Nucleic Acid requirements for library preparation
suspension buffer:
Samples may be suspended in different types of buffer, including 10mM Tris, dH2O, or Qiagen EB. We do however prefer when samples are suspended in dH2O. It will allow us to concentrate the samples when necessary without having to worry about concentrating the salts present in other types of buffer. High salt (EDTA) concentration can interfere with the various enzymes that are used during library preparation, especially for PacBio sequencing.
Plates and Tubes:
When submitting one sample, 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.
Should you submit more than one sample, please use polypropylene PCR plates. Plates should be labeled with the order number and sample number range clearly marked (e.g 4596_S01-S96), and should be sealed firmly with a foil plate seal capable of withstanding -80 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_S01-S96_Plate1, 4596_S97-S192_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!
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. Please DNAse treat RNA samples before submission.
| Library Type | Lib 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) | Illumina 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) | Illumina 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 µl | 9.5 | ≥ 10 ng |
| smRNA-Seq | QiaSeq miRNA Library Prep | total RNA | 1 ng - 10ng RIN ≥ 7 | 5ul | 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 |
| 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 |