Nanopore is a comparatively new sequencing platform and researchers are nonetheless making an attempt to optimize the protocol for their very own particular purposes. In our lab, we work primarily with metagenomic samples and use the 1D sequencing kits. Over the previous yr, we now have optimized this system. To verify the quality of the Nanopore library preparation we verify between steps using Qubit and Nanodrop measurements. Nanopore library quality principally is dependent upon the individual researcher’s technical expertise and pipetting method. In this article, we talk about some tips that could perform a successful NGS library preparation for metagenomics sequencing on the MinION platform.
- 1 Use High-Quantity Starting Materials
- 2 Use Excessive-High quality Nucleic Acids
- 3 Think about the optimum DNA fragment measurement
- 4 Improve Your Incubation Occasions
- 5 Use Recent Beads
- 6 Reduce Tube Modifications Throughout Nanopore Library Preparation
- 7 Combine Your Samples Completely… however Rigorously!
- 8 Optimize Your PCR
- 9 Prep Your DNA Before Ordering a New Flowcell
- 10 Make Positive You Have a Good Internet Connection
- 11 Widespread Practices to Maintain in Mind for Nanopore Library Preparation
Use High-Quantity Starting Materials
The really helpful starting material for DNA sequencing is <1 ug. Nevertheless, we now have observed in our experiments that with slightly greater quantity (around 1.5 occasions) of DNA, the final library concentration can be inside the desired vary. It is a well-known undeniable fact that larger the library focus the better the sequencing end result. We have now noticed the identical with our own experiments. However, it isn't all the time straightforward to get top quality DNA in enough focus. Pooling of samples, if attainable, might be finished totry and improve the focus. Alternatively, you need to use PCR adopted by purification of desired bands using gel extraction kits.
Use Excessive-High quality Nucleic Acids
We advocate using a fluorometer to measure DNA/RNA concentrations as Nanodrop readings for concentration shouldn’t be all the time dependable. Nevertheless, the absorbance ratios at 260/280 and 260/230 measured in Nanodrop are very helpful. The 260/280 ratio for DNA should all the time be within the vary of 1.7 – 1.9. The closer it is to 1.eight, the better the outcomes. The 260/230 ratio ought to larger than 2.zero. For RNA, the 260/280 ratio ought to be round 2.0 and the 260/230 ratio ought to be above 2.zero. Learn extra concerning the strengths and limitations of your nanodrop.
If attainable, the standard of DNA/RNA also needs to be measured using Bio-analyser/Tape station along with traditional strategies. As there are not any steps to examine the quality as soon as the library preparation starts, it’s higher to start out with a high-quality sample fairly than looking for issues in case of poor sequencing knowledge. Actual time PCR kits are available to verify library prep high quality but this provides to the price and time of experiment.
Think about the optimum DNA fragment measurement
The DNA fragment measurement also impacts the quality of the sequencing reads and we spent a number of our time determining the optimum fragment measurement for Nanopore sequencing.
We found that the optimum measurement was between 3 Kb and 8 Kb. Fragments smaller than 1-2 Kb enormously decreased the overall sequencing high quality, whereas larger molecular weight DNA fragments (10 Kb to 40 Kb that are widespread in guide DNA preparations) do not intrude with general high quality of knowledge. Some Nanopore users have reported clogging of pores when utilizing excessive molecular weight DNA and advocate the new move cell wash package to revive the clogged pores and put together for the subsequent run. Stream cell washing is beneficial after each run and latest package supposedly provides better yields when combined with a nuclease flush step.
The objective of Nanopore is lengthy learn sequencing however how long the reads ought to be (10 Kb) is up to the scientist to determine depending on their individual experiment parameters. It must be noted that the overall fragment measurement distribution amongst all the samples in a single batch ought to be comparable. Fragment size normalization and optimization will assist to scale back the sequencing bias of heterogeneous samples. Steps ought to be taken to take care of a uniform fragment length distribution and ought to be normalized across samples. Fragment measurement additionally affects the effectivity of PCR barcoding steps. Longer fragments are troublesome for PCR barcoding. End prep is another necessary step. Barcoding step impacts the general end result of the sequencing. Inadequate barcoding leads to lack of beneficial knowledge.
Improve Your Incubation Occasions
We now have noticed that incubating the samples with beads for a longer period provides better results. After mixing with the beads, we left the tubes idle for twice as long as instructed by the manufacturer’s protocol.
Use Recent Beads
AMPure XP beads makes use of an optimized buffer to selectively bind DNA fragments 100 bp and bigger to paramagnetic beads. Excess primers, nucleotides, salts, and enzymes could be removed utilizing a simple washing process. Prepare recent 70% ethanol each time to avoid dilution impact upon reuse because of the hygroscopic nature of 70% ethanol. We found that this easy step dramatically improves the bead utilization.
Reduce Tube Modifications Throughout Nanopore Library Preparation
We might advocate not transferring DNA from one tube to another until it’s absolutely mandatory. For example, when eluting DNA from beads, it’s essential to acquire the eluent in a recent new sterile tube. This is primarily to keep away from lack of pattern throughout library preparation. The tubes and ideas usually are not utterly immune to liquid sticking on the walls, and we really feel it is higher to keep away from this relatively than to attempt to retrieve the lost pattern. Good pipetting practices will aid you to attenuate quantity loss during such transfers. Additionally, we advocate utilizing DNA LoBind Eppendorf® tubes for all the steps.
Combine Your Samples Completely… however Rigorously!
The tube contents and reagents must be combined properly. Although vortexing shouldn’t be really helpful, mild vortexing could be finished within the initial steps. Probably the most applicable technique for mixing is flicking the tubes. Pipette mixing can be carried out, however this is not really helpful as repeated pipetting might end in shearing of the DNA fragments affecting the dimensions distribution. Once more, proper pipetting expertise are required to ensure that the sample is processed optimally.
Optimize Your PCR
In our experiments, we now have observed that for PCR barcoding, the variety of cycles might be doubled to 30 cycles. We did not get sufficient output with the beneficial number of cycles (12 to 15 cycles). This may increasingly range relying on the sample sort and protocol however there isn’t a hurt in programming further PCR cycles. All the time verify with Nanodrop and Qubit before and after such procedures to make sure consistency.
Prep Your DNA Before Ordering a New Flowcell
One of many main problems that we confronted, aside from the issues in Nanopore library preparation, was decrease pore rely of the MinION flowcell. Although the corporate claims that there are 2048 pores, by the point we obtain the flowcells, the rely of pores is right down to less than 1400. Having fewer pores immediately impacts the quality and quantity of sequences/reads within the output. Because the number of pores reduces, so does the arrogance on the quality and amount of the output knowledge. Each day we lose a bunch of pores as these are organic and never chemical in nature and the longer the flowcell sits within the fridge the decrease the capacity. So, we highly advocate getting ready your DNA samples first – before ordering a new flowcell. As soon as you obtain it, you must have the ability to begin the experiment instantly.
Make Positive You Have a Good Internet Connection
It have to be famous that the newest MinION flowcell can generate 30 GB raw knowledge and close to 5 GB FASTQ knowledge after basecalling. We have now confirmed these numbers from our personal experiments even at low pore counts of 1500 per flowcell. You want a high-speed broadband web connection for real time basecalling. We advocate this strategy as you’ll be able to constantly monitor the info generated and cease the run when adequate knowledge is collected. You possibly can instantly wash the flowcell and prime it for the subsequent batch. This manner you possibly can reuse the movement cells for no less than three library preps making it more economical in comparison with different sequencing platforms.
Widespread Practices to Maintain in Mind for Nanopore Library Preparation
Whereas engaged on the varied experiments with Nanopore MinION, we found out that the following practices drastically improved the final knowledge output from sequencing:
- The working bench, pipettes and all the consumables ought to be clear and mud free.
- The reagents stored in -20°C must be thawed on ice before use and must be immediately returned to the right storage space (-20°C) after use – use an ice bucket always.
- The magnetic beads must be normalised to room temperature and resuspended correctly earlier than use.
- Because the library preparation protocols will not be very lengthy, try to end the entire process within someday and start sequencing. Though there are steps after which the pattern might be stored and the protocol continued the subsequent day, it’s higher to keep away from this state of affairs if potential.
- Plan forward of time to make sure all of the gear is available, begin early and finish the complete course of in one go without stops.
We hope these optimization options assist you get previous the training curve a bit of quicker and will let you gather meaningful knowledge with extra ease.
So, what are you waiting for?
Get began with Nanopore library prep and sequencing.