Frequently Asked Questions About Our Products And Services

• General FAQs for Next Generation Sequencing >>

We recommend: New England Biolabs Monarch® Spin gDNA Extraction Kit, QIAGEN Genomic-tip-500/G, QIAGEN MagAttract HMW DNA Kit, QIAGEN Puregene Yeast/Bacteria Kit. In general, all extraction methods that allow the generation of HMW gDNA fulfilling the above requirements can be used. To ensure to extract HMW DNA we recommend

• Wash bacterial cell pellets before DNA extraction with PBS to remove potential inhibitors
• Add 1ml of PBS and resuspend cells by pipetting
• Pellet the cells by centrifugation and discard the supernatant
• Avoid vortexing and fast or unnecessary pipetting; use wide-bore tips only
• Elute in nuclease-free elution buffer, not water
• Avoid over-drying of gDNA
• Do not expose DNA to high temperatures (>37°C) for >1 hour
• Use buffer with appropriate pH 7.5- 8.5
• Avoid intercalating fluorescent dyes, or UV radiation
• Avoid freeze-thaw cycles; store gDNA at 4°C for 1-2 months

Short DNA fragments are typically not ideal for long-read sequencing, as they can result in poor assembly quality. We recommend that at least 50% of the DNA is above 15 kb in length. To improve sequencing outcomes, we recommend removing shorter DNA fragments from your sample before submission. One effective protocol involves using 4X diluted SPRISelect beads (35% volume by volume) to eliminate fragments smaller than 3-4 kb.

Please note that the DNA quantity will decrease following this process, so it’s essential to quantify the remaining DNA to ensure sufficient material is available for sequencing.

• Transfer each sample into a clean 1.5 ml Eppendorf DNA LoBind tube.
• Dilute SPRISelect beads with Elution Buffer to 35% (volume by volume).
• Resuspend the diluted SPRISelect beads by vortexing.
• Add 4x volume of the resuspended diluted SPRISelect Beads to each gDNA and mix by flicking the tube.
• Incubate for 5 minutes at room temperature.
• Prepare sufficient fresh 80% EtOH in nuclease-free water for all of your samples.
• Spin down the samples and pellet the beads on a magnet until the eluate is clear and colourless. Keep the tubes on the magnet and pipette off the supernatant.
• Keep the tube on the magnet and wash the beads with freshly prepared 80% EtOH without disturbing the pellet. The EtOH volume must be enough to cover the beads entirely. Remove the EtOH using a pipette and discard.
• Repeat the previous step.
• Briefly spin down and place the tubes back on the magnet for the beads to pellet. Pipette off any residual EtOH. Allow to dry for 30 seconds, but do not dry the pellets to the point of cracking.
• Remove the tubes from the magnetic rack and resuspend the pellet in e.g. 50 µL nuclease-free 1xTE or EB. Spin down and incubate for 10 minutes at 37°C with gentle agitation (700rpm) on a shaker.
• Pellet the beads on a magnet until the eluate is clear and colourless.
• Remove and retain the eluate into a clean 1.5 ml Eppendorf Safe Lock Tubes™

Alternatively, we recommend the DNA clean-up and size selection for long-read sequencing from Jones et al. 2021 (Jones A, Torkel C, Stanley D, Nasim J, Borevitz J, et al. (2021) High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing. PLOS ONE 16(7): e0253830. https://doi.org/10.1371/journal.pone.0253830; https://www.protocols.io/view/dna-clean-up-and-size-selection-for-long-read-sequ-261ge6ymol47/v4)

Purity:

• Phenol, chloroform and other related reagents will inhibit the library preparation and must be completely removed. Although spectrophotometric measurements may not detect all types of contamination, we highly recommend performing them to assess sample purity. Ideally, the 260/280 ratio should be above 1.8, and the 260/230 ratio should fall between 2.0 and 2.2. If the sample is contaminated and does not meet this metric, either re-extract the sample or clean up the sample to remove the contaminants using a Qiagen cleanup kit or AMPure XP beads.Must not contain RNA; we strongly recommend RNase treatment during extraction
• Must not contain denaturants (guanidinium salts, phenol, etc.) or detergents (SDS, Triton-X100, etc.)
• Must not contain residual contaminants from the organism/tissue (heme, humic acid, polyphenols, etc.)
• Must not contain insoluble material or be colored or cloudy

Quantitative assessment 

Preferred measurement method: fluorescence-based methods like Qubit® (Invitrogen, Life Technologies), Quant-iT™ (Invitrogen) or Quantifluor (Promega). 

We strongly discourage using Nanodrop for gDNA quantification.

HMW gDNA often requires extra homogenization effort (longer incubation time, increased incubation temperature, very extensive gentle mixing, etc.) to obtain accurate quantification. If separate DNA quantifications from the top and bottom of the sample are within 15% of each other, this is usually a good indication of adequate homogeneity.

If you have less than the requested amount of gDNA, we strongly recommend that you perform additional extractions to increase yield.

Qualitative assessment 

Preferred measurement method: capillary electrophoresis-based methods like Fragment Analyzer or Bioanalyzer. High molecular weight DNA is greater than 15 kb in size and shows minimal smearing. Contamination, damage and degradation are revealed through a low molecular weight smear and should be removed using alternative cleanup strategies as described above. 

No, this is not recommended. Optimal results can be achieved when the fragmentation is done directly before the library preparation. It is recommended to send unfragmented HMW DNA, as large as possible.

Depending on your needs we recommend the following coverage: 

• Germline/frequent variant analysis: 20-50x 
• Somatic/rare variants: 100x
• De novo assembly: 100x

Sequencing output is dependent on a variety of factors e.g. organism, extraction method, DNA quality, size, handling & storage. Therefore, successful EntryQC cannot guarantee high sequencing yield as not all inhibitors can be detected.

Plants, especially, can contain high levels of secondary metabolites that can interfere with the sequencing process. These compounds can be challenging to detect and remove during preprocessing, leading to potential issues in sequencing yield and data quality.

Based on our experience, you can generally expect around 50-80 Gb of data per PromethION flow cell and approximately 6-7 Gb per GridION flow cell. These yields are dependent on factors such as DNA quality and fragment length.

According to Oxford Nanopore's specifications for the chemistry and flow cells we currently use, raw read accuracy is >Q20 (>99%) and the consensus accuracy is typically greater than 99.99% respectively.

See https://nanoporetech.com/platform/accuracy

The prevalent error modes in Oxford Nanopore sequencing include deletions within homopolymer stretches, errors occurring at the central position of the Dcm methylation site CCTGG or CCAGG, and errors at the Dam methylation site GATC.

So please handle these regions with special care.

No, we do not have any guarantees for assembly quality. However, using high-quality input DNA with small and not overly complicated genomes, a single contig is usually generated.

Available on request.