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.
| 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 |
| 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 | 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 |
PacBio Sequencing:
| Library type | Lib Prep Kit we will use | Input Type | Input amount recommended by manufacturer | minimum volume | maximum volume |
| 15-20kb insert library | SMRTbell Template Prep Kit 2.0 | gDNA | ≥ 10 ug | 50 µl | 150 µl |
| 4-6kb insert for multiplexed bacterial samples | SMRTbell Template Prep Kit 2.0 | gDNA | min 8 ug for the pool (better 16 ug). If multiplexing 8 samples, we'll need 1ug for each sample. If multiplexing 4 samples, we'll need 2ug per sample... | 50ul per sample | 50 ul per sample |
| Amplicons <2kb in size | SMRTbell Template Prep Kit 2.0 | PCR amplicons | min 500ng | 50 µl | 50 µl |
| Amplicons >2kb in size | SMRTbell Template Prep Kit 2.0 | PCR amplicons | min 1ug | 50 µl | 50 µl |
| IsoSeq | NEB Next single cell/low input cDNA synthesis & amplification module followed by Express Template prep kit 2.0 | Total RNA | min 300ng, ideally 1ug RIN ≥ 7 | 10ul | 10 ul |
We do not accept prepared libraries for PacBio sequencing.
The PacBio template preparation process does not utilize amplification techniques. Therefore, the input DNA quality will be directly reflected in the sequencing results. Any DNA damage (e.g., basic sites, nicks, interstrand crosslinks) or contaminants (e.g., single-stranded DNA, RNA, proteins, dyes, or salts, phenol) present in the input material will impair performance of the system. PacBio recommends Qiagen MagAttract HMW gDNA extraction kits but depending how the organisms you are working with, other methods might be available. Here is a collection of publication describing various extraction methods for PacBio sequencing. gDNA samples need an OD 260/280 ratio of approximatively 1.8 to 2. Once extracted, the DNA should be stored at 4°C as much as possible since freeze thaw cycles may shear the DNA. It can be sent to use on ice packs.
For PCR products, gel cuts may be done, but the gel can not be exposed to ethidium bromide and UV light. Use instead CYBR green and blue light. PCR products must be cleaned prior to submission using either AMPure XP beads or Qiagen PCR cleanup spin columns.
Ensure that your DNA sample:
- Is double-stranded. Single-stranded DNA will not be made into a SMRTbell template and can interfere with quantitation and polymerase binding.
- Has undergone a minimum of freeze-thaw cycles. For HMW DNA it is best to keep the DNA at 4°C.
- Has not been exposed to high temperatures (>65°C for 1 h can cause a detectable decrease in sequence quality).
- Has not been exposed to pH extremes (<6 or >9).
- Does not contain insoluble material, and is not colored or cloudy.
- Does not contain RNA.
- Has not been exposed to intercalating fluorescent dyes or UV radiation.
- Does not contain chelating agents (e.g., EDTA), divalent metal cations (like Mg 2+), denaturants (like guanidinium salts, phenol), or detergents (like SDS, Triton-X100).
- Does not contain carryover contamination from the starting organism/tissue (like heme, humic acid, polyphenols).