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.

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!

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.

 

Single Molecule Sequencing and Genome Assembly of a Clinical Specimen of Loa loa

Scientists Apply Successful Single Molecule Sequencing and de novo Genome Assembly to a Parasitic Worm that Infects Human Eyes and Skin

Investigators at the Institute for Genome Sciences (IGS) at the University of Maryland School of Medicine and the Laboratory of Parasitic Diseases at the National Institute of Allergy and Infectious Diseases (NIAID) at the National Institutes of Health (NIH) used the long-read, single-molecule Pacific Biosciences platform for the successful genome sequencing and de novo assembly of Loa loa round worms from a clinical sample. Their research, which generated the most complete genome sequence of a filarial nematode produced to date, provides a more comprehensive reference genome for this parasite in the hopes of developing better molecular diagnostics to decrease morbidity from filarial nematodes. Their findings appear in today’s issue of BMC Genomics.

Click here to access the abstract and complete article.

GRC Awarded Contract to Expand FDA Microbial Genome Database

IGS and the GRC have been awarded a contract to assist the U.S. Food and Drug Administration (FDA) in the expansion and curation of a public database of microbial genome sequences and associated metadata. This will serve as a valuable reference to evaluate and assess high-throughput sequencing based diagnostic devices. In addition to all publicly available microbial genome sequences, the database will include more than 550 newly sequenced, assembled, and annotated genomes from under-represented branches of the phylogenetic tree. For more information on the project, please click here or contact the GRC.

GRC Posters Presented at AGBT 2014

This year we are highlighting some of the work we’ve done in the past year.

The first poster provides an overview of how changes to our PacBio pipeline have increased our sequencing yields and read lengths, resulting in finished, high-quality microbial genomes, assembled using only PacBio data.

The second poster demonstrates how Next Gen sequencing can be used to investigate host and pathogen associations in cases of pulmonary non-tuberculous mycobacterial (PNTM) infections.

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.

PacBio Pipeline Off to a Strong Start for 2014

It has been a busy January for our PacBio RSII instrument. We are excited to report a new record yield from a single SMRT cell – 896,457,524 passed filter bases! It seems we are not far off from hitting 1 G.

 

Some more stats from this cell:

                Mean Read Length: 8391 bp

                P50 Subread Length: 6187 bp

                P90 Subread Length: 12314 bp

                P95 Subread Length: 14032 bp

                Maximum Subread Length: 24585 bp

 

We have come a long way in the past year. Here is a comparison of yields and mean read lengths of our top 20 SMRT cells in January 2013, compared to our top 20 SMRT cells so far in 2014:

The increases in both SMRT cell yields and read lengths are making PacBio an attractive option for sequencing and finishing microbial genomes. We are excited to see where 2014 will take us!

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.

Finishing Genomes with the PacBio RS II – Read our Core Lab Profile

The GRC, which offers services from sequencing library prep through genome assembly and downstream analysis, is generating complete bacterial genome sequences and methylation profiles using PacBio SMRT sequencing on the RS II. Several advancements in the library prep, sequencer, sequencing protocols, and data analysis software have all contributed to this.

To learn more about these breakthroughs and other emerging applications of SMRT sequencing, please read the PacBio Core Lab Profile showcasing the research performed at GRC and IGS here.

GRC and IGS offer not only cutting-edge sequencing, but a complete menu of services including assembly, annotation, and custom analyses. For more information about services offered, visit our Laboratory Services and Analysis Services pages. Please contact us if you have any questions.