ASM SMRT Grant Winner: Squirrel Microbiome!

We’re pleased to announce the winner of this year’s SMRT Grant, which launched during the American Society for Microbiology annual meeting this summer. The grant program, co-sponsored by PacBio and the Institute for Genome Sciences (IGS), was very competitive, with over 100 submitted proposals. From this broad range of entries, our judges faced quite a task choosing just one recipient for the grant.

Congratulations to Jessica Sieber from the University of Minnesota Duluth, who impressed reviewers with her proposal, “Metagenomic analysis of the gut microbiota of the 13-lined ground squirrel, a model fat storing hibernator.”

Ground squirrels have been models for human health conditions from diabetes and obesity to longevity and hypothermia. These particular squirrels are scientifically interesting because they almost triple their weight before going into a six-month hibernation, during which they consume nothing. Sieber notes that the hibernation process involves reducing the squirrel’s body temperature to 4 degrees Celsius. While that should be a challenging environment for the animal’s gut microbes, in fact they appear to thrive and may be responsible for folate production to protect the squirrel’s brain during hibernation. A deeper understanding of the role these microbiota play in this process may have downstream implications for human health.

Sieber’s project involves using SMRT Sequencing to produce a high-resolution picture of these gut microbial communities, including how they withstand the cold hibernation temperature. We look forward to learning about the new insights she discovers as a result of this grant!

In addition to the grant winner, the submitters of the top 10 proposals will have the opportunity to conduct their projects through IGS with a 20% discount on SMRT sequencing. Thank you to all of the submitters who participated in the grant competition. We look forward to a number of exciting new projects in the coming months!

Do you have a project that could benefit from SMRT sequencing or any of our other sequencing and analysis services? If so, please contact us for a free project consultation.

Do you have this year’s SMRTest Microbe? Apply Now!

As announced at ASM Microbe last month, the GRC and PacBio are once again co-sponsoring the “SMRTest Microbe” Grant Program. We are accepting research proposals for one more week!

Submit a short application (<250 words) describing your microbe or microbial community and how it would benefit from the long reads, high accuracy and direct detection of epigenetic modifications generated by SMRT Sequencing. One winner will be selected by a panel of scientists and will receive up to four library preparations and a sequencing run using up to eight SMRT Cells. Sequencing and bioinformatics analysis will be performed by the GRC, a PacBio Certified Service Provider.

Are you interested in microbial genomes, complex populations, or methylomes? If so, submit your proposals by July 15, 2016.

Other restrictions apply. See official rules.

The PacBio Sequel is here! Join us for the PacBio User Group Meeting – June 8

We are excited to announce that our PacBio Sequel System has arrived! The newest instrument from PacBio joins the RS II system which has been with us since 2011. The Sequel uses redesigned SMRT Cells containing more than one million ZMWs (zero mode waveguides), a significant increase over the 150,000 ZMWs on RS II cells. The increased capacity of the cells means a potential for up to 6x as many reads per run compared to the RS II system. Over the past several weeks, we have begun evaluating and optimizing the Sequel. While initial supplies of the new SMRT Cells are limited, we expect improved cells and the next chemistry release in the coming months. Our goal is to offer sequencing on the Sequel as part of our services portfolio this summer.

If you are interested in hearing more about PacBio applications and the Sequel System at GRC, please join us as we host the PacBio East Coast User Group Meeting on June 8.

Pacbio Sequel

The PacBio Sequel System next to the Pacbio RS II.

 SMRT Cells

Evolution of PacBio’s SMRT Cell.  From Left to Right: SMRT Cell V2, SMRT Cell V3, and SMRT Cell 1M.

SMRTest Microbe Grant Winner: Identifying antibiotic resistance mechanisms with SMRT Sequencing

Congratulations to the winner of our recent “SMRTest Microbe” grant competition, Dr. Erin Price at the Menzies School of Health Research in Australia! The grant program, co-sponsored by PacBio and the Institute for Genome Sciences (IGS), was very competitive, with over 100 submitted proposals.

Dr. Price will receive SMRT® Sequencing and analysis from IGS — using up to 4 SMRTbell libraries and 8 SMRT Cells — to characterize the mechanisms behind the emergence of antibiotic resistance in Burkholderia pseudomallei, a highly pathogenic bacterium that causes the potentially deadly disease melioidosis. Dr. Price and colleagues have recently uncovered the development of meropenem resistance in local cases of B. pseudomallei infection, along with evidence that this resistance is linked to at least two mortalities in Australia so far.

Prior short-read based attempts to sequence and assemble these meropenum-resistant B. pseudomallei genomes have suffered from the inability to scaffold across highly repetitive and paralogous loci, low genome complexity, high GC content, and genomic inversions. Dr. Price plans to use the long reads generated by SMRT Sequencing to overcome these assembly issues and close the genomes. As noted in her proposal, complete genome sequences “will provide significant insights into the molecular basis of meropenem resistance in this dangerous pathogen.”

In addition to the grant winner, the submitters of the top 10 proposals will have the opportunity to conduct their projects through IGS with a 20% discount on SMRT sequencing. Thank you to all of the submitters who participated in the grant competition. We look forward to a number of exciting new projects in the coming months!

Do you have a project that could benefit from SMRT sequencing or any of our other sequencing and analysis services? If so, please contact us for a free project consultation.

 

IGS to Host Pacific Biosciences User Group Meeting

The Institute for Genome Sciences is pleased to once again host the PacBio Americas East Coast UGM and Workshops in Baltimore from June 16-18, 2015.

The UGM meeting is a day-long event on June 17th.  An optional half-day Bioinformatics workshop will be held on the afternoon of June 16th and the morning of June 18th, and an afternoon sample prep workshop will be held on June 16th. The bioinformatics workshop will cover large genome assembly with FALCON and analysis for the Iso-Seq™ method, among other topics. Special guests Jon Badalamenti and Sergey Koren will host on Tuesday and Thursday, respectively. The sample prep workshop will include talks on ultra-long DNA fragments, barcoding, targeted sequencing with solution-based capture, the Iso-Seq method, an intro to the SMRT Portal, and more.

View the agenda and register to attend.

GRC a PacBio Certified Service Provider; Co-sponsoring SMRTest Microbe Grant Program at ASM 2015

We are pleased to announce that the GRC is the first PacBio certified service provider on the East Coast. This recently announced program is a partnership between PacBio and select sequence providers who have completed the certification process and offer the highest quality sequencing and analysis services using the PacBio technology. We offer a full range of PacBio services, including whole genome sequencing, transcriptome sequencing via Iso-Seq, targeted amplicon sequencing, and other customized applications. Our analysis team has expertise in genome assembly and annotation, variant analysis, transcriptome analysis, and base modification detection. We look forward to continuing our strong partnership with PacBio and offering the highest quality sequencing and analysis to our customers and collaborators.

As part of this new partnership, the GRC is proud to co-sponsor the SMRTest Microbe Grant Program. One lucky winner will receive sequencing and analysis services from the GRC. To enter, submit a short grant application detailing your project and how it would benefit from the long reads and high consensus accuracy of SMRT Sequencing. The deadline for submissions is June 27, 2015.

For more information on our full range of sequencing and analysis services, visit our Laboratory Services and Analysis Services pages. Please contact us if you have any questions or would like a quote.

If you are attending ASM 2015 next week, please stop by the IGS booth (#776) to learn more about the grant program and all of our sequencing and analysis services. See you in New Orleans!

HiSeq 4000 Update

We are excited to announce that our HiSeq 4000 is here and running. We are off to the races! The run time for two flow cells is down to 3.5 days, which will allow us to complete projects faster than ever. Each dual flowcell run can sequence up to 12 human genomes, 180 exomes, or 100 transcriptomes.

Our first flow cell, a PE150 run, produced 2.9 billion clusters and more than 850 Gb of data.

Here are some basic stats from our first flow cell:

Lane Total Reads Total Bases %PF %Q20 %Q30
1 727,970,622 109,923,563,922 75.41 94.42 87.53
2 777,363,006 117,381,813,906 80.53 96.27 91.53
3 754,973,540 114,001,004,540 78.21 96.24 91.49
4 753,317,088 113,750,880,288 78.03 95.86 90.36
5 749,170,142 113,124,691,442 77.61 96.21 91.07
6 684,821,518 103,408,049,218 70.94 96.04 90.89
7 665,485,116 100,488,252,516 68.94 95.65 90.25
8 602,875,822 91,034,249,122 62.45 95.17 89.38

 

Do you have a project that could benefit from the combination of increased output and read length the HiSeq 4000 offers? If so, please contact us. We will discuss the details of your project with you and guide you through the process of planning your sequencing experiments.

Over 100 Publications Strong!

Less than eight years after beginning in an empty lab, our sequencing and analysis has contributed to more than 100 peer-reviewed publications. The first, published in Nature, was a comparison of the gut microbiome in obese and lean twins (DOI: 10.1038/nature07540). Since then, data generated by the GRC have been used for many other types of studies. Check out our list of publications.

If you have a publication resulting from data generated by the GRC that isn’t listed here, please let us know!

Thanks to all of our collaborators on these exciting projects for making the GRC a success. We look forward to the next 100 publications!

GRC Spring 2015 Update

The Genomics Resource Center (GRC) continues to expand its capabilities and project portfolio. As part of our contract with the U.S. Food and Drug Administration (FDA) to sequence, assemble, and annotate pathogens in support of the development and expansion of a comprehensive, curated public reference database, we are developing a new pipeline for Ebola virus sequencing and analysis. We have also initiated several new projects to sequence large animal and plant genomes using the Pacific Biosciences platform. These larger projects were made possible by our recent upgrade to the new P6-C4 chemistry. This new chemistry, combined with improved software, has increased read lengths by more than 30% and doubled overall throughput. In June, we will host the Pacific Biosciences East Coast User Group Meeting for the third consecutive year. Please join us to hear about this exciting technology and its expanding applications.

Our Illumina platform continues to improve as well. In April, we will take delivery of our first HiSeq4000. This sequencer, the newest announced by Illumina, will increase throughput by 50% while reducing run time by an additional 50%. Each HiSeq4000 will be capable of sequencing 24 human genomes per week. We have also expanded our MiSeq repertoire with the installation of a MiSeq Dx in our CLIA facility for clinical sequencing applications.

The GRC will be hosting a booth at the annual American Society for Microbiology (ASM) general meeting in New Orleans from May 30 – June 2, 2015. If you’re there, please stop by to visit and learn more about our services and capabilities!

Q&A with the Co-Directors of the GRC

How do I initiate a project with GRC?

It’s easy! Contact us via our website (www.igs.umarylande.edu/grc) or email (grc-info@som.umaryland.edu) and we will set up an initial consultation with you. During this consultation, we will discuss your project goals and expectations and advise on experimental design. From there, we develop a project plan that includes sample requirements, timelines, cost estimates, and deliverabes. For large, long-term projects, we schedule additional discussions to finalize the project plan and monitor progress.

How long does it take? How much will it cost?

These are the most common questions we hear, but often difficult to answer. Depending on the scope and scale of the project, the timeline can vary from a few weeks to months or even years. Similarly, costs can fall in a wide range. We treat each project separately and develop the best estimates of cost and timelines as part of our consultation with each investigator.

Do you offer analysis, or only sequencing?

We do it all – from project design through sequencing and analysis. We have bioinformatics teams specialized in genome assembly, variant analysis, metagenomics, transcriptomics, and epigenomic analysis. If you are interested in analysis, we include that as part of the project consultation and project plan.

Click here to find the full IGS Spring 2015 newsletter as well as previous editions.

Automated Preparation of Long Insert PacBio Libraries

The GRC sequences thousands of microbial samples each year. The high throughput of our sequencers and the small genome sizes of these samples means that a lot of libraries are needed to keep our sequencers running at full capacity. Automation of library preps is key to keeping instruments busy, reducing error, and maximizing the productivity of our lab staff.

We have used automation to prepare large batches of libraries for Illumina sequencing for several years. Earlier this year, we began testing preparation of long-insert libraries for our PacBio RS II platform.

Our Biomek FXP (BeckmanCoulter) is a Dual Multi-channel Span-8 with an integrated thermal cycler. The combination of a 96-channel pipetting head and Span-8 pipetting head (which has eight independently controlled channels) allows for the preparation of up to 96 libraries at a time and the minimization of master mix dead volumes.

A script for preparing SMRTbell libraries developed by Todd Hartley at NCI/SAIC-Frederick was downloaded from Pacific Biosciences SMRT Community. We modified to the protocol to accommodate the specific tips and reagents tubes our lab uses, and to optimize reaction mixing.

 

Deck Layout

PB_Biomek_Deck

Prior to being loaded on the Biomek, samples are sheared with g-TUBEs (Covaris, Woburn, MA), targeting an average fragment size of 20kb. Master mixes for each reaction are prepared and placed in the robot. The following steps are performed by the Biomek:

  • DNA damage repair
  • End Repair
  • Ampure clean up
  • SMRTbell Ligation
  • Exonuclease
  • Ampure clean up

Once the run is complete, the libraries are removed and size-selection is performed using the BluePippin (Sage Science, Beverly, MA).

  manual preps robotic preps
Number of libraries (n) 91 27
Average input amount (ng of sheared gDNA) 4945 4951
Average library size (bp) 18507 18910
Average library concentration (nM) 5.1 3.5
Average recovery (ng) 902.8 707.4
Average recovery (%) 18.7 14.2

Above are data comparing libraries prepped manually and on the Biomek, from March 2014 to date. While the yields from the Biomek preps are slightly lower than manual preps, the yield is comparable and sufficient for sequencing multiple SMRT cells per library.

For more information on our full range of sequencing and analysis services, visit our Laboratory Services and Analysis Services pages. Please contact us if you have any questions.