Oxford Nanopore NGS

Oxford Nanopore Sequencing

AGC Oxford Nanopore GridION, installed August 2020

The Advanced Genomics Core offers long-read DNA and RNA sequencing on the Oxford Nanopore Technology GridION X5 and PromethION platforms. While ONT technology allows sequencing of nucleic acid molecules with lengths greater than 100,000 bp, typical samples produce read lengths of ~ 40,000 bp.  The length of a ONT read is dependent on the quality of the sample and the preparation method. There are a variety of protocols available enabling sequencing of genomic DNA, PCR products, methylated DNA, cDNA, and RNA. ONT reports good results with high molecular weight gDNA when the average fragment size is >30kb.

Oxford Nanopore library preparation is sensitive to contaminants that can be revealed by Nanodrop readings. The following is quoted from ONT’s QC protocol:

• We recommend that sample DNA has a 260/280 ~1.80, and a 260/230 ~2.0-2.2
• A 260/280 which is higher than ~1.8 indicates the presence of RNA
• A 260/280 which is lower than ~1.8 can indicate the presence of protein or phenol
• A 260/230 significantly lower than 2.0-2.2 indicates the presence of contaminants, and the DNA may need additional purification

The following extraction methods are recommended:

• Circulomics
• Qiagen Genomic-Tip

The following extraction methods are NOT recommended:

• Phenol-chloroform
• Spin-column based methods (will reduce fragment length)

Other species- and sample-specific extraction methods developed for ONT sequencing can be found on the ONT community website (which does require you to log in with a free account) here: https://community.nanoporetech.com/extraction_methods

Flow Cells

We highly recommend you book a Virtual Office Hour Appointment to discuss your project and experiment design.

ONT Library Prep

Nanopore sequencing at the Advanced Genomics Core begins with the purchase of a ONT flow cell. Each flow cell can be expected to have a minimum of pores and a sequencing lifespan of approximately 48-72 hours when run with high-quality, chemically pure samples. Often, we can get more sequencing time out of the flow cell, so long as no contaminants were present in the original samples. Contaminants in samples can damage the pores irreversibly, significantly decreasing the sequencing life. With the 48+ hours of sequencing lifespan, you have several options, discussed in detail in the following sections. 

DNA sequencing

Ligation Sequencing 

Ligation sequencing is the most popular ONT library prep we offer. It can sequence gDNA, cDNA, or amplicons of any size. This PCR-free method generates 1D reads equal to the fragment length. For clients who are seeking long or full-length reads, this method is ideal. ONT currently rates this library prep method for one of their highest throughputs, so you will get the most amount of data using this kit; however, it is important to realize that long reads are negatively correlated with throughput due to physical complications with sequencing very high molecular weight DNA. 

For the longest reads, we recommend starting with 1-3 ug of gDNA, with fragment sizes >30 kbp. With 1 ug, we are usually able to produce enough library for a single run on a GridION flow cell, but with 3 ug we are usually able to produce enough for 2-3 runs from a single prep. Long fragments tend to clog the pores on the flow cell quickly, so having enough library generated to wash and reload improves output. Sometimes, we may use up the library generated from the first prep, but there will still be pores available for sequencing left on the flow cell. At this point, we will reach out and see if you would like us to re-prep more library, if the sample remains, and continue sequencing. 

For cDNA or amplicons, we recommend starting with 100-200 fmol, which typically produces plenty of library for 2+ runs on a GridION flow cell. Since shorter fragments do not clog flow cells as quickly as long fragments, we do not need to wash and reload as often, and therefore a single prep of cDNA or amplicons is usually enough to use up a flow cell’s sequencing life. 

Rapid Sequencing  

Rapid sequencing is the second most popular ONT library prep we offer. It is recommended for only gDNA with fragments >30 kbp. Unlike with ligation sequencing, this PCR-free kit generates libraries using a transposase, which cleaves the gDNA at random loci to attach adapters. Due to the difference in chemistry, you can expect 1D reads of slightly shorter length than the input. Some protocols do use this kit to generate ultra-long reads by starting with many micrograms of extremely long fragments; please reach out to the AGC prior to submission if you are interested in this. ONT rates this kit for intermediate throughput, so the expectation is lower than the ligation sequencing kit.  

For this kit, we recommend starting with 400 ng gDNA, which produces enough library for a single run on a GridION flow cell. The limiting factor in this kit is the transposase adapter, so we are not able to produce more than one run’s worth of library from a single prep. This does mean that read length is correlated to input amount; if we start with a large amount of long DNA, the fragments produced will be longer. 

RNA sequencing

Direct RNA Ligation (SQK-RNA002) 

The Direct RNA library preparation is the only RNA sequencing protocol that we offer currently. This library prep enables sequencing of RNA molecules directly. This sequencing method has two advantages – 1) end-to-end transcript sequencing allows quantification of long-range modifications like isoforms and fusion transcripts with ease, and 2) direct RNA sequencing allows estimation of base modifications, like pseudouridine, 5mC and m6A. This prep is PCR-free and targets only 3’ poly-A transcripts preferably >200 bp. The library prep method involves an optional reverse transcription that makes an RNA-cDNA hybrid for better throughput, however, only the RNA bases are read during the sequencing. We suggest ONT’s recommended RIN score of above 7 for total RNA, as higher RIN number directly correlates with longer read distribution. 

ONT recommends input of 500 ng poly-A enriched RNA in 9 ul for this library prep. However, we will also need to estimate quality with a RIN score for a good sequencing run. RIN score can only be estimated with whole RNA. Due to this reason, we recommend submitting total RNA that contains 500 ng poly-A enriched RNA. According to ONT, 1-5% of total RNA can be poly-A tailed mRNA. With that, we arrive at an estimation of 10-50 ug of total RNA as starting input to obtain 500 ng of poly-A enriched transcripts. We recognize that this is a huge amount to extract, so we ask that you submit as much total RNA as you can for us to ribo-deplete it to ONT’s recommendation. We will reach out to you about your RNA sample quality before sequencing. 

Multiplexing

For DNA sequencing, both the ligation and rapid sequencing methods allow for multiplexing of samples. You have two options for multiplexing: we can run all samples at once, or we can run them in a series of runs with a nuclease wash between them. The first option is most likely to get you an even number of reads between all samples, while the second option is good if your samples are significantly different from each other (different lengths, different species, etc), since they will not compete with each other for pores. 

For direct RNA sequencing, ONT at present does not offer any multiplexing solutions. So all samples will need to be sequenced either on multiple flow cells or one at a time on a single flow cell with a nuclease wash step between each sample. 

ONT Run Monitoring

Our technicians will monitor your flow cell during the run and adjust as necessary to improve output and performance. Below are a few common situations that arise during ONT sequencing: 

• When sequencing long fragments, we will see an accumulation of saturated pores that are “clogged” and unable to sequence. The only way we can “unclog” the flow cell is to perform a nuclease wash and reload with more library. If we do not have more library to reload with and there is more of the original sample remaining, we will reach out to you and ask if you would like us to prepare more library and continue sequencing. If we do not have more of the original sample remaining, we will reach out to you and ask if you would like to submit more sample to be sequenced.
• When sequencing shorter fragments, the flow cell will run out of fuel and the translocation speed drops. When this happens, we will pause the experiment, refuel, and resume without needing to reload with any additional library.
• For any size fragments, eventually the number of available pores will decline. Once the number of pores drop below 10-20%, we assume that the flow cell is exhausted. At that point, we will do a nuclease wash and see how many pores are recovered for sequencing. While this is handled case-by-case, generally if the pores stay below 200, we will declare the flow cell and run as finished. Alternatively, if the recovery was higher, we will reload more library and continue sequencing. If we do not have more library, we will reach out to you and let you know how many pores are left so you can decide how to proceed.