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).

 

 

AGBT 2014 – Summary of Day 1

AGBT 2014 Summary

The first day of the Advances in Genome Biology & Technology (AGBT) meeting kicked off with an introduction by Eric Green, Director of the National Human Genome Research Institute. He announced that this 15th annual meeting was the largest ever with 850 expected to attend. The opening plenary session certainly did not look like 850 people in attendance. Winter Storm Pax wreaked havoc on flights coming in from Atlanta and other cities, resulting in several speaker and general attendee cancellations.

The plenary session began with scheduled talks by Aviv Regev, Jeanne Lawrence, Wendy Winckler and Valerie Schneider. Jeanne Lawrence couldn’t make it, which was a shame particularly since she gave a brilliant talk at ASHG on using a single gene XIST to shut down the extra copy of chromosome 21 in Down syndrome. This work was nicely summarized in a publication that came out this summer titled: Translating dosage compensation to trisomy 21.          

Aviv Regev and Wendy Winckler’s talks were subject to a blog/tweet embargo (unclear whether Regev’s talk was completely under embargo or only the last half, we’re playing it safe and not discussing it here), leaving Valerie Schneider’s presentation the only one that was tweeted or written about. This instantly created great angst among those attending the lectures, those stuck in airports enroute to AGBT and those at home waiting for in depth coverage.

Single-cell sequencing, considered the “method of the year” by Nature Methods was the basis of the opening lecture. Aviv Regev offered an excellent view of the dendritic cell network based on cyclical perturbations and variations between single cells. Regev’s first half of her presentation titled, “Harnessing Variation Between Single Cells to Decipher Intra and Intercellular Circuits in Immune Cells” was largely covered by her publication in April, “Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells”.

The second talk, by Wendy Winckler was not allowed to be discussed or tweeted according to Winckler, courtesy of Novartis’s communications department. The title of her presentation “Next Generation Diagnostics for Precision Cancer Medicine” wasn’t revealing either. To get an idea of what she’s up to and the direction of her lecture, you can read these recent publications.

The final talk by Valerie Schneider, titled “Taking advantage of GRCh38” began with an analogy to an unwanted pair of socks one receives for Christmas that ends up being used and finally really liked. “It was time for an update….whether or not it was on your wish list”. We were reminded that centromeres are important specialized chromatin structures important for cell division, but because of repetitive regions, they are not represented in reference assemblies. Previous versions of the human reference assembly had centromeres represented by a 3M gap. The latest assembly, GRCh38 incorporates centromere models generated using whole genome shotgun reads as part of the Venter sequencing project. Since there are two copies of each centromere for each autosome, these centromere models represent an average of two copies. She concluded her presentation urging users to switch now: http://www.ncbi.nlm.nih.gov/genome/tools/remap.

 After a short break from the talks, the closing reception sponsored by Roche began outside. Halfway through, there was a brief yet sudden Florida thundershower that sent the entire AGBT community scurrying indoors for shelter. That was okay though because the conversations just continued indoors. Looking forward to tomorrow morning’s lectures. Several of the ones we’ve highlighted will be up.

 

3 Top Factors Researchers Consider When Selecting an NGS Provider

At Genohub, not only do we seek feedback from researchers, our development methodology is almost entirely based on this feedback. We receive this feedback via website forms as well as routine one-on-one conversations with some of the top researchers using next generation sequencing for their projects. Through this data and interaction, certain trends have begun to emerge which may be useful to an NGS provider seeking additional projects. This list is not based on a controlled experiment, however countless conversations indicate that these factors are extremely important:

  1. Turnaround time – this one is a toss up when compared with price, but we typically find turnaround time to be among the leading factors in a researcher’s decision to select an NGS provider. We have heard quite a few stories of researchers seeing turnaround times over several months for library prep and sequencing.
  2. Price – while this is one of the biggest factors for researchers, it must be qualified with established trust which is the next major factor.
  3. Trust – this one is a biggie for many researchers and often a non-starter if not established. The main reasons for this are that researchers are hesitant to ship their precious samples (ie human brain tissue) to an NGS provider for quite often costly sequencing if they are not confident in their abilities. Researchers have told us some of the things they look for which lend to building their confidence:
    • Referrals & Reviews – researchers seek out colleagues who have done similar projects and look for recommendations. Word of mouth is one of the biggest methods researchers rely on to select an NGS provider.
    • Publications – providers who are listed in publications involving similar projects.
    • What kind of QC will be run on the sample.
    • Overall experience indicators such as time in business and volume of samples regularly handled.
    • Data and sample security.
    • Location – this factor is considerably important if previous trust is not established. Some researchers have absolutely no problem shipping samples across the globe, while others might physically drive their samples to a local provider to ensure sample integrity.

We would love to hear your feedback on this topic whether you are an NGS provider, or a researcher actively using next sequencing. What other decision driving criteria have you found as a provider, or what are some other factors important to you as a researcher?

In a Nutshell: Life Tech Exome Certified Service Provider Program

Life Technologies announced yesterday that they launched the Ion AmpliSeq Exome Certified Service Provider Program.

What the program is in a nutshell:

  • Goals: Offer a network of next gen sequencing providers able to help researchers get a high quality exome sequence at a reduced cost with fast turnaround times and low amounts of input material
  • Exome sequencing inputs: as little as 50ng of customer DNA
  • Library kit used: Ion AmpliSeq Exome kit
  • NGS Instrument used: Ion Proton
  • Exome sequencing outputs: high quality data, which of course can be used with Ion Reporter Software for mutation validation, annotation, and reporting

The Service Provider Program is intended to fill exome sequencing market demand which Life Tech argues has been under-serviced with exome sequencing currently going for $1,000+ , long turnaround times up to 8 weeks, and requiring up to 3mg of DNA. Dr. Candace Johnson, Deputy Director and the Wallace Chair of Translational Research at Roswell Park Cancer Institute states “Exome sequencing will be central to discoveries made in clinical research”. If the Exome CSP delivers as promised, it could have a major impact in accelerating discoveries made in clinical research.

For more information on the Life Tech Provider Program please see the entire press release.

Targeted Resequencing (TPS/WES) Tops Next Gen Sequencing Survey

Oxford Gene Technology (NGS provider currently listed on Genohub) recently presented the results of their next gen sequencing survey which demonstrated targeted resequencing as the top use for next generation sequencing. The results are based on a survey of 596 researchers who responded regarding their current and expected use of NGS services. When compared to the results for whole genome sequencing the popularity of targeted resequencing is possibly attributed mostly to the lower cost of targeted resequencing. This infographic depicts the results:

OGT NGS Survey Results

OGT NGS Survey Results

Other interesting results point to a general data problem with 38% of respondents saying they lack trust in bioinformatics data. Bioinformatics also leads the field when researchers were asked about the biggest barrier to NGS usage (see below).

Barriers to NGS Usage

Barriers to NGS Usage

Undoubtedly this presents an immense opportunity for the bioinformatics sector to increase confidence in data accuracy and interpretation which could have a positive impact on the use of next gen sequencing as a whole.

You can find many more interesting survey results on the excellent infographic titled Oxford Gene Technology – NGS Survey 2013.

First XPRIZE Cancelled Due To Unexpected Innovation in Next Gen Sequencing

For the first time ever, an XPRIZE has been cancelled. The reason — unexpected innovation in next generation sequencing. The Archon Genomics XPRIZE announced in 2006, had promised to award $10 mil to the first team that was able to accurately sequence 100 whole human genomes at a cost of $10,000 or less per genome in a short period of time. The competition was cancelled as XPRIZE CEO Peter Diamandis and team felt it was not serving its intended purpose to incentivize technological innovation in gene sequencing.

As stated by Peter Diamandis, “Every XPRIZE is carefully designed to address a market failure and hopefully create a new industry to achieve breakthroughs and solutions once thought to be impossible.” Although the Archon Genomics XPRIZE was conceived according to this criteria, the XPRIZE team felt that innovation in gene sequencing has been progressing independently of the XPRIZE incentive, therefore voiding the need for the competition.

The rapid innovation in next generation sequencing has caused sequencing times to decrease and prices to plummet to around $5,000 per genome. The XPRIZE team feels as if the targets laid out by the competition will be met in the very near future with or without their incentive, and have opted to cancel the XPRIZE and return the money to sponsors. The announcement by Peter Diamandis can be read in its entirety on the Huffington Post.

The logic behind the XPRIZE cancellation seems clear, however it remains to be seen what backlash, if any, arises from scientists who may have spent considerable time and effort devoted to meeting this challenge. Although next gen sequencing instruments are developed by large companies such as Illumina ($1.15B revenue), which may not be driven by a competition like the XPRIZE, innovation in this field must also be attributed to the wider research community, of which a team may have conceivably won the competition independent of any large commercial enterprise. In fact, in his cancellation announcement, Peter Diamandis thanks George Church and the Wyss Institute at Harvard for registering for the competition. Does the XPRIZE lose some of its ability to incentivize future competitions because of this cancellation? We welcome your comments on the matter.

How to Select the Best Next Generation Sequencing Platform For Your Project

We often get questions from researchers on selecting the best next generation sequencing platforms for specific projects. We are glad to offer free consultation to researchers on inquiries such as these among others. We also offer a sequencing guide to get you started.

Another great resource is a YouTube talk by Dr Elaine Mardis, a professor of Genetics and Molecular Biology and the co-director of the Genome Institute at the Washington University School of Medicine. She begins by discussing next gen sequencing instrument similarities, such as library amplification, nucleotide detection, read lengths, and paired end sequencing. She then dives into the factors unique to individual next gen sequencing instruments by Roche 454, Life Technologies, Illumina, Ion Torrent, PacBio, and Oxford Nanopore. The discussion focuses on the benefits and dissimilarities among them associated with:

  • Paired end reads: linear vs circularized fragments
  • Sequencing technologies: DNA polymerase, DNA ligase, synthesis H+ detection, syntheses, and nanopore
  • Library amplification methods: emPCR, bridge amplification, and its absence in some 3rd gen platforms
  • Run times
  • Error rates
  • Read lengths

Below, you can view the video in its entirety which goes into quite some detail and discusses the best types of projects for each NGS platform.