Nanopore is a relatively new sequencing platform and researchers are still trying to optimize the protocol for their own specific applications. In our lab, we work primarily with metagenomic samples and use the 1D sequencing kits. Over the past year, we have optimized this technique. To check the quality of the Nanopore library preparation we check between steps using Qubit and Nanodrop measurements. Nanopore library quality mostly depends on the individual researcher’s technical skills and pipetting technique. In this article, we discuss some pointers to perform a successful NGS library preparation for metagenomics sequencing on the MinION platform.
Use High-Quantity Starting Material
The recommended starting material for DNA sequencing is <1 ug. However, we have observed in our experiments that with a little higher quantity (around 1.5 times) of DNA, the final library concentration will be within the desired range. It is a well-known fact that higher the library concentration the better the sequencing result. We have observed the same with our own experiments. Nevertheless, it is not always easy to get high quality DNA in sufficient concentration. Pooling of samples, if possible, can be done totry and improve the concentration. Alternatively, you can use PCR followed by purification of desired bands using gel extraction kits.
Use High-Quality Nucleic Acids
We recommend using a fluorometer to measure DNA/RNA concentrations as Nanodrop readings for concentration is not always reliable. However, the absorbance ratios at 260/280 and 260/230 measured in Nanodrop are very useful. The 260/280 ratio for DNA should always be in the range of 1.7 – 1.9. The closer it is to 1.8, the better the results. The 260/230 ratio should higher than 2.0. For RNA, the 260/280 ratio should be around 2.0 and the 260/230 ratio should be above 2.0. Read more about the strengths and limitations of your nanodrop.
If possible, the quality of DNA/RNA should also be measured using Bio-analyser/Tape station along with traditional methods. As there are no steps to check the quality once the library preparation starts, it is better to start with a high-quality sample rather than searching for issues in case of poor sequencing data. Real time PCR kits are available to check library prep quality but this adds to the cost and time of experiment.
Consider the optimal DNA fragment size
The DNA fragment size also impacts the quality of the sequencing reads and we spent a lot of our time determining the optimal fragment size for Nanopore sequencing.
We found that the optimal size was between 3 Kb and 8 Kb. Fragments smaller than 1-2 Kb greatly reduced the overall sequencing quality, whereas higher molecular weight DNA fragments (10 Kb to 40 Kb which are common in manual DNA preparations) do not interfere with overall quality of data. Some Nanopore users have reported clogging of pores when using high molecular weight DNA and recommend the new flow cell wash kit to revive the clogged pores and prepare for the next run. Flow cell washing is recommended after every run and latest kit supposedly gives better yields when combined with a nuclease flush step.
The objective of Nanopore is long read sequencing but how long the reads should be (< 10 Kb or >10 Kb) is up to the scientist to decide depending on their individual experiment parameters. It should be noted that the overall fragment size distribution among all the samples in a single batch should be similar. Fragment length normalization and optimization will help to reduce the sequencing bias of heterogeneous samples. Steps should be taken to maintain a uniform fragment length distribution and should be normalized across samples. Fragment size also affects the efficiency of PCR barcoding steps. Longer fragments are difficult for PCR barcoding. End prep is another important step. Barcoding step affects the overall outcome of the sequencing. Insufficient barcoding leads to loss of valuable data.
Increase Your Incubation Times
We have observed that incubating the samples with beads for a longer duration gives better results. After mixing with the beads, we left the tubes idle for twice as long as suggested by the manufacturer’s protocol.
Use Fresh Beads
AMPure XP beads utilizes an optimized buffer to selectively bind DNA fragments 100 bp and larger to paramagnetic beads. Excess primers, nucleotides, salts, and enzymes can be removed using a simple washing procedure. Prepare fresh 70% ethanol each time to avoid dilution effect upon reuse due to the hygroscopic nature of 70% ethanol. We found that this simple step dramatically improves the bead utilization.
Minimize Tube Changes During Nanopore Library Preparation
We would recommend not transferring DNA from one tube to another unless it is absolutely necessary. For example, when eluting DNA from beads, you must collect the eluent in a fresh new sterile tube. This is primarily to avoid loss of sample during library preparation. The tubes and tips are not completely resistant to liquid sticking on the walls, and we feel it is better to avoid this rather than to try and retrieve the lost sample. Good pipetting practices will help you to minimize volume loss during such transfers. Additionally, we recommend using DNA LoBind Eppendorf® tubes for all the steps.
Mix Your Samples Thoroughly… but Carefully!
The tube contents and reagents should be mixed properly. Even though vortexing is not recommended, light vortexing can be done in the initial steps. The most appropriate method for mixing is flicking the tubes. Pipette mixing can also be done, but this is not recommended as repeated pipetting may result in shearing of the DNA fragments affecting the size distribution. Again, proper pipetting skills are required to ensure that the sample is processed optimally.
Optimize Your PCR
In our experiments, we have observed that for PCR barcoding, the number of cycles can be doubled to 30 cycles. We did not get enough output with the recommended number of cycles (12 to 15 cycles). This may vary depending on the sample type and protocol but there is no harm in programming additional PCR cycles. Always check with Nanodrop and Qubit before and after such procedures to ensure consistency.
Prep Your DNA Before Ordering a New Flowcell
One of the major problems that we faced, other than the issues in Nanopore library preparation, was lower pore count of the MinION flowcell. Even though the company claims that there are 2048 pores, by the time we receive the flowcells, the count of pores is down to less than 1400. Having fewer pores directly impacts the quality and quantity of sequences/reads in the output. As the number of pores reduces, so does the confidence on the quality and quantity of the output data. Every day we lose a bunch of pores as these are biological and not chemical in nature and the longer the flowcell sits in the fridge the lower the capacity. So, we highly recommend preparing your DNA samples first – before ordering a new flowcell. As soon as you receive it, you should be able to start the experiment right away.
Make Sure You Have a Good Internet Connection
It must be noted that the most recent MinION flowcell can generate 30 GB raw data and close to 5 GB FASTQ data after basecalling. We have confirmed these numbers from our own experiments even at low pore counts of 1500 per flowcell. You need a high-speed broadband internet connection for real time basecalling. We recommend this approach as you can continuously monitor the data generated and stop the run when sufficient data is collected. You can immediately wash the flowcell and prime it for the next batch. This way you can reuse the flow cells for at least three library preps making it more economical compared to other sequencing platforms.
Common Practices to Keep in Mind for Nanopore Library Preparation
While working on the various experiments with Nanopore MinION, we figured out that the following practices greatly improved the final data output from sequencing:
- The working bench, pipettes and all the consumables should be clean and dust free.
- The reagents stored in -20°C should be thawed on ice before use and should be immediately returned to the proper storage area (-20°C) after use – use an ice bucket at all times.
- The magnetic beads should be normalised to room temperature and resuspended properly before use.
- As the library preparation protocols are not very long, try to finish the complete procedure within one day and start sequencing. Though there are steps after which the sample can be stored and the protocol continued the next day, it is better to avoid this scenario if possible.
- Plan ahead of time to ensure all the equipment is readily available, start early and finish the entire process in one go without stops.
We hope these optimization suggestions help you get past the learning curve a little faster and allow you to collect meaningful data with more ease.
So, what are you waiting for?
Get started with Nanopore library prep and sequencing.