina Assay||Min Recommended Input||Min Volume||Quality and Concentration Minimums|
|Poly-A (standard) Library Prep||50 ng||25 µl||RIN > 7 and 2 ng/µl|
|Poly-A (low input) Library Prep||1 ng||25 µl||RIN > 7 and 0.05 ng/µl|
|Total RNA* (standard) Library Prep||10-40 ng||25 µl||
DV200 >70% – 0.80 ng/µl (10 ng total)
DV200 50-70% – 0.8 ng/µl (20 ng total)
DV200 30-50% – 1.6 ng/µl (40 ng total)
DV200 < 30% not recommended
|Total RNA* (low input) Library Prep||1-4 ng||25 µl||
DV200 >70% – 0.04 ng/µl (1 ng total)
DV200 50-70% – 0.08 ng/µl (2 ng total)
DV200 30-50% – 0.16 ng/µl (4 ng total)
DV200 < 30% not recommended
|small RNA Library Prep||10 ng||15 µl||if submitting total RNA RIN > 7|
|DNA (standard) Library Prep||100 ng||25 µl||4 ng/µl|
|DNA (low input) Library Prep||0.5 ng||25 µl||0.02 ng/µl|
|User-made Library||10nM||25 µl||< 1% adapter dimer|
*The ribosomal depletion probes used during Total RNA library prep are selected based on the species indicated in the submission form. If both bacterial and human/mouse/rat probes are required, you must include both species in your form or indicate that it is mixed species. If your samples are isolated from blood and you require globin depletion, please indicate that in the submission notes.
- RNA (DNase treated) – molecular biology grade water (NOT DEPC)
- DNA – EB buffer (10mM TRIS (pH= 8.0-8.4)) or molecular biology grade water
- Illumina Libraries – EBT (10mM TRIS + 0.1%Tween20), EB (10mM TRIS)
Note: The presence of EDTA can inhibit enzymatic activity and lead to reduced final yield, failure of library prep, or low and uneven coverage when analyzed bioinformatically.
Oxford Nanopore Input Recommendations
Oxford Nanopore library preparation is sensitive to contaminants that can be revealed by Nanodrop readings. Note that the rapid chemistry is optimized for samples >30kb, while ligation sequencing can accommodate both HMW DNA and amplicons.
|ONT Assay||Recommended Input||Min Concentration||Min Volume||Quality|
|Ligation Sequencing (with or without Native Barcoding)||1-2µg||25 ng/µl||30-50 µl||260/280 ~1.8-2.0
|Rapid Barcoding Sequencing||500ng (≤12 samples)
100ng (>12 samples)
|55 ng/µl (≤12 samples)
6 ng/µl (>12 samples)
|10-15 µl||260/280 ~1.8-2.0
- 10 mM TRIS (pH=8.0-8.4)
Note: We require that you provide us with a minimum 20ul aliquot of your elution buffer, so that we can use it to get accurate nanodrop readings when assessing the purity of your samples. This aliquot should be labeled with your Service Request ID and “EB”.
Other Assays Input Recommendations
|Other Assay||Min Recommended Input||Min Volume|
|Quality Control||NA||3-5 µl|
|BeadArray Genotyping||500 ng||10-15 µl|
|Single Cell/Single Nuclei (targeting 10K)
- 10x Genomics – 1X PBS (calcium and magnesium free) containing 0.04% weight/volume BSA (400 μg/ml). If necessary, PBS can be replaced with most common cell culture buffers.
Note: Nanodrop can overestimate the amount of nucleic acid in a sample since it is not DNA/RNA-specific and can be influenced by the presence of contaminants. Fluorescence-based concentrations can be as much as 50% lower than those determined by nanodrop. If using nanodrop to quantify your samples prior to submitting to the AGC, be sure to at least double the amount of material being submitted.