PacBio vs. Oxford Nanopore sequencing

Long-read sequencing developed by Pacific Biosciences and Oxford Nanopore overcome many of the limitations researchers face with short reads. Long reads improve de novo assembly, transcriptome analysis (gene isoform identification) and play an important role in the field of metagenomics. Longer reads are also useful when assembling genomes that include large stretches of repetitive regions.

Currently there are two long read sequencing platforms. To help a researcher choose between which platform has greater utility for their application, we compare overall instrument specifications offered by PacBio and Oxford Nanopore, and published applications in the next-generation sequencing space.

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Oxford Nanopore charges an access fee that gives users one MinION/PromethIon instrument, a starter pack of consumables, certain data services, and community-based support

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Although both PacBio and Oxford Nanopore generate longer reads compared to short read Illumina or Ion sequencing, the higher error rate of both the PacBio and Oxford Nanopore sequencers remain an issue needs addressing. Whereas PacBio reads a molecule multiple times to generate high-quality consensus data, Oxford Nanopore can only sequence a molecule twice. As a result PacBio generates data with lower error rates compared to Oxford Nanopore. PacBio has slightly better overall performance for applications such as discovery of transcriptome complexity and sensitive identification of isoforms. On the other hand, MinION provides higher throughput as nanopores can sequence multiple molecules simultaneously. Hence, it is best suited for applications that require a larger amounts of data9

As long reads can provide large scaffolds, de novo assembly is one of the main applications of PacBio sequencing5. Though the error rate of PacBio data is higher than that of short read Illumina or Ion sequencing, increased coverage or hybrid sequencing can greatly improve accuracy of genome assembly. PacBio sequencing has been successfully used to finish the 100-contig draft genome of Clostridium autoethanogenum DSM 10061, a Class III, the most complex genome classification in terms of repeat content and repeat type. It has a 31.1% GC content and contains repeats, prophage, and nine copies of rRNA gene operons. Using a single PacBio library and sequencing it with two SMRT cells, an entire genome can be assembled de novo with a single contig. When short read Illumina or Ion sequencing  was used alone with the same genome, >22 contigs were needed, and each of the assemblies contained at least four collapsed repeat regions, PacBio assemblies had none10.

PacBio sequencing has also been used to assemble the chloroplast genome of Potentilla micrantha11, Saccharomyces cerevisiae, Aradopsis thaliana and Drosophila melanogaster using fewer contigs and CPU time for assembly compared to assemblies using Illumina sequencers12.

PacBio sequencing of PCR products can be used to improve the quality of current draft genomes by closing gaps and sequencing through hairpin structures and areas of high GC content more efficiently than Sanger sequencing13.

Pacific Biosciences has developed a protocol, Iso-Seq, for transcript sequencing. This includes library construction, size selection, sequencing data collection, and data processing. Iso-Seq allows direct sequencing of transcripts up to 10 kb without use of a reference genome. Iso-Seq has been used to characterize alternative splicing events involved in the formation of blood cellular components14. This is essential for interpreting the effects of mutations leading to inherited disorders and blood cancers, and can be applied to design strategies to advance transplantation and regenerative medicine.

Another major application of PacBio sequencing is in epigenetics research. Recent studies demonstrate that investigation of intercellular heterogeneity in previously undetectable genome DNA modifications (such as m6A and m4C) is facilitated by the direct detection of modifications in single molecules by PacBio sequencing15.

Compared to PacBio, the Oxford Nanopore MinION is small (size of a USB thumb drive), affordable, utilizes a simple library prep and is field portable16. This is useful in situations such as a virus outbreak where a mobile diagnostic laboratory can be set up using MinIONS. In remote regions such as parts of Brazil and Africa where there are logistical issues associated with shipping samples for sequencing, MinION can provide immediate and real-time data to scientific investigators. The most notable clinical use of MinION has been the analysis of Ebola samples on-site during the viral outbreak in West Africa17,18.

The low cost of sequencing and portability of the MinION sequencer also make it a useful tool for teaching. It has been used to provide hands-on experience to students, most recently at Columbia University and the University of California Santa Cruz, where every student performed their own MinION sequencing19.

Perhaps the most ambitious MinION application is its potential to detect and identify bacteria and viruses on manned space flights. In a proof-of-concept experiment, Castro-Wallace et al. demonstrated successful sequencing and de novo assembly of a lambda phage genome, an E. coli genome, and a mouse mitochondrial genome. They observed that there was no significant difference in the quality of sequence data generated on the IInternational Space Station and in control experiments that were performed in parallel on Earth22.

Recently, Oxford Nanopore developed a bench-top instrument, PromethION, that provides high-throughput sequencing and is modular in design. It contains 48 flow cells that can be run individually or in parallel. The PromethION flow cells contain 3000 channels each, and produce up to 40 Gb of data.

 

References:

  1. Pacific Biosciences – AllSeq. Available at: http://allseq.com/knowledge-bank/sequencing-platforms/pacific-biosciences/.
  2. Jain, M., Olsen, H. E., Paten, B. & Akeson, M. The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community. Genome Biol. 17, 239 (2016).
  3. Lu, H., Giordano, F. & Ning, Z. Oxford Nanopore MinION Sequencing and Genome Assembly. Genomics. Proteomics Bioinformatics 14, 265–279 (2016).
  4. Rhoads, A. & Au, K. F. PacBio Sequencing and Its Applications. Genomics, Proteomics Bioinforma. 13, 278–289 (2015).
  5. MinION. Available at: https://nanoporetech.com/products/minion.
  6. PromethION Early Access Programme. Available at: https://nanoporetech.com/community/promethion-early-access-programme.
  7. Oxford Nanopore in 2016. Available at: http://blog.booleanbiotech.com/nanopore_2016.html.
  8. Weirather, J. L. et al. Comprehensive comparison of Pacific Biosciences and Oxford Nanopore Technologies and their applications to transcriptome analysis. F1000Research 6, 100 (2017).
  9. Brown, S. D. et al. Comparison of single-molecule sequencing and hybrid approaches for finishing the genome of Clostridium autoethanogenum and analysis of CRISPR systems in industrial relevant Clostridia. Biotechnol. Biofuels 7, 40 (2014).
  10. Ferrarini, M. et al. An evaluation of the PacBio RS platform for sequencing and de novo assembly of a chloroplast genome. BMC Genomics 14, 670 (2013).
  11. Berlin, K. et al. Assembling large genomes with single-molecule sequencing and locality-sensitive hashing. Nat Biotech 33, 623–630 (2015).
  12. Zhang, X. et al. Improving genome assemblies by sequencing PCR products with PacBio. Biotechniques 53, 61–62 (2012).
  13. Chen, L. et al. Transcriptional diversity during lineage commitment of human blood progenitors. Science (80-. ). 345, (2014).
  14. Feng, Z., Li, J., Zhang, J.-R. & Zhang, X. qDNAmod: a statistical model-based tool to reveal intercellular heterogeneity of DNA modification from SMRT sequencing data. Nucleic Acids Res. 42, 13488–13499 (2014).
  15. Jain, M., Olsen, H. E., Paten, B. & Akeson, M. Erratum to: The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community. Genome Biol. 17, 256 (2016).
  16. Quick, J. et al. Real-time, portable genome sequencing for Ebola surveillance. Nature 530, 228–232 (2016).
  17. Hoenen, T. et al. Nanopore sequencing as a rapidly deployable Ebola outbreak tool. Emerg. Infect. Dis. 22, 331–334 (2016).
  18. Citizen Sequencers: Taking Oxford Nanopore’s MinION to the Classroom and Beyond – Bio-IT World. Available at: http://www.bio-itworld.com/2015/12/9/citizen-sequencers-taking-oxford-nanopores-minion-classroom-beyond.html. (Accessed: 24th May 2017)
  19. Castro-Wallace, S. L. et al. Nanopore DNA Sequencing and Genome Assembly on the International Space Station. bioRxiv (2016).

 

 

New Short, Long and High Throughput Sequencing Reads in 2016

 

Nanopore sequencing

Nanopore sequencing

An exciting wave of newly released DNA sequencing instruments and technology will soon be available to researchers. From DNA sequencers the size of a cell phone to platforms that turn short reads into long-range information, these new sequencing technologies will be available on Genohub as services that can be ordered. Below is a summary of the technology you can expect in Q1 of 2016:

10X Genomics GemCode Platform

The GemCode platform from 10X Genomics partitions long DNA fragments up to 100 kb with a pool of ~750K molecular barcodes, indexing the genome during library construction. Barcoded DNA fragments are made such that all fragments share the same barcode. After several cycling and pooling steps, >100K barcode containing partitions are created. GemCode software then maps short Illumina read pairs back to the original long DNA molecules using the barcodes added during library preparation. With long range information, haplotype phasing and improved structural variant detection are possible. Gene fusions, deletions and duplications can be detected from exome data.

Ion Torrent S5, S5 XL

The S5 system was developed by Ion to focus on the clinical amplicon-seq market. While the wait for delivery of Proton PII chips continues, Ion delivered a machine with chip configurations very much similar to past PGM and Proton chips. 520/530 chips offer 200-400 bp runs with 80M reads and 2-4 hour run times. Using Ion’s fixed amplicon panels, data analysis can be completed within 5 hours. The Ion chef is required to reduce hands on library prep time, otherwise libraries and chip loading needs to be performed manually. Ion looks to have positioned their platform toward clinical applications. With stiff competition from Illumina and their inability to deliver similar read lengths and throughput, this is a smart decision by Ion. Focusing their platform on a particular application likely means future development (longer and higher throughput reads) has been paused indefinitely.

Pacific Biosciences Sequel System

Announced in September 2015, the Sequel System uses the same Single Molecule, Real Time (SMRT) technology as the RSII,   but boasts several tech advancements. At around one third the cost of a RSII, the Sequel offers 7x more reads with 1M zero –mode waveguides (ZMWs) per SMRT cell versus the previous standard of 150K. The application of Iso-Seq or full length transcript sequencing is especially promising as 1M reads crosses into the threshold where discovery and quantitation of transcripts becomes interesting. By providing full length transcript isoforms, it’s no longer necessary to reconstruct transcripts or infer isoforms based on short read information. Of course, the Sequel is ideal for generating whole genome de novo assemblies. We’l follow how the Oxford Nanopore’s ONT MinIon competes with the Sequel system in 2016.

Oxford Nanopore’s (ONT) MinIon

In 2014, Oxford Nanopore started it’s MinIon Access Program (MAP) delivering over 1,000 MinIons to users who wanted to test the technology. These users have gone on to publish whole E. Coli and Yeast genome assemblies. Accuracy of the device is up to 85% per raw base and there are difficulties in dealing with high G+C content sequences. There remains a lot of work left to improve the technology before widespread adoption. The workflow is simple and uses typical library construction steps of end-repair and ligation. Once the sample is added to the flow cell, users can generate long reads >100 kb and can analyze data in real time. Median reads are currently in the 1-2 kb length. Combined alongside with MiSeq reads, publications have shown MinIon output can enhance contiguity of de novo assembly. Lower error rates generated by Two Direction reads produced with recent updated MinIon chemistry does give cause for optimism that greatly reduced error rates can be achieved in the near future. This along with a low unit cost and the ability to deploy the USB sized device in the field make this a very exciting technology.

Illumina HiSeq X

While HiSeq X services have been available on Genohub for over a year, Illumina’s announcement of its expansion to non-human whole genomes was well received. However there are still several unanswered questions. Illumina states,

The updated rights of use will allow for market expansion and population-scale sequencing of non-human species in a variety of markets, including plants and livestock in agricultural research and model organisms in pharmaceutical research. Previously, it has been cost prohibitive to sequence non-human genomes at high coverage.

You can now sequence mouse, rat and other relatively large sized genomes economically on the HiSeq X. This makes the most sense for high coverage applications, e.g. 30x or above. While smaller sized and medium sized genomes can be sequenced on a HiSeq X, the low level of barcoding and high coverage you’d obtain makes these applications less attractive. According to Illumina, as of 12/20/2015, metagenomic whole genome sequencing was not a compatible application on the HiSeq X. The instrument is still restricted to WGS only. RNA-Seq, Exome-seq and ChIP-Seq applications will have to wait. Perhaps by the time the HiSeq X One is released access will be opened to these non-WGS applications.

While these new instruments make their way onto Genohub’s Shop by Project page, you can make inquiries and even order services by placing a request on our consultation page.