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Oxford Nanopore sequencing in a research-based undergraduate course

Background Nanopore sequencing is a third generation genomic sequencing method that offers real time sequencing of DNA samples. Nanopore sequencing is an excellent tool for teaching because it involves cutting-edge sequencing methods and also helps students to develop a research mindset, where students can learn to identify and resolve problems that arise during an experiment. Results We, as a group of undergraduate biology students, were able to use nanopore sequencing to analyze a sample of pupfish DNA. We were able to accomplish this without computer science backgrounds and only some basic DNA extraction training. Although there were issues, such as inconsistent results across runs, we found it useful as a research learning experience and an application of the skills we learned. Conclusions As students, it was exciting to be able to experience this technology first hand and apply what we learned in the classroom. Nanopore sequencing holds potential for DNA sequencing of large fragments in real time. It allows students to be acquainted with novel technologies and the theories behind them. However, as with all new techniques, it does not have the same established support, and when students run into difficulties while using nanopore sequencing, it is often difficult to identify what went wrong.


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Background: 29 Nanopore sequencing is a third generation genomic sequencing method that offers real time 30 sequencing of DNA samples. Nanopore sequencing is an excellent tool for teaching because it 31 involves cutting-edge sequencing methods and also helps students to develop a research mindset, 32 where students can learn to identify and resolve problems that arise during an experiment. 33 Results: 34 We, as a group of undergraduate biology students, were able to use nanopore sequencing to 35 analyze a sample of pupfish DNA. We were able to accomplish this without computer science 36 backgrounds and only some basic DNA extraction training. Although there were issues, such as 37 inconsistent results across runs, we found it useful as a research learning experience and an 38 application of the skills we learned. 39 Conclusions: 40 As students, it was exciting to be able to experience this technology first hand and apply what we 41 learned in the classroom. Nanopore sequencing holds potential for DNA sequencing of large 42 fragments in real time. It allows students to be acquainted with novel technologies and the Introduction 51 52 When I first decided to take a seafood mislabeling class I didn't expect to be able to test 53 developing third-generation DNA sequencing technology or learn about unique evolutionary 54 adaptations from two new species of pupfish from the Bahamas. My course was aimed towards 55 giving undergraduate biology majors a chance to get hands on experience in a research 56 environment. Oxford nanopore sequencing can be applied in a much broader biological context 57 and even dips into computer programming. So, what exactly is nanopore sequencing and why is 58 it important? 59 DNA sequencing has come a long way from traditional Sanger sequencing. Although 60 Sanger sequencing is still considered the gold standard for accuracy, it requires gel 61 electrophoresis and the use of ddNTPs to identify the sequences of amplified segments of DNA 62 (Obenrader 2003). Sanger sequencing is incredibly accurate but requires lots of processing and 63 thus the cost of sequencing DNA of even relatively short fragments is very high (Goodwin 2016). 64 Next generation sequencing which replaced traditional methods drastically reduced prices of 65 whole genome sequencing (Goodwin 2016). Although these methods made DNA sequencing 66 more accessible, they are generally less accurate (99%) than methods such as Sanger sequencing 67 which can have accuracies as high as 99.999% (Shendure 2008;Morozova 2008). Another major 68 constraint in next generation, or second generation, sequencing is the short read length (50 -250 69 bp on Illumina platforms) which makes genome assembly and alignment very difficult (Goodwin 70 2016). 71 We are currently approaching the third generation of genomic sequencing which builds 72 further upon the advancements of second generation sequencing. Third generation sequencing 73 addresses the main weakness of second generation sequencing because many platforms can 74 produce reads over 10 kb to even 100 kb or more (Lee 2016). Third generation sequencing does 75 not require PCR amplification as previous generations have. Due to the read lengths and high 76 accuracies, the current third generation sequencing technologies, Pacific Biosciences (PacBio) 77 Single Molecule Real Time (SMRT) sequencing, Illumina Tru-seq Synthetic Long-Read 78 technology, and Oxford Nanopore Technologies sequencing have been able to fill in previous 79 gaps in genomes (Lee 2016). Of the three, Illumina Tru-Seq Synthetic Long Reads is considered 80 to be the most accurate but has much shorter read lengths, requires more DNA, and is more 81 expensive (Lee 2016). PacBio's SMRT and Oxford Nanopore Technologies sequencing 82 techniques both have high raw error rates, but with certain algorithm techniques both methods 83 can greatly increase their accuracy to almost 99.999% (Lee 2016). The most recent of third 84 generation technologies, Oxford Nanopore Technologies, is greatly limited in the accuracy of 85 their reads in comparison to other third generation techniques but its strength is in its portability. 86 Oxford Nanopore Technologies' MinION is a device slightly larger than a USB drive that can be 87 plugged into any modern laptop to provide real-time data. Currently in development is an even 88 more portable version that can be run on a smartphone called the SmidgION. 89 Nanopore sequencing is a third-generation genomic sequencing technique that was only 90 recently commercialized in 2014. The nanopore is a protein with a pore that is only a nanometer 91 wide (Clarke 2009). This tiny pore only allows single molecules such as individual DNA strands 92 to pass through one at a time. These nanopores are embedded into a sheet that has a current 93 applied through it. The current is extremely sensitive to the molecules that pass through it as 94 each nucleotide creates a unique change in the resistance of the nanopore which allows 95 individual nucleotides to be identified individually as they pass through (Deamer 2016). 96 Nanopore sequencing can read DNA sequences more than 100 kb long and the entire run 97 completes within 48 hours. This potentially makes it a low-cost method to sequence genomes, as 98 each kit (including the USB MinION sequencing machine) costs $1,000 for two samples (Meller   99   2000). Although currently nanopore sequencing is not as accurate in comparison to other third-100 generation sequencing techniques, what makes this developing technology so exciting is that it 101 offers a possible tool in the future to sequence entire genomes on any lab bench. In fact, it has 102 already proven to be useful in the identification and genome sequencing of diseases in the field, 103 such as Ebola virus (Quick 2016; Schmidt 2017). However, as the newest of the third-generation 104 sequencing technologies, nanopore technologies are continuing to rapidly improve as more 105 accurate and robust nanopores are being developed (Oxford Nanopore Technologies 2017). 106 Not much has been done in a classroom setting exploring nanopore sequencing or the 107 techniques involved and thus it is a unique opportunity for undergraduates to explore developing 108 technologies. The portability and ease of preparation makes nanopore sequencing ideal in a 109 classroom, because all that students need to analyze data obtained from prepared samples is a 110 working laptop. Previously, only one upper-level class has been well documented which focused 111 on the analysis of DNA sequences produced from nanopore sequencing to identify unknown 112 samples, as well as unknown human DNA (Zaaijer 2016). However, the students did not actually 113 extract the DNA or prepare the sequencing libraries. Analyzing human DNA also presents 114 ethical concerns and falls under institutional review board policies. However, the application of 115 nanopore sequencing can be developed further by teaching many basic laboratory techniques 116 such as proper pipetting and DNA extraction which are necessary to prepare samples. Thus, 117 nanopore sequencing can be used for de novo assemblies in the classroom and to introduce 118 students to important laboratory skills and exciting developments in genomic sequencing. 119 We chose the nanopore sequencing project as our independent research project topic 120 because of its novelty as a portable option for genomic sequencing. We sequenced a sample of 121 muscle tissue from the molluscivore pupfish, Cyprinodon brontotheroides, a species of pupfish 122 endemic to San Salvador Island in the Bahamas (Box 1). The genomes of other species of pupfish 123 have already been sequenced so we would also be able to test whether the DNA sequences we 124 got from nanopore sequencing were accurate enough to identify the pupfish sample. Our goal 125 was to use nanopore sequencing to sequence the pupfish genomic DNA and compare the 126 sequence with known data using the Basic Local Alignment Search Tool (BLAST) by the 127 National Center for Biotechnology Information, both as a test to see the accuracy of nanopore

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We found that it was entirely possible for undergraduate biology students to run nanopore 134 sequencing. From our successful run we generated a massive amount of raw DNA sequences 135 ranging from smaller segments to our longest read of 170 kb (see Table 1). 136 We ran a total of three experiments. We recorded the number of active nanopores before 137 conducting each experiment (Table 2). We conducted the quality control experiment using 138 Lambda DNA provided as suggested on the first flow cell before using sample DNA. Our second 139 experimental run using extracted pupfish DNA was successful and the results are reported in     My fellow students expressed great interest in nanopore sequencing and took initiative to learn 168 how to use the MinION. It was an opportunity to apply the basic techniques we learned in the 169 class, such as DNA extraction, to nanopore sequencing. Although, as undergraduates, we had no 170 problem prepping and running the library with what was provided with the MinION, we did find 171 multiple problems while running the experiment. 172 We had difficulties with some of the basics such as opening the files, which are in .fast5 173 format. HDFView by the HDF Group is a program that can open the files and will present the 174 sequences in FASTA format which can be easily run through BLAST. Our main obstacle, 175 however, was sorting through the raw data and identifying which sequences to BLAST. With 176 computer science backgrounds, it's possible to tackle this problem, but as none of us had much 177 experience with programming we had to look for alternatives. Beyond Metrichor, the MinION 178 has a lot of user made programs, each with their own pros and cons, most notably Poretools (an 179 extensive list by Next Gen Seek can be found in the references). Poretools is a toolkit that can be 180 used to go through the large amount of raw data and sort the reads for easier analysis (Loman 181 2014). This allows users who would like to view high quality sequences to access them quickly. 182 Metrichor, the analytical component of the MinION, can currently only recognize 183 organisms such as bacteria. In identification of pupfish this makes the results from Metrichor less 184 useful as a fish is a multicellular organism. We found it difficult to select high quality reads from 185 the data we generated to BLAST as nanopore sequencing created such large amounts of data and 186 currently has very little user friendly supporting programs for analysis. Luckily, currently Oxford 187 Nanopore is working to continue developing Metrichor so it can search for more organisms from 188 the NCBI database which would resolve the current issue of identifying species from the data 189 obtained. In the end, the easiest method for us was to use HDFView to open the files before 190 using BLAST for individual sequences. 191 Another problem was that MinION flow cells are advertised to be able to run about three 192 experiments each but active nanopores degrade rapidly, especially after the long runs needed for 193 the experiments. In reality each flow cell can only handle one run, before too many nanopores 194 become degraded. Originally, we had hoped to run more experimental runs, but the experience of 195 the first failed runs taught us how to troubleshoot and improve our methods for the future as we Metrichor's What's In My Pot application offers a large variety of options students can explore. 224 One activity students can do is to take environmental swabs of various locations and identify 225 which organisms they find in their samples. Students can choose locations that interest them, 226 such as public transportation, bathrooms, or water from local streams. They can take their 227 samples and purify and extract DNA. Using nanopore sequencing to read their DNA they can 228 then analyze and identify which bacterial species they found in their samples using WIMP. 229 However, until WIMP's database is updated, the species that can be identified will be limited to 230 bacterial and fungal.

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Identification of an unknown sample: 233 Students can also use nanopore sequencing as a tool to determine the identity of an unknown 234 sample. Students may be interested in identifying the ingredients of the food they normally 235 consume or teachers could set up a more structured lab for students to identify various samples. 236 For example, if students were interested in figuring out which type of fish were used in their 237 sushi, they could use nanopore sequencing to identify the extracted DNA from a slice of sashimi. 238 Alternatively, teachers could prepare samples of DNA and have students identify which 239 unknown organism they were given.

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De novo genome assembly: 242 As a third generation sequencing technology, nanopore sequencing is also entirely capable of 243 generating incredibly long reads that are necessary for genome assembly. This approach would 244 require some experience with genome assembly. Students could use results from nanopore 245 sequencing and other sequencing methods to assemble the genome of a sample.

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Conclusion: 248 We found nanopore sequencing to be an incredibly interesting path for our independent project 249 and found that even with very little guidance beyond what is provided online that we could 250 properly run the experiments. Although some basic knowledge of laboratory techniques is 251 necessary, such as pipetting procedures and DNA extraction, the procedure itself for running 252 nanopore sequencing is very straight forward. As we had very little computer science 253 background, our main issue was actually analyzing the data we obtained.       flow cell we completed two runs, one of a control DNA and one of our samples. The first run we 576 did was the suggested control run using lambda DNA provided in the kit for the MinION that 577 acquainted us with how to prepare and tagment DNA and how to prepare that library for loading 578 into the machine. We also tried an experimental run but while running quality control counts of 579 active nanopores on the flow cells we found that the most of the pores had degraded. As 580 expected our first experimental run using pupfish DNA failed, however interestingly enough the 581 control run also failed. 582 MinKNOW can provide data in real time although the sample runs take about 48 hours to 583 complete. Within the first half hour there was already a lot of data displayed on the automatically 584 updating charts. MinKNOW is a fairly straight forward program where all the user has to do is 585 enter the flow-cell id, an identifier for the experiment run, and to choose whether they are 586 running the control experiment or a sample experiment. The data is also updated to Metrichor 587 where it can be analyzed. 588 The data generated from Metrichor indicates that all the reads collected from both our 589 control and the first experimental run failed minimum quality filters that indicate how confident 590 the program is in the accuracy of the sequence reads. We attempted to troubleshoot what may 591 have gone wrong. After carefully rereading all the minimum quality requirements for the 592 extracted DNA, we went back and double-checked the quality of the pupfish DNA using Qubit. 593 Here we noticed that we did not dilute the experimental DNA to the proper concentration. After 594 diluting the sample to the proper concentration, we decided to use a fresh flow cell as the drastic 595 decrease in active pores in the previous experimental run indicated less nanopore activity. 596 However, as the control run also failed to meet minimum quality filters, we decided to also 597 increase the attention to detail in preparation of the library for loading into the flow cell. We took 598 extra care with pipetting small amounts of reagents and also used a centrifuge to insure the 599 solution was completely mixed. . This run ended up successful and generated massive amounts 600 of data. Metrichor can track the quality of the reads and record the length of each sequence over 601 time 602 We also needed to use BLAST to identify random sequences we pulled from the folder  u  e  n  c  e  f  i  s  h  C  t  e  n  o  p  h  a  r  y  n  g  o  d  o  n  i  d  e  l  l  a  c  l  o  n  e  G  C  F  L  -0  4  0  5  E  6  M  H  C  c  l  a  s  s  I  a  n  t  i  g  e  n  (  C  i  t  d  -U  B  A  )  g  e  n  e  ,  c  o  m  p  l  e  t  e  c  d  s  ;  a  n  d  t  a  p  a  s  i  n  (  t  p  s  n  )  g  e  n  e  ,  p  a  r  t  i  a  l  c  d  s  G  r  a  s  s  c  a  r  p