Please read and follow the instructions for sample requirements. We will return submitted samples that do not adhere to the following requirements. We will not replate or process orders that do not adhere to the following:
Suspension Buffer:
The best suspension liquid is dH2O followed 10mM Tris and Qiagen EB. High salt (e.g. EDTA) concentration will interfere with the various enzymes that are used during library preparation and will most likely need to be cleaned for an extra charge..
Plates and Tubes:
One sample:
Submit in a 1.5mL Eppendorf tube. Make sure to write the sample number listed on your order sample sheet on the top the tube.
Multiple samples:
Submit more than one sample in a clear semi-skirted polypropylene PCR plate. We will not accept full-skirted, deep well, round-bottom, or flat-bottom plates.
Load samples on the plate in columns and not in rows (Sample 1 = A1 should be in well A1, sample 2 = B1, sample 3 = C1, etc).
Please label plates (e.g. Olympus 96-Well PCR Plate, Semi-Skirted (Genesee Scientific # 27-108) with the order number and sample number range clearly marked (e.g 4596_S01-S96) and seal firmly with a foil plate seal capable of withstanding -80o C temperatures (e.g. Thermo Scientific™ Adhesive PCR Foil Plate Seal (Fisher AB-0626)). If you have more than one plate, please also number the plates (e.g. 4596_S01-S96_Plate1, 4596_S97-S192_plate2).
Any plate submitted must be clear and semi-skirted. We will not accept full-skirted, deep well, round-bottom, or flat-bottom plates.
Samples should be loaded on the plate in columns and not in rows. Sample 1 should be in well A1, sample 2 in well B1, sample 3 in C1, etc.
Sample Concentration and Volume:
Illumina Sequencing:
All samples undergo quality check (QC) before going into library preparation. Make sure to include enough volume to allow for 5 uL to be used in QC. The inputs listed below should remain after the completion of this QC.
| Library Type | Library Prep Kit Used | Input Type | Input amount recommended by manufacturer | Minimum Volume | Maximum Volume | Recommended Amount |
|---|---|---|---|---|---|---|
| DNA-Seq | Kapa Hyper Prep | Genomic DNA | 1 ng - 1 ug | 20 µl | 50 µl | ≥ 500 ng |
| DNA-seq (full volume reaction) | llumina DNA Tagmentation -96 | genomic DNA; blood/saliva (extra lysis steps) | 1 - 500 ng | 35 µl | 65 µl | ≥ 100 ng |
| DNA-Seq (1/8 volume reaction) | llumina DNA Tagmentation -384 samples | genomic DNA; blood/saliva (extra lysis steps) | n/a - we start with 12.5 - 62.5 ng | 8 µl | 20 µl | ≥ 12 ng |
| Stranded mRNA-Seq | Kapa Stranded mRNA-seq kit | Total RNA | 100 ng - 4 ug RIN ≥ 7 | 20 µl | 50 µl | ≥ 500 ng |
| Stranded Total RNA-Seq with Ribo-zero | Illumina Tru-Seq Stranded Total RNA-seq kit W/ Ribo-Zero | Total RNA | 100 ng - 1 ug RIN <7 is acceptable | 10 µl | 10 µl | ≥ 500 ng |
| Low RNA input RNA-Seq - Not stranded | Clontech SMARTer Ultra-low input mRNA-seq kit followd by Kapa Hyper Prep | Total RNA | 10pg - 10ng RIN ≥ 7 | 9.5 | 9.5 | ≥ 10 ng |
| smRNA-Seq | QiaSeq miRNA Library Prep | Total RNA | 1 ng - 10ng RIN ≥ 7 | 5 µl | 5 µl | |
| Mate-Pair | Nextera Mate-Pair Sample Prep | Genomic DNA | 4 ug | 20 µl | 48 µl | ≥ 4 ug |
| Blood mRNA-Seq | Nugen Universal Plus mRNA-seq | Total RNA from blood | 10 ng - 1 ug RIN ≥ 7 | 10 µl | 50 µl | ≥ 500 ng |
| ChIP-Seq | Kapa Hyper Prep | ChIP enriched DNA | 1 ng - 1 ug | 10 µl | 50 µl | |
| IDT Human Exome (12 plex) | contact sequencing@duke.edu | |||||
| Twist Mouse whole genome | contact sequencing@duke.edu | |||||
| Agilent SureSelect XT Mouse All Exon | contact sequencing@duke.edu | |||||
| ImmunoSeq Adaptive - Survey Mode | Immunoseq hsTCRB kit + Qiagen Multiplex PCR Plus Kit | Sorted T-cell, PBMCs, Whole blood, Bone marrow, BMMCs, Lymphoid and Non-Lymphoid Tissue, FFPE Tissue, gDNA | <Human TCRB Kit Overview> | 40 µl | 65 µl | <Sample Preparation Guidelines: Human TCRB Kit> |
| ImmunoSeq Adaptive - Deep Mode | Immunoseq hsTCRB kit + Qiagen Multiplex PCR Plus Kit | Sorted T-cell, PBMCs, Whole blood, Bone marrow, BMMCs, Lymphoid and Non-Lymphoid Tissue, FFPE Tissue, gDNA | <Human TCRB Kit Overview> | 75 µl | 100 µl | <Sample Preparation Guidelines: Human TCRB Kit> |
| Methyl-Seq | contact sequencing@duke.edu | |||||
| TruSeq RNA Exome (3 plex) for RNA-seq on FFPE samples | contact sequencing@duke.edu | |||||
| NuGen mRNA-Seq with Anydeplete Globin96 samples | same as blood mRNA-seq | |||||
| Tapestri single cell Targeted DNA-seq library prep | contact sequencing@duke.edu | |||||
| Oxford Nanopore | contact sequencing@duke.edu |
Blood Samples for RNA extractions
All blood samples (2.5ml) should be collected in PAXgene Blood RNA Tubes. Immediately after blood collection, gently invert the PAXgene Blood RNA Tubes 8–10 times. Store the PAXgene Blood RNA Tubes upright at room temperature (18−25 ̊C) for a minimum of 2 hours and a maximum of 72 hours before transferring to freezer. The PAXgene Blood RNA Tubes can be stored at −20o C and below. If tubes are to be kept at temperatures below −20o C freeze them first at −20o C for 24 hours, then transfer them to −80o C.
Tubes should be delivered to us frozen. Note that the frozen PAXgene Blood RNA Tubes are very fragile and break easily.
Tubes containing material for isolation must be clearly labeled with your DUGSIM order number and sample number (“4596_S001”). For human blood samples, please provide us with your IRB number to confirm that your project has the appropriate IRB approval.
Cell pellets for RNA extractions
Cell pellets should have between 1-3 million cells.
Cells should be pelleted in Sarstedt 2.0 mL tubes - cat # 72.693 or 72.608
We accept prepared libraries for sequencing; however, we do not guarantee the sequencing results for any customer prepared libraries.
For MiSeq and NextSeq:
Prepared libraries should be sent at ≥10 nM. A minimum volume of 15 ul is required. Concentration should ideally be determined using fluorometric means (picogreen/Qubit). If you are pooling your libraries, the final pool should also be at at ≥10 nM with a volume of at least 15 ul.
For NovaSeq:
If you are submitting libraries for us to pool, each library to be pooled should be at ≥10 nM and have a volume of 15 ul or more.
If you are submitting already pooled libraries, concentration and volume requirements will change depending on the flow cell you will sequence your library pool on.
| S-Prime and S1 full flow cells | S2 full flow cell | S4 full flow cell | Sequencing by the lane regardless of flow cell type |
|
|---|---|---|---|---|
| Molarity required (in nM) | 20nM | 20nM | 20nM | 10nM |
| Volume required (in ul) | 30ul | 45ul | 100ul | 15ul |
One library
When submitting one library or a few pool of libraries, please submit in a 1.5mL Eppendorf tube. Make sure to write the sample number listed on your order sample sheet on the top the tube.
Multiple libraries
Should you submit multiple libraries for us to pool, please use polypropylene PCR plates. Plates should be labeled with the order number and library number range clearly marked (e.g 4596_L01-L96), and should be sealed firmly with a foil plate seal capable of withstanding -20o C temperatures. We recommend the Thermo Scientific™ Adhesive PCR Foil Plate Seal (Fisher AB-0626). If you have more than one plate please also number the plates (e.g 4596_L01-L96_Plate1, 4596_L97-L192_plate2). For the plates, we recommend the Olympus 96-Well PCR Plate, Semi-Skirted (Genesee Scientific # 27-108).
Any plate submitted must be clear and semi-skirted. We will not accept full-skirted, deep well, round-bottom, or flat-bottom plates.
Libraries should be loaded on the plate in columns and not in rows. Library 1 should be in well A1, Library 2 in well B1, Library 3 in C1, etc.