DNA library prep kits we offer:
We offer library prep for gDNA and amplicons with the following kits:
- Ligation Sequencing kit V14 (SQK-LSK114) - gDNA/amplicon library prep w/o barcodes
- Ligation Sequencing Native Barcoding kit 96 V14 (SQK-NBD114.96) - gDNA/amplicon library prep with barcoding (96 indexes)
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Rapid Sequencing Kit V14 (SQK-RAD114) - gDNA library prep w/o barcodes
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Rapid Barcoding Kit 96 V14 (SQK-RBK114.96) - gDNA library prep with barcoding (96 indexes)
Library prep protocols
Users may choose between adapter ligation based or transposase based preps. Both preps are PCR-free, and are available with and without barcoding.
Ligation prep
The Ligation Sequencing Kit V14 offers a flexible method of preparing sequencing libraries from dsDNA. The library preparation method is straightforward: DNA ends are repaired and dA-tailed before the sequencing adapters are ligated onto the prepared ends.
The ligation kit is recommended for users who want to obtain duplex data, and require control over read length.
Rapid prep
The Rapid Sequencing Kit V14 generates sequencing libraries by using a transposase which simultaneously cleaves template molecules and attaches tags to the cleaved ends. Rapid Sequencing Adapters are then added to the tagged ends for sequencing on a flow cell. The Rapid Sequencing Kit is optimized for samples which contain long fragments (>30 kb). Note: This kit is able to generate Q20+ and duplex data but has been optimized for speed and simplicity. If users require high accuracy, we recommend using our Ligation Sequencing Kit V14 (SQK-LSK114).
Sample Requirements
We do not perform DNA isolation, and users therefore need to deliver gDNA or amplicons to the sequencing center that meets the criteria listed in the following section.
Sample Integrity
Gel: Average fragment size, as measured by pulsed-field gel analysis ( >30 kb). To determine the fragment size of the sample you could use: gels (e.g. Bio-Rad CHEF system), Agilent devices (e.g. Bioanalyzer or Tapestation), or FEMTO Pulse from AATI.
Sample Purity
Purity as measured using Nanodrop:
- A260/280 = 1.8
- A260/230 = 2.0-2.2
The majority of RNA should be removed by RNase digestion.
Sample concentrations
| Application | Prep type | Sample amount | Recommended conc. | Min. volume |
|---|---|---|---|---|
|
gDNA |
Rapid | >700 ng | 30 ng/ul | 30 ul |
| Rapid w/barcodes | >1 ug | 35 ng/ul | 30 ul | |
| Ligation | 5 ug | 50 ng/ul | 100 ul | |
| Ligation native barcodes | 3 ug | 100 ng/ul | 30 ul | |
|
Amplicon |
Ligation | 500-1000 fmol | 10 fmol/ul | 100 ul |
| Ligation native barcodes | 800-1000 fmol | 30 fmol/ul | 30 ul |
Accepted buffers
For long-term storage of high molecular weight (HMW) gDNA we recommend the use of TE buffer.
Accepted tubes/plates and labeling
Each tube/plate must be marked with the user’s name and date, in addition to sample name. When submitting >16 samples, use 96-well PCR plates, not 8-well stripes or 0.5 ml tubes, (filling in columns – see figure below). Lower numbers of samples should be submitted in 1.5-ml Safelock Eppendorf tubes (please don`t tape Parafilm around the tubes). We do not accept precipitated/dried samples.
Correct layout for filling samples in 96-well PCR plates (i.e. filled by columns from left side, not by rows). Do not leave empty wells or gaps!
Failure to follow these guidelines voids any guarantees on library preparation or run yields!