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Join us on April 28 2016, 08:00 am PST (10:00 am CST / 11:00 am EST / 04:00 pm BST) for the discussion, and make sure to bring your questions for the panel! ----- Applying PacBio Long-Read Sequencing for Human Biomedical Research
Jonas Korlach
“Applications of Single Molecule, Real-Time (SMRT) Sequencing in Cancer Research”-- Giancarlo Russo
“Non-invasive, early detection of colorectal cancer driving mutations: a peek at the future of single molecule sequencing”
Adam Ameur
“Making SMRT sequencing ready for diagnostic use”------ http://www.frontlinegenomics.com/3644/webinar-applying-pacbio-long-read-sequencing-human-biomedical-research/?platform=hootsuite
Does Pacific Biosciences of California (NASDAQ:PACB) Look Attractive? MKM Partners Starts Coverage
by Linda Rogers — April 15, 2016-- Why MKM Partners Gives Pacific Biosciences of California (NASDAQ:PACB) $16.50 Price Target
http://www.clintongazette.com/does-pacific-biosciences-of-california-nasdaqpacb-look-attractive-mkm-partners-starts-coverage/
Friday, April 15, 2016-- Japanese Scientists Find Gene Fusion Driving B Cell Leukemia-- In a new Nature Genetics paper, scientists from the University of Tokyo and several other Japanese institutes and hospitals present results of a sweeping study of gene fusions driving a form of leukemia in teenagers and young adults. They used SMRT Sequencing to validate the gene fusion.
“Recurrent DUX4 fusions in B cell acute lymphoblastic leukemia of adolescents and young adults” comes from lead author Takahiko Yasuda and senior author Hiroyuki Mano, along with many collaborators. The team embarked on the search for new oncogenes responsible for acute lymphoblastic leukemia (ALL) in subjects from 15 to 39 years of age because the mechanisms responsible for this cancer “remain largely elusive,” they write.
From a large RNA-seq analysis, they found frequent insertion of a D4Z4 repeat that includes the DUX4 gene into the IGH locus, creating a DUX4-IGH gene fusion that produces high expression levels of an aberrant form of DUX4. The scientists transplanted this gene fusion into mice, where it led to the generation of B cell leukemia. They report that fusion-driven oncogenes are more important for causing ALL in this age range than previously thought. “Our data thus show that DUX4 can become an oncogenic driver as a result of somatic chromosomal rearrangements and that [ALL in adolescents and young adults] may be a clinical entity distinct from ALL at other ages,” Yasuda et al. write.
The team used SMRT Sequencing to confirm the full sequence of the gene fusion, which could not be done with short-read sequencing. “Given that the average read length in our next-generation sequencing approach was 104 bp, it was difficult to determine how many copies of DUX4 had been inserted into the IGH locus,” they report. They performed whole genome sequencing of a B cell line cultured from a 19-year-old ALL patient, generating about 69 Gb of data.
“Our analysis confirmed that one full-length and one partial copy of D4Z4 were translocated to the IGH locus, accompanied by minor rearrangements within the IGHD2-15 and IGHVII-60-1 regions,” the scientists report. This figure shows SMRT Sequencing data confirming the presence of the DUX4-IGH gene fusion.
To learn more about applying SMRT Sequencing to cancer research, check out our AACR conference preview. http://www.pacb.com/blog/japanese-scientists-find-gene-fusion-driving-b-cell-leukemia/?platform=hootsuite
Wednesday, April 13, 2016--- Genome and Transcriptome Analysis Help Scientists Deconstruct Cancer Complexity-- At Cold Spring Harbor Laboratory, scientists used SMRT® Sequencing to decode one of the most challenging cancer genomes ever encountered. Along the way, they built a portfolio of open-access analysis tools that will help researchers everywhere make structural variation discoveries with long-read sequencing data.
When Mike Schatz realized a few years ago that his PacBio® System had reached the throughput needed to process human genomes, he decided to give it a real challenge: the incredibly complicated, massively rearranged SK-BR-3 breast cancer cell line. The genome consists of 80 chromosomes, and that’s just the tip of the complexity iceberg.
“We were really interested in sequencing a human genome that would be maximally impactful and that was aligned with our research interest in cancer genomes, where it’s been well documented that structural variations play a major role,” says Schatz, now an associate research professor of computer science at Johns Hopkins University and an adjunct associate professor of quantitative biology at Cold Spring Harbor Laboratory, where the analysis took place. He notes that despite its importance, structural variation has not been thoroughly studied because short-read sequencers cannot reliably identify these large genomic elements. “One of the really special properties about the PacBio Sequencer is, in addition to being able to call SNPs or small variants, we also get to look for large variants such as structural variation,” he says.
But as Schatz and his collaborators at Cold Spring Harbor Laboratory and the Ontario Institute for Cancer Research delved into this work, they realized that existing variant callers were tailored to short-read data. To make the most of the large amount of long-read information they were generating, the team wrote a suite of new analysis tools optimized for SMRT Sequencing data. “The tools catering to short-read data just aren’t made to capture the awesome information that we can now take advantage of,” says Maria Nattestad, a graduate student in Schatz’s lab who wrote several of the new algorithms. “Building our own tools was really the only way to go here.”
Those tools, which are especially important for understanding structural variation, are now being publicly released to fuel further SMRT Sequencing studies of human genomes. Also coming out soon is the team’s detailed analysis of the SK-BR-3 genome and transcriptome, which includes a high-quality assembly as well as a new understanding of gene fusions, the evolutionary history of this cell line, and more.
De novo sequencing and assembly were the first steps in making sense of the SK-BR-3 genome. With 72-fold SMRT Sequencing coverage, “we got an outstanding assembly of this genome even though it’s so complicated,” Schatz says, citing a contig N50 size of 2.5 Mb compared to a state-of-the-art short-read assembly with a contig N50 of just 3 kb. “That’s nearly a thousand-fold more contiguous going from short-read to long-read assemblies, and it’s through that improved assembly that the majority of structural variants were detected.”
Using custom-built analysis tools, including variant callers Sniffles, by Schatz lab member Fritz Sedlazeck, and Assemblytics, by Nattestad, the scientists found more than 10,000 structural variants in the SK-BR-3 genome ranging in size from 50 bases to millions of base pairs long. Another major discovery involved meticulously characterizing the complicated process that led to the cell line’s Her2 oncogene amplification.
The team also used the Iso-Seq™ method to analyze the full transcriptome of SK-BR-3, finding as much complexity at the RNA level as they saw in the DNA. “In the Iso-Seq analysis, we see many tens of thousands of novel isoforms,” Schatz says. “That’s a really strong testament to the long reads, which fully capture an isoform in one sequence — unlike short reads, where you have to infer isoform structure.”
To learn more about the project, which included novel findings about gene fusions in cancer, check out the full case study. http://www.pacb.com/blog/genome-and-transcriptome-analysis-help-scientists-deconstruct-cancer-complexity/
Bio-IT World Conference & Expo comes early this year (April 4-7th), where more than 3,000 researchers, life science, pharmaceutical, clinical, and IT professionals descend upon Boston. If you’re headed to Bio-IT World, please stop by the DNAnexus booth (#317) to explore and discuss our latest projects, new datasets, and tools.
This year, Big Data is a featured theme at the event. Our friend and collaborator, Dr. Taha Kass-Hout (Chief Health Informatics Officer and Director of the FDA’s Office of Health Informatics) kicks off the Big Data: Sharing vs. Privacy vs. Security in Healthcare session (Track 3, Thursday at 2:00pm) with an overview of precisionFDA, the new community platform for NGS assay evaluation and regulatory science exploration. Additional fascinating talks follow: John E. Mattison (Co-Chair, eHealth Workgroup, Global Alliance for Genomics and Health) will present emerging initiatives from the GA4GH and Robert Grossman (Director, Center for Data Intensive Science) will discuss large-scale data commons for genomic and clinical data. The final session includes those experts along with John M. Conley (Robinson Bradshaw & Hinson), Andrew K. Porter (Merck & Co.), and Mollie Shields-Uehling (SAFE-BioPharma Assoc.) for a panel discussion looking at data sharing innovations and the regulatory environment.
Best_Practices_Logo_2016_finalist-blue (1)Precision medicine continues to be a strong theme for 2016, bolstered by President Obama’s recent Precision Medicine Initiative (PMI) Summit. At DNAnexus, we like to think of genetic data as the original Big Data, and we are passionate about Big Data’s role in advancing medical discovery and treatments. We are proud to announce that precisionFDA is a Bio-IT World Best Practice Award finalist. The Best Practice Award highlights outstanding examples of how technological innovation can be powerful forces of change in the life sciences. The precisionFDA platform was developed under contract for the FDA by DNAnexus, and currently provides over 1,000 users from nearly 500 organizations in the genomics community a sandbox for NGS assay evaluation and regulatory science exploration. Be sure to be there when the winners are announced live on Wednesday, April 6th at 9:30am.
Dr. Taha Kass-Hout, Chief Health Informatics Officer and Director of FDA’s Office of Health Informatics, will be making a cameo appearance at the DNAnexus booth to answer questions and showcase the precisionFDA platform. Learn how precisionFDA is leveraging community participation to advance regulatory science in the area of next-generation sequencing – DNAnexus is proud to have collaborated with the FDA to develop the community platform.
PrecisionFDA Demo & Dr Taha Meet & Greet at the DNAnexus Booth (#317)
Wednesday, April 6th at 10:00am-11:00am
Wednesday, April 6th at 5:30pm-6:30pm
DNAnexus Booth (#317) Activities
Unite your workflows. DNAnexus offers end-to-end tailored solutions. We’ll be demoing a few upstream and downstream partner options at our booth: Sapio
Sciences LIMS, Pacific Biosciences tools, and WuXi NextCODE Cloud platform.
Wednesday, April 6th
3:25pm Sapio’s Exemplar NGS LIMS Demo
See how the integration of the DNAnexus platform with Sapio Sciences’ LIMS solution is enabling seamless integration with bioinformatics operations, enabling secure scalability and flexible workflows.
Thursday, April 7th
10:00am WuXi NextCODE Cloud Demo
Learn how the WuXi NextCODE Cloud — powered by DNAnexus — is accelerating the use of genomics to benefit patients worldwide. China cloud solution now available.
1:20pm PacBio de novo Assembly & SV Analysis Tools Demo
Demo the latest PacBio tools on the DNAnexus platform. See how complex bioinformatics is made simple.
Must-See Presentations
We’re proud to support our customers who push the envelope in advancing genomic science and medicine. Be sure not to miss these talks!
Track 3 Software Applications & Services: Regeneron Genetics Center’s Use of the DNAnexus Annex in the Amazon Cloud for Large-Scale NGS Downstream Compute
Presenter: Christopher Sprangel, Director, IT, Human Genetics, Genome Informatics, Regeneron Genetics Center and Nathan Wallace, Founder & CEO, Turbot
When: Wednesday, April 6 at 2:25pm
Track 3 Software Applications & Services: Featured Presentation: precisionFDA
Presenter: Taha A. Kass-Hout, MD, MS, Chief Health Informatics Officer & Director, Office of Health Informatics, FDA
When: Thursday, April 7 at 2:00pm
Track 3 Software Applications & Services: Panel Discussion: How will data sharing innovations fare in the regulatory environment?
Moderator: John M. Conley, JD, PhD, Robinson Bradshaw & Hinson
Panelists: Robert Grossman, PhD, University of Chicago, Taha A. Kass-Hout, MD, MS, FDA, John E Mattison, MD, Kaiser Permanente, Andrew K. Porter, Merck & Co., and Mollie Shield-Uehling, SAFE-BioPharma Association
When: Thursday, April 7 at 3:30pm
To learn more about DNAnexus and see a demo of the DNAnexus Platform, visit booth #317 in the exhibit hall at the Bio-IT World Conference & Expo.-- http://blog.dnanexus.com/2016-04-01-at-bio-it-world-technological-innovation-advancing-genomic-science-and-medicine/
The sex of your baby could be determined by a 1.5 million year old VIRUS
Read more: http://www.dailymail.co.uk/sciencetech/article-3517772/The-sex-baby-determined-1-5-million-year-old-VIRUS.html#ixzz44X9ODL2K
Follow us: @MailOnline on Twitter | DailyMail on Facebook
(Sex of a baby? Ancient virus makes the call) "Pacific Biosciences of Menlo Park provided the technology used in the discovery".--------http://news.yale.edu/2016/03/30/sex-baby-ancient-virus-makes-call#.Vv2GwDgwdH0
Anthera Pharmaceuticals Inc (NASDAQ:ANTH) Given Consensus Rating of “Strong Buy” by Analysts
Posted by Taylor Nule on Mar 3rd, 2016-- Shares of Anthera Pharmaceuticals Inc (NASDAQ:ANTH) have earned a consensus broker rating score of 1.00 (Strong Buy) from the two brokers that provide coverage for the company, Zacks Investment Research reports. Two analysts have rated the stock with a strong buy rating.
Analysts have set a 1 year consensus price objective of $12.33 for the company and are forecasting that the company will post ($0.28) earnings per share for the current quarter, according to Zacks. Zacks has also assigned Anthera Pharmaceuticals an industry rank of 71 out of 265 based on the ratings given to its competitors.
Anthera Pharmaceuticals (NASDAQ:ANTH) traded up 4.24% during mid-day trading on Thursday, reaching $3.69. The company’s stock had a trading volume of 437,261 shares. The company’s 50-day moving average price is $3.05 and its 200 day moving average price is $5.35. Anthera Pharmaceuticals has a 52 week low of $2.28 and a 52 week high of $11.65. The firm’s market capitalization is $147.14 million.
ANTH has been the topic of a number of research reports. Zacks Investment Research raised shares of Anthera Pharmaceuticals from a “hold” rating to a “buy” rating and set a $7.00 target price on the stock in a research note on Wednesday, November 4th. Jefferies Group reaffirmed a “buy” rating and set a $9.00 price target (down previously from $12.00) on shares of Anthera Pharmaceuticals in a research report on Friday, February 19th. Finally, Citigroup Inc. reduced their price target on shares of Anthera Pharmaceuticals from $15.00 to $11.00 in a research report on Friday, February 19th. http://lulegacy.com/?p=904123
On Rare Disease Day, Celebrating the Contributions of Genomics
Monday, February 29, 2016
Today we are celebrating Rare Disease Day with like-minded folks all over the world. The tribute kicked off in 2008 and has gathered so much momentum that people in more than 80 countries are expected to participate in 2016. Each disease is rare — affecting fewer than 1 in 1,500 people — but because there are so many of these diseases, together they affect millions of people globally.
Here at PacBio, many of our team members have their own stories about dealing with rare disease, and we imagine the same is true of our blog readers. We’re so proud that leading scientists have already begun using SMRT Sequencing to make important new DNA and RNA discoveries about the genetics and disease mechanisms of rare diseases. In the future, we anticipate even more of these studies will lead to novel breakthroughs as scientists expand their use of PacBio sequencing for human disease studies. Together, we can have a real impact in helping families struggling with these diseases.
Here are some examples of how researchers have shed light on rare diseases with SMRT Sequencing:
Baylor’s Jim Lupski, who studies and has been diagnosed with Charcot-Marie-Tooth neuropathy, recently spoke about a de novo PacBio assembly of his genome that found much more structural variation — especially copy number changes — than previous assemblies from short-read data. He also described how long reads are able to better resolve and characterize break points associated with these disease-causing structural variants, and also resolve sequence context to provide base-level resolution of specific genotypes.
In a separate presentation, Richard Gibbs from Baylor College of Medicine noted that just 25% of Mendelian disorders have been solved with short-read sequence data, and suggested that the success rate may be limited by the inability of these platforms to detect structural variation, repeat regions, and complex events. With SMRT Sequencing and structural variation analysis algorithms created at his genome center, scientists may be able to uncover the genetic basis of many more Mendelian disorders using low-coverage, long-read PacBio sequencing.
Paul Hagerman from the University of California, Davis, led the first team in the world to completely sequence a fully expanded pathogenic ‘CGG repeat allele’ in the FMR1 gene on the X chromosome that is associated with Fragile X Syndrome. Previously thought to be “unsequenceable,” PacBio sequencing of repeat expansions in the FMR1 gene is shedding new light on pathogenic variants and interruptions that are meaningful for screening and carrier counseling, and that may lead to improved diagnostic and intervention strategies for families affected by Fragile X syndrome.
In a related project, follow-up work from Flora Tassone and other UC Davis researchers applied the Iso-Seq method to characterize alternative splicing in the FMR1 gene for a different disorder called Fragile X-associated tremor/ataxia syndrome (FXTAS). They found differential expression for certain gene isoforms suggesting a functional relevance for these in the pathology of FMR1-associated disorders.
Scientists in North Carolina generated the first high-quality sequence of MUC5AC, a gene that has been implicated in a range of diseases, including cystic fibrosis. The gene had long been represented as a gap in the human reference genome because of its complex and highly repetitive central exon. Characterization of the MUC5AC gene and the sequence variation in the central exon will facilitate genetic and functional studies for this critical airway mucin.
In a recent talk at AGBT, Bobby Sebra from the Icahn School of Medicine presented results from the recent targeted PacBio sequencing of the C9orf72 loci, which contains a GGGGCC repeat expansion now known to cause familial ALS (also known as Lou Gherig’s Disease). He presented sequencing data from both the PacBio RS II platform and the new Sequel System, showing the ability to fully characterize the sequence of this locus and provide novel insights into the genetics underlying this debilitating disease.
Tetsuo Ashizawa and Karen McFarland from the University of Florida are making progress understanding the genetics of spinocerebellar ataxia type 10 (SCA10). In a recently published study, they describe sequencing through a pentanucleotide repeat allele known to cause this disorder, and characterizing various repeat interruption motifs associated with different SCA10 clinical phenotypes.
Shinichi Morishita’s lab at the University of Tokyo has described similar methods for characterizing tandem repeats associated with the SCA31 brain disease using a hybrid long- and short-read approach.
At Stanford University, Ayal Hendel is working in collaboration with John Day and the Myotonic Dystrophy Foundation to study the CTG/CAG repeat tracts that represent the genetic basis for myotonic dystrophy type 1 (DM1), and explore the cellular and molecular pathological mechanisms involved in DM — including aberrant alternative splicing.
We’d like to congratulate these scientists, along with all the others around the world who are working hard to make a difference in the lives of people burdened by rare disease. Whether you’re using our technology or any other, we thank you and wish you all the best!
PacBio is proud to be an official partner of Rare Disease Day. Get involved with global efforts or US-based initiatives to honor those dealing with rare diseases. http://www.pacb.com/blog/on-rare-disease-day-celebrating-the-contributions-of-genomics/
Rhenman & Partners Asset Management Has $1,160,000 Position in Anthera Pharmaceuticals Inc (ANTH)
Posted by Logan Wallace on Feb 25th, 2016--- Rhenman & Partners Asset Management maintained its stake in Anthera Pharmaceuticals Inc (NASDAQ:ANTH) during the fourth quarter, according to its most recent Form 13F filing with the Securities and Exchange Commission. The fund owned 250,000 shares of the biopharmaceutical company’s stock at the end of the fourth quarter. Rhenman & Partners Asset Management owned about 0.63% of Anthera Pharmaceuticals worth $1,160,000 at the end of the most recent quarter.
Anthera Pharmaceuticals Inc (NASDAQ:ANTH) traded down 2.34% during mid-day trading on Thursday, hitting $2.92. The company had a trading volume of 335,731 shares. The firm’s market capitalization is $116.44 million. Anthera Pharmaceuticals Inc has a 12 month low of $2.28 and a 12 month high of $11.65. The company has a 50-day moving average price of $3.12 and a 200 day moving average price of $5.59.
ANTH has been the subject of a number of analyst reports. Zacks Investment Research upgraded Anthera Pharmaceuticals from a “hold” rating to a “buy” rating and set a $4.25 price target for the company in a research report on Monday, January 11th. Jefferies Group restated a “buy” rating and issued a $9.00 price target (down previously from $12.00) on shares of Anthera Pharmaceuticals in a research report on Friday, February 19th. Finally, Citigroup Inc. reduced their price target on Anthera Pharmaceuticals from $15.00 to $11.00 in a research report on Friday, February 19th. One research analyst has rated the stock with a hold rating and four have issued a buy rating to the stock. The stock presently has an average rating of “Buy” and a consensus price target of $10.45.--- http://www.tickerreport.com/banking-finance/1432922/rhenman-partners-asset-management-has-1160000-position-in-anthera-pharmaceuticals-inc-anth/
Anthera Pharmaceuticals Inc (ANTH) PT Lowered to $11.00
February 20th, 2016 ----- Anthera Pharmaceuticals Inc (NASDAQ:ANTH) had its target price dropped by equities research analysts at Citigroup Inc. from $15.00 to $11.00 in a report released on Friday, StockTargetPrices.com reports. Citigroup Inc.’s price objective would indicate a potential upside of 263.04% from the company’s previous close.
An institutional investor recently raised its position in Anthera Pharmaceuticals stock. Sio Capital Management LLC raised its position in Anthera Pharmaceuticals Inc (NASDAQ:ANTH) by 61.0% during the fourth quarter, according to its most recent Form 13F filing with the SEC. The firm owned 1,378,443 shares of the biopharmaceutical company’s stock after buying an additional 522,371 shares during the period. Anthera Pharmaceuticals makes up 2.8% of Sio Capital Management LLC’s holdings, making the stock its 12th largest position. Sio Capital Management LLC owned 3.46% of Anthera Pharmaceuticals worth $6,396,000 as of its most recent SEC filing.
Other analysts also recently issued reports about the company. Jefferies Group restated a “buy” rating and set a $9.00 price objective on shares of Anthera Pharmaceuticals in a report on Friday. Zacks Investment Research downgraded Anthera Pharmaceuticals from a “buy” rating to a “hold” rating in a report on Thursday, November 5th. One research analyst has rated the stock with a hold rating and four have given a buy rating to the stock. The company presently has a consensus rating of “Buy” and an average target price of $10.45.--http://www.owler.com/iaApp/article/56c965e1e4b075e7a6489d48.htm?utm_source=twitter&utm_medium=social&utm_campaign=sectorNews_Pharmaceuticals
Streamed live on Feb 12, 2016
PacBio AGBT 2016 Workshop Live
Co-hosted by Roche-----
Anthera Pharmaceuticals, Inc. (NASDAQ:ANTH): Zacks Growth Score
February 15, 2016 11:02 am------- Zacks Group follows a process of giving Growth Style Scores to listed companies in the industry. The entity has placed a positive score on Anthera Pharmaceuticals, Inc. (NASDAQ:ANTH). This study takes into account the different parameters like firm’s financial numbers and the growth predictions. While conducting a financial study, the firm takes all the parameters including Balance Sheet and others. Once these parameters are analyzed, a score is given that can be anywhere between ‘A’ and ‘F’. A stock with an ‘A’ rating has a higher growth potential to achieve strong returns on the market.
Zacks frequently conducts a survey of market analysts to get price target and earnings forecast on any firm. Following the latest poll, the research group has given a mean growth prediction of N/A for Anthera Pharmaceuticals, Inc.. This is based on the anticipated growth numbers for next few years.
For the short-term, the research groups have a price target of $13.333. This figure records recommendations of 3 brokerages. The experts who think that the stock can give better returns over market has a positive target of $14 while the conservative target is $12.
There are many brokerage firms functioning in the market, and all of them have a different methodology for preparing a research report. Therefore, it becomes confusing for the investors to make an investment decision. In order to eliminate this confusion, Zacks uses a scale of 1-5, wherein investors keep their holdings if rating is ‘3’, opt for a buy if rating is 1 or 2 and can go for sell if rating is 4 or 5. Anthera Pharmaceuticals, Inc. has scored a rating of 1.
Anthera Pharmaceuticals, Inc. (NASDAQ:ANTH) is likely to unveil its quarterly report on 2016-03-21. For the period ended on 2016–1-2-31, the firm posted quarterly EPS of $-0.29 and this time it is projected at $-0.28.-- http://theenterpriseleader.com/stock-watch/anthera-pharmaceuticals-inc-nasdaqanth-zacks-growth-score/69638/
"DaleYuzuki: JK: Points out ability to read mutations impt in cancer, but not accessible to ILMN"--- http://m.g3journal.org/content/5/12/2801.long?view=long&pmid=26497143
Zacks: Anthera Pharmaceuticals Inc (NASDAQ:ANTH) Given $13.33 Average Target Price by Brokerages
Posted on February 9, 2016--- Shares of Anthera Pharmaceuticals Inc (NASDAQ:ANTH) have been assigned an average broker rating score of 1.00 (Strong Buy) from the three brokers that provide coverage for the stock, Zacks Investment Research reports. Three equities research analysts have rated the stock with a strong buy rating.
Analysts have set a 12 month consensus target price of $13.33 for the company and are anticipating that the company will post ($0.28) earnings per share for the current quarter, according to Zacks. Zacks has also assigned Anthera Pharmaceuticals an industry rank of 70 out of 265 based on the ratings given to related companies.
Anthera Pharmaceuticals (NASDAQ:ANTH) opened at 2.46 on Thursday. The stock has a 50 day moving average of $3.64 and a 200 day moving average of $6.21. Anthera Pharmaceuticals has a one year low of $2.28 and a one year high of $11.65. The firm’s market cap is $98.09 million.
Separately, Zacks Investment Research downgraded shares of Anthera Pharmaceuticals from a “strong-buy” rating to a “hold” rating in a research note on Monday, October 12th.
In other news, insider Colin Hislop sold 12,500 shares of the firm’s stock in a transaction on Friday, November 13th. The shares were sold at an average price of $5.31, for a total transaction of $66,375.00. Following the completion of the transaction, the insider now owns 19,147 shares of the company’s stock, valued at $101,670.57. The transaction was disclosed in a legal filing with the Securities & Exchange Commission, which is available through this hyperlink.
Anthera Pharmaceuticals Inc (NASDAQ:ANTH) is a biopharmaceutical company focused on developing and commercializing products to treat serious diseases associated with inflammation and autoimmune diseases. The Company’s primary Phase 3 product candidate blisibimod targets elevated levels of Bcell activating factor (BAFF) which has been associated with a variety of Bcell mediated autoimmune diseases including systemic lupus erythematosus or lupus IgA nephropathy lupus nephritis multiple myeloma and others.
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http://sleekmoney.com/zacks-anthera-pharmaceuticals-inc-nasdaqanth-given-13-33-average-target-price-by-brokerages/927448/
Anthera Pharmaceuticals Inc (ANTH) Sets New 1-Year Low at $2.28
Posted by Joseph Griffin on Feb 8th, 2016 --- Shares of Anthera Pharmaceuticals Inc (NASDAQ:ANTH) reached a new 52-week low during trading on Monday , Marketbeat reports. The company traded as low as $2.28 and last traded at $2.34, with a volume of 591,284 shares. The stock had previously closed at $2.67.
Separately, Zacks Investment Research downgraded Anthera Pharmaceuticals from a “buy” rating to a “hold” rating in a report on Thursday, November 5th. One analyst has rated the stock with a hold rating and four have issued a buy rating to the company. Anthera Pharmaceuticals currently has a consensus rating of “Buy” and an average price target of $11.85.
The stock’s market capitalization is $98.09 million. The firm’s 50-day moving average price is $3.64 and its 200-day moving average price is $6.21.
In other news, insider Colin Hislop sold 12,500 shares of the company’s stock in a transaction dated Friday, November 13th. The stock was sold at an average price of $5.31, for a total value of $66,375.00. Following the transaction, the insider now owns 19,147 shares in the company, valued at approximately $101,670.57. The sale was disclosed in a filing with the Securities & Exchange Commission, which is available through the SEC website.
A hedge fund recently raised its stake in Anthera Pharmaceuticals stock. California State Teachers Retirement System boosted its position in Anthera Pharmaceuticals Inc (NASDAQ:ANTH) by 1.7% during the fourth quarter, according to its most recent 13F filing with the Securities and Exchange Commission. The institutional investor owned 73,476 shares of the biopharmaceutical company’s stock after buying an additional 1,263 shares during the period. California State Teachers Retirement System owned approximately 0.18% of Anthera Pharmaceuticals worth $341,000 at the end of the most recent reporting period.
Anthera Pharmaceuticals Inc (NASDAQ:ANTH) is a biopharmaceutical company focused on developing and commercializing products to treat serious diseases associated with inflammation and autoimmune diseases. The Company’s primary Phase 3 product candidate blisibimod targets elevated levels of Bcell activating factor (BAFF) which has been associated with a variety of Bcell mediated autoimmune diseases including systemic lupus erythematosus or lupus IgA nephropathy lupus nephritis multiple myeloma and others.
http://www.tickerreport.com/banking-finance/1364912/anthera-pharmaceuticals-inc-anth-sets-new-1-year-low-at-2-28/
California State Teachers Retirement System Increases Stake in Anthera Pharmaceuticals Inc (ANTH)
February 6th, 2016 • California State Teachers Retirement System raised its stake in Anthera Pharmaceuticals Inc (NASDAQ:ANTH) by 1.7% during the fourth quarter, according to its most recent filing with the Securities and Exchange Commission. The institutional investor owned 73,476 shares of the biopharmaceutical company’s stock after buying an additional 1,263 shares during the period. California State Teachers Retirement System owned 0.18% of Anthera Pharmaceuticals worth $341,000 at the end of the most recent reporting period.
Shares of Anthera Pharmaceuticals Inc (NASDAQ:ANTH) opened at 2.67 on Friday. Anthera Pharmaceuticals Inc has a 12 month low of $2.51 and a 12 month high of $11.65. The firm’s market capitalization is $106.47 million. The firm’s 50 day moving average is $3.72 and its 200-day moving average is $6.33.
Separately, Zacks Investment Research downgraded shares of Anthera Pharmaceuticals from a “buy” rating to a “hold” rating in a research note on Monday, January 18th. One equities research analyst has rated the stock with a hold rating and four have given a buy rating to the stock. The company presently has an average rating of “Buy” and an average target price of $11.85.
In other news, insider Colin Hislop sold 12,500 shares of the company’s stock in a transaction on Friday, November 13th. The shares were sold at an average price of $5.31, for a total transaction of $66,375.00. Following the transaction, the insider now directly owns 19,147 shares of the company’s stock, valued at approximately $101,670.57. The transaction was disclosed in a filing with the SEC, which is accessible through the SEC website.
Anthera Pharmaceuticals Inc (NASDAQ:ANTH) is a biopharmaceutical company focused on developing and commercializing products to treat serious diseases associated with inflammation and autoimmune diseases. The Company’s primary Phase 3 product candidate blisibimod targets elevated levels of Bcell activating factor (BAFF) which has been associated with a variety of Bcell mediated autoimmune diseases including systemic lupus erythematosus or lupus IgA nephropathy lupus nephritis multiple myeloma and others.
12 Month Chart for NASDAQ:ANTH
Receive News & Ratings for Anthera Pharmaceuticals Inc Daily - Enter your email address below to receive a concise daily summary of the latest news and analysts' ratings for Anthera Pharmaceuticals Inc and related companies with MarketBeat.com's FREE daily email newsletter. http://www.americanbankingnews.com/2016/02/06/california-state-teachers-retirement-system-increases-stake-in-anthera-pharmaceuticals-inc-anth/
Menlo Park: Biotech firm's invention puts it at the forefront of DNA-related research
By Kevin KellyDaily News Staff Writer
POSTED: 02/05/2016 06:00:00 AM PST0 COMMENTS
A biotech company in Menlo Park is getting widespread attention through a cutting-edge tool it has developed for genetic research.
Pacific Biosciences, which operates in a historically industrial section of Belle Haven, markets a tool that is essentially a high-powered microscope for analyzing DNA.
The tool, whose most recent iteration looks a bit like a vending machine with an iPad for a keypad, gives researchers the ability to perform long DNA reads in the fields of human biomedical research, microbiology and plant and animal sciences.
For instance, the company's tools were used to map a Fragile X syndrome mutation, something researchers previously couldn't do. Fragile X is a common form of cognitive impairment in humans, more common in men than women, with roughly 1 in every 3,000 males born severely mentally impaired. Prior to Pacific Biosciences' RS II System and the newer Sequel System -- basically a 2.0 version of RS II -- which began shipping in December, scientists could only point to the spot on the human genome where the defect was occcuring.
"It's unsequenceable with any other technology," said Jonas Korlach, chief scientific officer for Pacific Biosciences, citing the tool's unique ability to read long sections of genetic code at once. "Fragile X (has) a repeat region that's very long ... and if you don't have the long read, you can't figure it out."
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The fully mapped mutation, announced by a UC Davis School of Medicine scientist in 2013, could allow quicker and better screening procedures for Fragile X and other similar genetic disorders.
More recently, the company's technology is being used to study how influenza spreads through a population, in the hopes of creating more effective vaccines. Like the Fragile X researchers before them, scientists from New York University and the University of Hong Kong are using Pacific Biosciences' tool for its singular ability to study long lines of genetic code, allowing minor variants in flu patients to be studied for the first time.
"We can look for a broader range of the type of variation from one individual to the other than other technologies that we compete with," said Pacific Biosciences CEO Michael Hunkapiller. "We have a more complete database, in terms of the changes being looked at. .... There's so much variation from one individual to another, and so much that is dependent on what your environment is.
"An example is there are as many cells in your body that ain't human as there are that are and (microorganisms) have a big influence on your health ... and when you have cancer, those cells can get really messed up."
The tool is also being used in the UC Santa Cruz-led Genome 10K Project, a conservation-minded effort that aims to collect DNA sequences representing the genomes of 10,000 vertebrate species, roughly one for every vertebrate genus.
The RS II and Sequel tools use technology that speed-reads through millions of molecules to find pertinent information. The newer Sequel, which was launched in October, is one-third the size and half the cost of the RS II, which was developed seven years ago and first entered the market in 2011. Hunkapiller said the Sequel can essentially take a 50-million piece jigsaw puzzle and turns it into a 50-piece puzzle.
"If you think of a genome, it's got 3 billion base pairs times two -- one from the mother, one from the father," he said. "You can't read that from one end to the other, there's no technology that does that right now, so you have to break it apart into a lot of little pieces, understand what the sequence of each of those little pieces and then you have to put it all back together and create a whole.
"The thing that our technology does is that it allows the scientists to not break the pieces of DNA into such small elements, so it's easy to take a complex situation and put it back together."
The more compact, lower-cost Sequel is propelling the company forward.
The company last month announced it has already received orders for 49 Sequels. With just 160 systems currently in place worldwide, that counts as a large leap.
Roche Holding AG, a Swiss health-care conglomerate that partners with Pacific Biosciences for in-vitro diagnostics has in recent weeks been exploring a possible acquisition of the company, Reuters reported Tuesday. Many analysts, including Economic News Daily, are suggesting Pacific Biosciences' stock as one to watch in 2016. The biotech's shares are anticipated to rise by 30 percent over the next five years.
"There are now over 1,000 research papers that have been published using this technology, (which is) what you set out to do as a method developer," said Korlach, citing advances in understanding how organisms work to create methane and how particular fungi attack crops. "The whole hope is that it's useful for a lot of people to ultimately improve the human condition. ... We want to improve health care, we want to figure out how to feed 9 billion people in 2050, we want to prevent infectious disease so you don't have to worry about the flu anymore."
Pacific Biosciences is planning to move to a new site at the end of 2017. It operates out of five buildings at Menlo Science & Technology Park, which was purchased by Facebook last February. Pacific Biosciences has received a use permit from the city of Menlo Park to convert a building at 1315 O'Brien Drive for a new headquarters, one with 8,000 additional square feet of workspace. As part of a deal with Facebook, the biotech is giving up two five-year extension options for a $20 million payout from the social network to leave early.
Email Kevin Kelly at kkelly@dailynewsgroup.com or call him at 650-391-1049. http://www.mercurynews.com/peninsula/ci_29472343/menlo-park-biotech-firms-invention-puts-it-at
Single Molecule Real Time Sequencing - Pacific Biosciences-----
Human Genome Turns 15: Mike Hunkapiller, CEO, PacBio
Mike Hunkapiller, CEO, Pacific Biosciences
Chapters:
0:00 When did you first encounter the idea to sequence the entire human genome?
5:09 Were you pushing Craig, or was he pushing you?
7:47 Definition of sequencing has changed in the past 15 years
14:40 How big a deal are long reads?
19:15 Will PacBio take over the research market?
24:32 What’s been the biggest surprise of your career?
We’re all familiar with the announcement in the year 2000 by US President, Bill Clinton, and the UK’s Prime Minister, Tony Blair, that scientists had completed the first draft of the human genome. It was a big deal. But the actual publications didn’t happen until the next year, February of 2001. Which means that this February is the fifteenth anniversary of the publication of the first human genome. For our commemorative show we’re joined by Mike Hunkapiller, the CEO of Pacific Biosciences.
Mike and his team at PacBio are coming off a great year. Their stock is up. Their long read sequencing technology is used for over a thousand scientific publications. And last year they launched a new better, faster, cheaper instrument, the Sequel, which are sold out through the first half of this year. PacBio is cool again.
How much were tool makers in the driving seat of the genomic revolution? And how much further can sequencing improve? Before asking Mike this, we explore some of his memories of those wild days when sequencing the human genome got presidents and prime ministers on the phone with their speech writers.
- See more at: http://mendelspod.com/podcasts/human-genome-turns-15-mike-hunkapiller-ceo-pacbio/#sthash.AIGUi9iX.6XBVg5Iw.dpuf
Submitted by Ayanna Monteverdi on Thu, 02/04/2016 - 09:36
- See more at: http://mendelspod.com/podcasts/human-genome-turns-15-mike-hunkapiller-ceo-pacbio/#sthash.AIGUi9iX.6XBVg5Iw.dpuf
Zacks: Anthera Pharmaceuticals Inc (NASDAQ:ANTH) Receives Average Recommendation of “Strong Buy” from Brokerages
February 4th, 2016 - By Mark Watkins - 0 comments
Anthera Pharmaceuticals logo. Shares of Anthera Pharmaceuticals Inc (NASDAQ:ANTH) have been assigned a consensus broker rating score of 1.00 (Strong Buy) from the three analysts that cover the company, Zacks Investment Research reports. Three analysts have rated the stock with a strong buy recommendation.
Brokers have set a 1-year consensus price target of $13.33 ----------http://www.dakotafinancialnews.com/?p=791923
Anthera Pharmaceuticals: Buy Low Opportunity On Potential Drug Approval
Feb. 2, 2016 8:51 AM ET| About: Anthera Pharmaceuticals, Inc. (ANTH)
Dave Dierking Dave Dierking+ Follow(713 followers)
Value, dividend investing, ETF investing, growth at reasonable price
Send Message|Website(201 clicks)
Summary
Anthera's lupus drug, Blisibimod, is in stage 3 clinical trial with results expected in the second half of 2016.
It also has its cystic fibrosis drug, Sollpura, in the clinical trial pipeline.
The stock was hurt late in 2015 following its second additional stock offering in the calendar year.
Zenyaku, its licensing and development partner in Japan, backed out of its deal in September 2015.
Citigroup recently reiterated its buy rating and $15 price target on Anthera's stock.
Anthera Pharmaceuticals (NASDAQ:ANTH) is a tiny biotech that trades primarily on hope at this point. The company's stock price moved significantly in 2015 and looked like it would be a top performer as it shot higher from $2 at the start of the year to a peak of over $11 in July as it announced it would release additional data on patient-reported outcomes of phase 2b PEARL-SC clinical trial of its leading drug candidate Blisibimod. The drug looks to treat the symptoms related to the onset of lupus.
The stock is currently trading back in the $3 range after the momentum of biotechs began cooling and the company needed to complete a second stock issue within the calendar year in order to raise funds for ongoing operations. It's a bit of a typical story for tiny biotech companies but if you begin looking towards what's in store throughout the remainder of 2016 there's a lot to like about Anthera.
Two drugs and five clinical studies underway
Anthera is currently developing two drugs.
Its lupus drug, Blisibimod, is in the midst of its phase 3 CHABLIS-SC1 clinical study after surpassing its 400 patient enrollment target in July of last year. The drug will also enter the phase 3 CHABLIS 7.5 trial shortly to study the effects of Blisibimod in the treatment of severe lupus symptoms not typically controlled with traditional medications. Anthera also has Blisibimod in the middle of a third middle stage clinical trial for treating IgA Nephropathy.
In September, Anthera initiated the phase 3 SOLUTION clinical study for its other drug, Sollpura, in treating cystic fibrosis patients. Another early stage clinical trial for the drug in treating pediatric cases is also being initiated.
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http://seekingalpha.com/article/3855466-anthera-pharmaceuticals-buy-low-opportunity-potential-drug-approval
On the fly -- Pacific Biosciences seen as likely to be bought after 'game changing' launch! (" Citing historical transactions and comparable multiples, the analyst thinks that it is reasonable for the company to be acquired in the $18-$33 per share range.") -- http://thefly.com/onthefly.php?id=2306287
Q2 2016 @PacBio will update his chemistry, the mean length of the reads will be increased to 32kb -- https://twitter.com/search?q=pacbio&src=tyah
Comprehensive
Genome
and
Transcriptome
Structural
Analysis
of
a
Breast
Cancer
Cell
Line
using
PacBio
Long
Read
Sequencing --http://schatzlab.cshl.edu/presentations/2015/2015.10.29.GI.SKBR3.pdf
22 Dec 2015
PATRIC December 2015 Release Features over 20,000 New Genomes and PacBio Assembly Support
New Genomes and Annotation System Enhancements
In this release, we have added 23,052 new genomes from NCBI GenBank, bringing the total number of genomes in PATRIC to over 54,000. The full list of available bacterial genomes can be accessed from the Genomes Tab for all bacteria, and from the Genomes Data Landing Page.
As part of this Data Release, we have implemented an enhanced annotation system and process that allows us to annotate and release new genomes continuously—in a near real-time mode. With this enhancement, we will be able to maintain currency with other public genome sources such as NCBI, as well as quickly incorporate new genomes from other sources.
Other recent enhancements to the annotation system include support for the following key features:
Public/private flag for any genome submission
Efficient submission of large batch of genomes
Submission of GenBank files
Parsing of minimum metadata, i.e., genome name, taxon id, genetic code, directly from GenBank files
Parsing features from original GenBank files and preserving them as ID synonyms
Carrying forward additional “misc” features such as repeats, binding sites, miscellaneous RNAs, etc. annotated in the GenBank file to PATRIC annotations
Assigning functions based on the latest build of k-mers from Core-SEED
Assigning new protein families—PLFams and PGfams
Computation of MLSTs
Automated parsing of genome metadata from NCBI BioProject and BioSample records
Automated parsing of antibiogram metadata from NCBI BioSample records
Loading antibiogram metadata into the corresponding new PATRIC Solr database core
Automated incorporation of strain name into genome name, if not already present
Automated incrementing of genome counts for corresponding taxonomy nodes (for public genomes)
Assembly Service Enhancements
Also in this release, we have enabled experimental support for assembly of genomes sequenced using PacBio technology and Oxford Nanopore technologies. PATRIC users can now take advantage of the long reads generated on these platforms and assemble them into longer contigs, and in some cases complete genomes. Both raw bax.h5 read files and filtered FASTQ files from PacBio and FASTQ from Oxford Nanopore are accepted as input. The PATRIC Genome Assembly Service can be accessed at https://www.patricbrc.org/app/Assembly. NOTE: You must have a PATRIC user account and be logged in to use the services and workspace.
http://enews.patricbrc.org/4865/patric-december-2015-release-features-over-20000-new-genomes-and-pacbio-assembly-support/?utm_source=twitterfeed&utm_medium=twitter
NCTC 3000: A joint collaboration between Public Health England, Pacific Biosciences and the Wellcome Trust Sanger Institute to complete the sequencing of 3,000 bacterial strains from PHE's National Collection of Type Cultures (NCTC) using Pacific Biosciences' Single Molecule, Real-Time (SMRT) sequencing technology. http://www.sanger.ac.uk/resources/downloads/bacteria/nctc/
A meeting with @Roche to plan first project of " 6kb long read capture" based on @nimblegen technology and @PACBIO https://twitter.com/search?q=pacbio&src=tyah
IsoSeq analysis and functional annotation of the infratentorial ependymoma tumor tissue on PacBio RSII platform--Available online 23 November 2015 ---
Abstract
Here, we sequenced and functionally annotated the long reads (1-2 kb) cDNAs library of an infratentorial ependymoma tumor tissue on PacBio RSII by Iso-Seq protocol using SMRT technology. 577 MB, data was generated from the brain tissues of ependymoma tumor patient, producing 1,19,313 high-quality reads assembled into 19,878 contigs using Celera assembler followed by Quiver pipelines, which produced 2952 unique protein accessions in the nr protein database and 307 KEGG pathways.
Additionally, when we compared GO terms of second and third level with alternative splicing data obtained through HTA Array2.0. We identified four and twelve transcript cluster IDs in Level-2 and Level-3 scores respectively with alternative splicing index predicting mainly the major pathways of hallmarks of cancer. Out of these transcript cluster IDs only transcript cluster IDs of gene PNMT, SNN and LAMB1 showed Reads Per Kilobase of exon model per Million mapped reads (RPKM) values at gene-level expression (GE) and transcript-level (TE) track. Most importantly, brain-specific genes-PNMT, SNN and LAMB1 show their involvement in Ependymoma.
Keywords
Ependymoma;
Isoseq;
PacBio;
Annotation
(lot more reading)http://www.sciencedirect.com/science/article/pii/S2214540015000705
yesterday at 8:21am
William Blair: Conclusions From Meetings at Large Genome Center
• We recently hosted a group of investors at a large genome center. In this note, we
provide our conclusions and our latest models for Illumina and Pacific Biosciences;
our Pacific Biosciences model includes our revised revenue build following the
company’s launch of the Sequel.
• Stock Thoughts. It is fairly clear that Illumina’s technology is not going anywhere
anytime soon, especially in these large production centers. The stock popped after it
replaced Sigma-Aldrich in the S&P 500 and is now trading at 50 times our 2016 EPS
estimate and seems on its way back to trading on total addressable market (TAM).
Estimates for 2016 look achievable, but we would like to see more conservatism
factored into 2017 and 2018 estimates to position the company better to beat
estimates and raise guidance. Illumina remains well positioned in a large addressable
market that is far from mature. Thus, we maintain our Outperform rating on the
stock. While it is possible that Illumina has a sizable product launch at the upcoming
JPMorgan conference, perhaps if the company focuses more on content development,
this would be a catalyst to drive a rebasing of longer-term estimates, offering
investors a better entry point to position long in the name into 2016.
• With regard to Pacific Biosciences, this center has not yet had any inbound projects
for long-read sequencing, but it sounds like a large project might be percolating. At a
minimum, it seems the center may bring a new Sequel into R&D. Our estimates
included below are a first guess of what product sales could look like over the next
two to three years. Demand could be higher or lower than our assumptions (e.g., we
assumed a doubling of annual placements of the Sequel relative to the RSII, but could
placements be as high as a NextSeq?), but we do not have enough data at this point to
see how far off we are. At roughly $10, the stock has reached where we believed it
could trade based on our revised income statement (revenue of about $150 million in
2018) and using appropriate multiples. Investors who have been in the stock since $4
to $5, however, could consider taking some off the table and leverage any volatility
around the launch (as it ramps up margins, etc.) to rebuild positions. PacBio’s stock
has a lot of momentum currently, however, and we have received increasing inbound
calls on the name. The demand picture remains quite strong and we believe the
price/specs of the Sequel fundamentally change the demand picture for PacBio’s
products; we reiterate our Outperform rating.
• Conclusions From the Genome Center Tour. The Genome Center’s X Tens are
primarily being used for human whole genome sequencing, although the center is
also sequencing a few other organisms’ genomes on the X since Illumina has opened
the platform to nonhuman species. While other service providers may be more
focused on price, this center has opted to compete on quality and service.
• As a service provider, the center’s instrument capital expenditure decisions are
usually based on project dynamics. The center has seen an influx of larger project
sizes and thus is not sample-limited. A lower per-genome price point should help
drive continued growth in large-scale projects that are funded. At present, the X
platforms are running close to full capacity. Thus, it seems feasible the center could
expand its X Ten fleet by a few machines over the next year or so.
• The center’s HiSeq 2500s are being used for exome sequencing, ChiP-seq, Rna-Seq, metagenomics, and epigenetics
(bisulfite sequencing). Given the center’s focus on larger-scale projects, the 2500s are running below capacity; thus, it is
unlikely the center will upgrade to HiSeq 3000/4000s.
• Given the ecosystem that has evolved to support Illumina technology (including sample prep, informatics and analysis
tools, application-specific workflows), Illumina is likely to continue to be the platform leveraged in production
environments—even if another platform is commercialized that is cheaper (while a platform that is 2 times cheaper with
the same specs might chip away at Illumina’s installed based, a platform would have to be at least 3 times cheaper with
similar specs to drive a meaningful replacement cycle).
• For some applications, there may be advantages to long-read/long-range technologies; however, this will ultimately
depend on the price of the sequence in aggregate compared with Illumina derived sequence only and depends on the
specific application. The center has yet to have meaningful inbound demand for these types of technologies. As the center
continues to ramp up resources, it may be able to shift more focus to R&D-type activities, which would most likely be
some sort of long-range/long-read technologies (i.e., PacBio’s Sequel and/or 10x Genomics’ GemCode Platform).
• One area that could be further addressed by the platform vendors is true sample-to-answer solutions (meaning
integration of DNA extraction and library prep); while the NeoPrep could help address some of this, current throughput of
16 samples may limit its use at a larger center.
• Ultimately, clinical continues to be considered a key future source of growth. To fuel a clinical market that is a factor of 10
times larger than the research market, however, would require distribution of platforms into smaller labs via true sampleto-
answer solutions. How do we get there? The most obvious way is microfluidics and miniaturization.
Read more: http://bridgeandtunnelinvestor.com/thread/2055/halts-revolocity-launch-complete-genomics?page=1#ixzz3sZf2HHi0
Nov 23, 2015-- Inside the Bizarre Genome of the World’s Toughest Animal
Tardigrades are sponges for foreign genes. Does that explain why they are famously indestructible?
The toughest animals in the world aren't bulky elephants, or cold-tolerant penguins, or even the famously durable cockroach. Instead, the champions of durability are endearing microscopic creatures called tardigrades, or water bears.
They live everywhere, from the tallest mountains to the deepest oceans, and from hot springs to Antarctic ice. They can even tolerate New York. They cope with these inhospitable environments by transforming into a nigh-indestructible state. Their adorable shuffling gaits cease. Their eight legs curl inwards. Their rotund bodies shrivel up, expelling almost all of their water and becoming a dried barrel called a “tun.” Their metabolism dwindles to near-nothingness—they are practically dead. And in skirting the edge of death, they become incredibly hard to kill.
In the tun state, tardigrades don't need food or water. They can shrug off temperatures close to absolute zero and as high as 151 degrees Celsius. They can withstand the intense pressures of the deep ocean, doses of radiation that would kill other animals, and baths of toxic solvents. And they are, to date, the only animals that have been exposed to the naked vacuum of space and lived to tell the tale—or, at least, lay viable eggs. (Their only weakness, as a researcher once told me, is “vulnerability to mechanical damage;” in other words, you can squish ‘em.)
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Scientists have known for centuries about the tardigrades’ ability to dry themselves out. But a new study suggests that this ability might have contributed to their superlative endurance in a strange and roundabout way. It makes them uniquely suited to absorbing foreign genes from bacteria and other organisms—genes that now pepper their genomes to a degree unheard of for animals.
Thomas Boothby from the University of North Carolina at Chapel Hill made this discovery after sequencing the first ever tardigrade genome, to better understand how they have evolved. Of the 700 species, his team focused on Hypsibius dujardini, one of the few tardigrades that’s easy to grow and breed in a lab.
At first, Boothby thought his team had done a poor job of assembling the tardigrade’s genome. The resulting data was full of genes that seemed to belong to bacteria and other organisms, not animals. “All of us thought that these were contaminants,” he says. Perhaps microbes had snuck into the samples and their DNA was intermingled with the tardigrade’s own.
But the team soon realized that these sequences are bona fide parts of the tardigrade’s genome.
By expelling their water, tardigrades have ironically become a sponge for foreign genes.
That wouldn't be unusual for bacteria, which can trade genes with each other as easily as humans might swap emails. But these “horizontal gene transfers” (HGT) are supposedly rare among animals. For the longest time, scientists believed that they didn't happen at all, and reported cases of HGT were met with extreme skepticism.
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Recently, more and more examples have emerged. Ticks have antibiotic-making genes that came from bacteria. Aphids stole color genes from fungi. Wasps have turned virus genes into biological weapons. Mealybugs use genes from many different microbes to supplement their diets. A beetle kills coffee plants with a borrowed bacterial gene. Some fruit flies have entire bacterial genomes embedded in their own. And one group of genes, evocatively called Space Invaders, has repeatedly jumped between lizards, frogs, rodents, and more. But in all of these cases, it's usually one or two genes that have jumped across. At most, the immigrants make up 1 percent or so of their new native genome.
But Boothby found that foreign genes make up 17.5 percent of the tardigrade's genome—a full sixth. More than 90 percent of these come from bacteria, but others come from archaea (a distinct group of microbes), fungi, and even plants. “The number of them is pretty staggering,” he says.
Claims like these have been debunked before, so the team took extra care to confirm that the sequences did indeed come from outside sources.
For a start, they re-sequenced the genome using PacBio—a system that decodes single unbroken strands of DNA without first breaking them into smaller fragments. This revealed that the foreign genes are physically linked to the tardigrade’s native ones. They are all part of the same DNA strands, which means they couldn't have come from other contaminating microbes. They have also gained several features that are characteristic of animal genes, like an animal gloss over their fundamental bacterial character. John Logsdon from the University of Iowa, who studies genome evolution, is certainly convinced. “It’s a very interesting and technically robust paper,” he says.
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So, how did these genes get into the tardigrade's genome in the first place? Boothby thinks that the answer lies in three quirks of tardigrade biology. First, they can dry themselves out, a process that naturally splits their DNA into small pieces. Second, they can stir back to life by rehydrating, during which their cells become leaky and able to take in molecules from the environment—including DNA. Finally, they are extremely good at repairing their DNA, sealing the damage that occurs when they dry out.
“So we think tardigrades are drying out, and their DNA is fragmenting along with the DNA of bacteria and organisms in the environment,” explains Boothby. “That gets into their cells when they rehydrate. And when they stitch their own genomes together, they may accidentally put in a bacterial gene.” By expelling their water, tardigrades have ironically become a sponge for foreign genes.
Do these genes do anything? So far, the team have found that the tardigrades switch on several of their borrowed genes, which, in other organisms, are involved in coping with stressful environments. That's pretty tantalizing: It suggests that these animals might owe at least part of their legendary durability to genetic donations from bacteria.
Boothby imagines something like this: Ancient tardigrades could dry themselves out to an extent, which allowed some foreign genes to enter their genome. If some of these genes made them more tolerant to drying, the animals would have become even more susceptible to horizontal gene transfers. “This positive feedback loop builds up over time,” says Boothby. “That’s speculation on our part.”
It certainly bolsters his case that another microscopic animal—a rotifer—can also dry itself out during tough times, and also shows signs of extensive horizontal gene transfer. Almost 10 percent of its genes came from foreign sources. Boothby’s team now wants to check for similar genetic infiltrations in other animals that tolerate desiccation, including some nematode worms, fish, and insects. They are also planning to gradually inactivate the tardigrade’s borrowed genes to see if that compromises its fabled invincibility.
Ralph Schill from the University of Stuttgart also points out that Hypsibius dujardini is something of a wuss among tardigrades, and isn't actually very good at surviving desiccation. Perhaps the genomes of its hardier relatives—the ones that shrug off extreme cold, extreme heat, and open vacuums—will yield even bigger surprises.
http://www.theatlantic.com/science/archive/2015/11/tardigrades-worlds-toughest-animals-borrowed-a-sixth-of-their-dna-from-microbes/417243/
Public Health England reference collections
This project aims to provide annotated and assembled genomes for 3,000 bacteria and 500 viruses as part of a new eResource
The project is split into two parts:
NCTC 3000: A joint collaboration between Public Health England, Pacific Biosciences and the Wellcome Trust Sanger Institute to complete the sequencing of 3,000 bacterial strains from PHE's National Collection of Type Cultures (NCTC) using Pacific Biosciences' Single Molecule, Real-Time (SMRT) sequencing technology.
NCPV 500: A collaboration between PHE and Sanger to produce 500 viral genomes from PHE's National Collection of Pathogenic Viruses (NCPV) using the Illumina sequencing platform.
Collectively, the data generated will be housed in a publically accessible web-based eResource that integrates metadata and genome sequences for type and reference strains of biomedically important bacterial and viral pathogens. This resource will integrate accession, taxonomy and authentication information with publications, genome sequences, comparative analysis databases and other resources at EMBL and NCBI.
This is a community resource project. Data will be available from here, and from the NCTC. We will submit assembled, annotated sequences to the International Sequence Databases as they become available. We request that you cite this webpage in any publication using the data, and would appreciate it if you contact us to discuss the use of this data.
Data Downloads
•Download annotated assemblies#
•BLAST server
Please note: these are pre-submission assemblies that should not be treated as final versions. Assemblies contain both chromosomal and plasmid contigs.
Background
The Wellcome Trust Sanger Institute will generate PacBio sequencing data and provide assembled and annotated genomes for the 3,000 bacteria from the NCTC collection.
The NCTC is one of the world's premier collections for bacterial strains, but most bacteria in NCTC currently have no genome references. Biological Resource Centres, such as NCTC and NCPV, are a vital part of the infrastructure underpinning life sciences, providing biomaterials of known provenance. Type and reference strains act as landmarks for mapping microbial diversity and measuring unique changes in properties and patterns of infection and response to clinical interventions. Combining reference genomes with the wealth of historical and biological information existing for these strains will generate a data set of enormous value for basic and clinical microbiology.
Pacific Biosciences' Single Molecule, Real-Time (SMRT) Sequencing technology achieves very long reads and high consensus accuracy, greatly improving the ability to finish bacterial genomes. And, because the technology can directly detect base modifications, the epigenomes for bacteria can also be obtained with no additional data acquisition, and this data will also be provided.
NCTC Strains
As bacterial strains from the collection progress through the project, links to the raw sequence data, epigenome data and annotated assemblies will appear here.
Current Statistics
•Number of species sequenced: 158
•Number of samples:719
•Number of samples manually assembled:298
•Number of samples automatically assembled:81
http://www.sanger.ac.uk/resources/downloads/bacteria/nctc/#t_2
Scientists Use the Iso-Seq Method to Study Genes Linked to Prostate Cancer
Monday, November 16, 2015
A team of scientists from Australia, Canada, and the US published fascinating new work that may help explain gene expression patterns seen in prostate cancer. In the course of the project, they used SMRT Sequencing and found a novel fusion transcript linking two genes with high sequence identity.
“Identification of a novel fusion transcript between human relaxin-1 (RLN1) and human relaxin-2 (RLN2) in prostate cancer” was published in Molecular and Cellular Endocrinology by lead author Gregor Tevz, senior author Colleen Nelson, and a number of collaborators. In it, the scientists attempted to untangle expression signals from two relaxin genes, which were formed by a duplication event sometime before humans and apes branched off. The genes play a role in reproduction and are most highly expressed in ovaries and prostate. “Outside normal physiology, RLN2 is a promoter of cancer progression in several different types of cancers,” the scientists note.
Previous studies were unable to distinguish between the two genes, so this team deployed long-read sequencing and the Iso-Seq method from PacBio to sort out reads from RLN1 and RLN2 in LNCaP cells. Using their results along with publicly available data, they made a number of discoveries. For one thing, they found that most prostate cancer cell lines underrepresent RLN1, which is highly expressed in both normal and cancerous tissue. “LNCaP cells best reflect the RLN1 expression observed in [prostate cancer] and is the most relevant cell line for the use in further studies of RLN1 biology,” the team reports.
They also detected a novel fusion transcript that incorporates large swaths of both RLN1 and RLN2, but were able to design primers to distinguish the fusion from the genes. “The fusion transcript encodes a putative RLN2 with a deleted secretory signal peptide indicating a potentially biologically important alteration,” the scientists write. They determined that RLN1 and the fusion transcript are inversely regulated by androgens, and suggest that follow-up studies will be helpful to elucidate the mechanisms governing this response.
While we’re on the subject of cancer, don’t forget that the abstract deadline for the 2016 AACR annual meeting is coming up on December 2. We’re already looking forward to hearing about more great discoveries at that conference!
http://www.pacb.com/blog/scientists-use-the-iso-seq-method-to-study-genes-linked-to-prostate-cancer/
The economics of PacBio sequencing a microbe
Published Mon November 2 2015
("This fiscal year we are also discontinuing the Illumina "Eukaryotic Standard Draft". Recent analysis by Alicia shows that we can also get better assemblies switching to 10kbp+ PacBio libraries for eukaryotes rather than continuing with the long mate pair and fragment approach. The extra costs associated with preparing two different libraries versus a single PacBio library which also produces a more contiguous assembly means we will do half our fungal draft sequencing on the PacBio for the next financial year.
This then leads into the Sequel announcement - given that we already use ~5 SMRT cells for a eukaryotic genome, switching to the PacBio Sequel will likely mean that this product type will become even more cost effective. This is calculated using the announced numbers: ~7X the GBp per SMRT cell at 2-3X the SMRT cell cost would mean approximately ~2X cost saving.") -- --
This post is a condensed version of a discussion in we had in my office after PacBio announced their new Sequel long-read sequencer. This contains some hand-wavy statements and calculations, however the aim is to provide some insight into how we make decisions about sequencing here at the JGI. I wrote this blog post jointly with Alex Copeland the QC and Assembly group lead, and Alicia Clum the Genome Assembly and Analysis lead. Alicia recently joined twitter and you should definitely follow her. She may have mentioned that she wanted everyone to send her all their genome assembly questions but I could have misheard her.
The PacBio Sequel specifications indicate ~7x the output per SMRT cell at half the instrument price of the RSII ($300k vs. $750k). The caveat being that the individual SMRT cell price will be 2-3X more expensive. Keith Bradnam has a round up of all the news and blogs discussing the announcement. The Joint Genome Institute (JGI) will be receiving one of the Sequels in November as part of the early access program. Therefore we are already in the process of considering how to integrate the Sequel into our existing pipelines.
The JGI offers several assembly products for prokaryotes and Eukaryotes. Each product is a combination of different DNA extraction, library preparation, sequencing and informatics post-processing. Our microbial isolate products relevant to the PacBio announcement and this discussion are:
•Prokaryotic Minimal Draft - A cultured Bacteria or Archaea, with a typically simple genome. Sequenced as a 2x150bp, 275bp insert fragment library on an Illumina 1T HiSeq with 48x pooling. We sequenced 1050 of these last financial year.
•Prokaryotic Improved Draft - Library preparation using AMPure bead purification to select fragments greater than 10kbp. Sequenced on the PacBio RSII usually requiring only a single SMRT cell. A "better" product than the prokaryotic minimal draft because this often produces a single contig assembly, this is used for more complex prokaryotic genomes such as Actinomycetes. We sequenced 250 of these last financial year, and expect to produce ~500 this year.
•Eukaryotic Minimal Draft - A cultured eukaryote, usually fungal. Similar to the prokaryotic minimal draft this is sequenced as a 2x150bp, 275bp insert fragment library on an Illumina 1T HiSeq with 8x pooling. Typically we sequence eukaryotes with more tractable genomes, such as Aspergillus, using this protocol. We sequenced 100 of these in the last financial year.
•Eukaryotic Standard Draft - Two libraries generated and sequenced using the ALLPATHS-LG recipe: one standard Illumina fragment, and one 4kb long mate pair library. The long mate pairs using ALLPATHS-LG allow us to generate a better assembly than the minimal draft. We sequenced ~100 of these in the last fiscal year.
•Eukaryotic Improved Draft - Same as a prokaryotic improved draft except that this is larger eukaryotic genome and requires ~5 SMRT cells for a ~40Mbp haploid genome. Extra attention is required in assembly due to the presence of organelles. We sequenced less than 10 of these in the last financial year. For the last few years this product has been a combined draft of one Illumina fragment library, one Illumina long mate pair library and one PacBio library. This financial year we will now switch to a 20kbp Blue Pippin library which has higher labour and reagent costs, compared with an AMPure PacBio library, but which produces a better assembly.
Eukaryotic Sequencing
You may notice that there is no 'Prokaryotic Standard Draft' in this list, i.e. no combined standard fragment and long mate pair sequencing of prokaryotes. The reason is that producing the long mate pair library and sequencing on Illumina costs us about the same as sequencing on the PacBio RSII, while a PacBio 10kbp library produces a better assembly overall with the same current throughput.
For the same reason this fiscal year we are also discontinuing the Illumina "Eukaryotic Standard Draft". Recent analysis by Alicia shows that we can also get better assemblies switching to 10kbp+ PacBio libraries for eukaryotes rather than continuing with the long mate pair and fragment approach. The extra costs associated with preparing two different libraries versus a single PacBio library which also produces a more contiguous assembly means we will do half our fungal draft sequencing on the PacBio for the next financial year.
This then leads into the Sequel announcement - given that we already use ~5 SMRT cells for a eukaryotic genome, switching to the PacBio Sequel will likely mean that this product type will become even more cost effective. This is calculated using the announced numbers: ~7X the GBp per SMRT cell at 2-3X the SMRT cell cost would mean approximately ~2X cost saving.
Prokaryotic Sequencing
The second point, and the reason to write this blog post, is what effect could the PacBio Sequel have on current our prokaryotic sequencing? Considering our existing PacBio prokaryotic sequencing, switching to the Sequel would mean more data that we do not necessarily require because, in most cases, one SMRT cell is sufficient to get a complete assembly for the average Bacteria/Archaea. Using one Sequel SMRT cell would make each prokaryotic assembly 2-3X more expensive than it already is. Therefore, it is unlikely we would do a straight switch over to the Sequel for prokaryotes if cost was the only consideration. There are however two caveats to this.
The first caveat is that we are currently in the middle of sequencing 1000 Actinomycetes - bacteria that we sequence on the PacBio RSII as prokaryotic improved drafts. These are high GC, repeat-rich genomes, with especially long repeats, meaning 2-3 RSII SMRT cells are usually needed to produce sufficient numbers of reads long enough to span repeats. Therefore in the cases where we are already using multiple SMRT cells, switching to the Sequel would make sense for the same cost-saving described above. An added advantage, and worth mentioning in this context, is that PacBio sequencing displays no GC-bias, so this is an additional advantage for organisms with very high or low GC genomes. An example is the JGI recently completed sequencing and assembly of a Piromyces fungal genome with <20% GC which, for the last decade, has resisted all previous attempts at sequencing and assembly.
The second caveat is that the increased Sequel capacity makes pooling important. If you are unfamiliar with pooling, this is the process of combining multiple different DNA libraries together and then sequencing them all together, usually in an Illumina flowcell. A unique oligonucleotide 'barcode' is added to each library during preparation which allows the FASTQ data to be separated back into the original library after sequencing. If we could take advantage of the extra Sequel capacity and sequence 7 microbes per SMRT cell using barcodes, then the same cost savings would apply.
An additional possibility that would streamline laboratory preparation, and therefore cost, is to skip barcoding entirely. The reason being that the long-read overlaps are unambiguous enough that the genomes would simply assemble together out of the pool, similar to that of a metagenome. This would however require the production planning to ensure that the pairwise genome distance between any two organisms is large enough to ensure no cross-assembly. I had a short twitter conversation and this approach with Mick Watson who outlined some possible problems.
Why don't we sequence everything on PacBio?
In the process of discussing the Sequel the question arose that we have considered multiple times in the past: why don't we sequence all microbes on the PacBio? One reason is that an Illumina prokaryotic minimal draft costs around 1/10th as much as a PacBio prokaryotic improved draft. Sequencing 1000 microbes on Illumina instead of PacBio RSII is the difference in approximately $360,000 a year.
This is a pure cost-only comparison and if cost was the only factor, then we would sequence everything on Illumina 1T using a standard fragment library. However, assembly quality is extremely important, and is why we don't only do this. If the costs drop in future with the new PacBio Sequel platform , we will reevaluate opportunities for applying PacBio to prokaryotic isolate sequencing.
http://www.bioinformaticszen.com/post/economies-of-sequencing-a-microbe/
Welcome to Silicon Valley, Festival of Genomics!
Monday, November 2, 2015
FOG logo CAThis week the Festival of Genomics comes to the West Coast, and we’re excited to be a founding sponsor of the Front Line Genomics organization. Not only is it our first chance to show off the new Sequel System in our home state, but there will also be a number of great talks reporting SMRT Sequencing results. Here are the some of the presentations to consider if you’re attending the event:
Wednesday, November 4
11:30 a.m.
Ali Bashir, Icahn School of Medicine at Mount Sinai
Uncovering Neglected Regions of the Human Genome: Assembly and Architecture via Single Molecule Technologies
12:00 p.m.
Will Salerno, Baylor College of Medicine
Delivering One Genome
4:30 p.m.
Robert Sebra, Icahn School of Medicine at Mount Sinai
Leveraging Multiple NGS Technologies for Developing Niche Diagnostic Assays
Thursday, November 5
11:05 a.m.
Tyson Clark, Pacific Biosciences
Enrichment of Unamplified DNA and Long Read SMRT Sequencing to Unlock Repeat Expansion Disorders
11:30 a.m.
Maria Nattestad, Cold Spring Harbor Laboratory
Long-Read Sequencing and Analysis of a Breast Cancer Cell Line as a Pilot Study for Future Diagnostics
We hope you’ll stop by booth #11 to get the tour of our new Sequel System. You can also see the PacBio team lacing up our running shoes for a good cause on Wednesday morning at 10:00 a.m.: once again we’ll be participating in the Race the Helix event, a fundraiser for the Greenwood Genetic Center. (At the first Festival of Genomics, held in Boston in June, our Race the Helix team won the best-dressed award — quite the feat when you’re sprinting on a treadmill for all you’re worth!)
http://www.pacb.com/blog/welcome-to-silicon-valley-festival-of-genomics/
Interview: J.Craig Venter--28 October 2015
Founder, J. Craig Venter Institute, Synthetic Genomics Inc and Human Longevity Inc.
(JV" We need a combination of the cost and the throughput of the Illumina sequencers, with the quality and long sequence reads on single molecules that we get with PacBio.") http://www.frontlinegenomics.com/2328/j-craig-venter-from-sequencing-the-first-human-genome-to-sequencing-all-the-human-genomes/
("The new Sequel’s SMRT cells each contain one million zero-mode waveguides (ZMWs)—a significant increase over the cells of the PacBio RS II, which contained 150,000 ZMWs.We expect to make similar, substantial performance improvements each year for the Sequel system. says CEO Mike Hunkapiller.") -- Published on October 26, 2015-- Pacific Biosciences of California Inc, Menlo Park, Calif, has launched a new nucleic acid sequencing platform called Sequel. The new system got its public debut earlier this month in Baltimore, at the annual meeting of the American Society of Human Genetics. Compared to the company’s previous sequencer, the PacBio RS II system, the Sequel system provides higher throughput, more scalability, a reduced footprint, and lower sequencing project costs—all while maintaining the existing benefits of the company’s single molecule, real-time (SMRT) technology. The core advancement embodied in the Sequel system resides in the capacity of its redesigned SMRT cells. At launch, Sequel’s SMRT cells each contain one million zero-mode waveguides (ZMWs)—a significant increase over the cells of the PacBio RS II, which contained 150,000 ZMWs. Immobilized within the ZMWs are active individual polymerases, providing windows to observe and record DNA sequencing in real time. The Sequel system is able to perform about seven times as many reads per SMRT cell as the PacBio RS II was able to perform. According to the company, customers should be able to realize lower costs and shorter timelines for sequencing projects, with approximately half the upfront capital investment compared to the previous technology. The US list price for the Sequel system is $350,000. Pacific Biosciences’ products enable scientists to perform a number of operations essential for resolving genetically complex problems: De novo genome assembly, finishing genomes in order to more fully identify, annotate, and decipher genomic structures. Full-length transcript analysis, to improve annotations in reference genomes, characterize alternatively spliced isoforms in important gene families, and find novel genes. Targeted sequencing to more comprehensively characterize genetic variations. DNA base modification identification to help characterize epigenetic regulation and DNA damage. According to Pacific Biosciences, the company’s SMRT technology provides industry’s highest consensus accuracy over the longest read-lengths, in combination with the ability to detect real-time kinetic information. The Sequel system, including consumables and software, provides a simple, fast, end-to-end workflow for SMRT sequencing. Although the Sequel system occupies a smaller footprint and is less than one-third the size and weight of its predecessor, it offers access to the key attributes associated with SMRT sequencing, including long reads, high consensus accuracy, uniform coverage, and integrated methylation information. Since the new system is built on the company’s established SMRT technology, most aspects of the sequencing workflow are unchanged. Michael Hunkapiller, PhD, Pacific Biosciences. “The system’s lower price and smaller footprint represent our continued commitment to leveraging the scalability of our technology and the unique characteristics of SMRT sequencing,” says Michael Hunkapiller, PhD, CEO of Pacific Biosciences. “Moreover, with its lower cost of goods (approximately a quarter of that of the PacBio RS II) we expect to be able to achieve substantial gross margin improvement and move more quickly toward profitability.” “We will continue to support our PacBio RS II customers, and we expect to introduce improvements in sample prep, sequencing chemistry, and software that will extend the performance of that system,” says Hunkapiller. “We expect to make similar, substantial performance improvements each year for the Sequel system. In addition, the Sequel architecture provides the ability to scale throughput by substantially varying the number of ZMWs on future SMRT cells, thereby optimizing throughput and operating costs for specific applications.” The Sequel system is designed for projects such as rapidly and cost-effectively generating high-quality, whole-genome de novo assemblies. It can characterize a wide variety of genomic variation types, including those in complex regions not accessible with short-read or synthetic long-range sequencing technologies, while simultaneously revealing epigenetic information. Using the company’s Iso-Seq protocol, the system can also be used to generate data for full-length transcriptomes and targeted transcripts. “We are excited to support the human genetics community as they pursue the generation of higher quality whole human genomes, and move beyond SNPs to sequence the full size-spectrum of human genetic variation,” says Jonas Korlach, chief scientific officer at Pacific Biosciences. “With the introduction of our Sequel platform, SMRT sequencing will be available to more scientists seeking to find the underlying heritability of genetic diseases.” According to the company, the Sequel system’s increased throughput should also facilitate applications of SMRT technology in metagenomics and targeted gene applications, for which interrogation of larger numbers of individual DNA molecules is important. The Sequel system was developed as part of the company’s collaboration with F. Hoffman-La Roche Ltd, Basel, Switzerland, which is ultimately aimed at providing a nucleic acid sequencing system for use in human in vitro diagnostics. Under that agreement, Roche agreed to pay Pacific Biosciences $40 million in milestone payments related to the development of the Sequel system. The company previously reported that it has earned $20 million to date, and now expects to earn the remaining $20 million during the fourth quarter of 2015. Dan Zabrowski, Roche. “This new sequencing platform has significant advantages over existing commercial platforms, and will be used as the basis for the Roche sequencing instrument being developed initially for clinical research, followed later by an IVD instrument launch,” says Dan Zabrowski, head of Roche sequencing and tissue diagnostics. “We anticipate the initial launch in the second half of 2016.” Pacific Biosciences expects to begin limited US shipments of the Sequel system during the fourth quarter of 2015, and will begin scaling the manufacturing process for Sequel systems and new SMRT cells during early 2016. Shipments outside the United States are expected to commence thereafter. A portion of the initial group of Sequel instruments will be delivered to Roche to expand its internal assay development program. - See more at: http://www.clpmag.com/2015/10/pacific-biosciences-launches-new-sequencing-platform-based-on-its-smrt-technology/#sthash.pYVTpUEg.FYZIm2pb.dpuf
Guest Blog: Rich Roberts Urges Scientists to ‘Think Methylation’ in Microbial Sequencing
Wednesday, October 21, 2015
Richard-Roberts-croppedRichard Roberts, Nobel Laureate and Chief Scientific Officer of New England Biolabs, offers his thoughts on the utility of methylation data for understanding prokaryotes. In his words:
“Please run SMRT Analysis to detect methylation in your prokaryotic PacBio data.
Most bacteria and archaea encode DNA methylases, many of which are known components of restriction-modification systems. Usually, these are quite specific in terms of the sequences they recognize; the restriction component becomes a key defense mechanism preventing phages, plasmids, and other DNA elements from infecting the cell.
Until recently, it was quite difficult to determine the recognition sequences of these methylases. For most organisms, we had no idea whether the genes we could detect in the genome were active or not. Now, thanks to the properties of the DNA polymerase used during SMRT Sequencing, we can accurately locate the positions of m6A and m4C along the genome and sometimes can deduce the position of m5C. By analyzing the sequence context of these modified bases, we can deduce motifs that are the recognition sequences for the various methylases encoded in the genomes. Increasingly, we can then accurately match the genes with the motifs they produce to enable precise, experimentally-determined annotation for those genes.
Further progress in this area will depend on our ability to gather as much experimental data as we can; to improve the algorithms for calling the motifs accurately from the raw PacBio reads; and to improve our ability to match the DNA methylase genes in a genome with the PacBio motifs that are found experimentally. The public availability of motif data produced by running SMRT Analysis after each PacBio run can be enormously beneficial. Even better, if the raw sequence reads are also available, then this can help the development of better algorithms for data interpretation.
There is another terrific use of the methylation data for anyone interested in trying to transform these strains: While the presence of methylated motifs — and hence methylase genes — does not mean that an active restriction system is present, very often it does, offering some information about how one might protect DNA to be used for transformation before it is introduced into the cell.
I encourage everyone to think ‘methylation’ when using PacBio systems to sequence bacterial and archaeal genomes. The current results of such methylation analysis can be found in REBASE by clicking on the blue PacBio icon. This also has a link through which you can submit your methylation motifs to REBASE.”
http://www.pacb.com/blog/guest-blog-rich-roberts-urges-scientists-to-think-methylation-in-microbial-sequencing/
Scientists Reveal Recent Autosome-to-Y Duplication Event in Drosophila
Tuesday, October 20, 2015
Drosophila_melanogaster1Following on the heels of characterizing 18 Mst77Y genes that were tandemly duplicated within a 96 kb region (Krsticevic FJ, et al., 2015), scientists from institutes in Brazil, Austria, and the United States recently published a study in which they also used the Drosophila melanogaster data release from PacBio to characterize a region of the Y chromosome that had never before been accessible.
In a paper published in PNAS, entitled “Birth of a new gene on the Y chromosome of Drosophila melanogaster,” lead author Antonio Bernardo Carvalho, senior author Andrew Clark, and collaborators detail their find of a gene duplicated from an autosome. “We emphasize the utility of PacBio technology in dealing with difficult genomic regions,” the authors write. “PacBio produced a seemingly error-free assembly of the FDY region, something that has eluded us for years of hard work.”
The 55 kb region, which consists of several pseudogenes as well as the newly discovered functional FDY gene, has been challenging to sequence and assemble since it exists only on the Y chromosome and is full of highly repetitive sequence. Some 75% of its length, the scientists report, is made up of transposable elements.
Their discovery was worth the wait. Unlike mammalian Y chromosomes, which are thought to evolve primarily by gene loss, the Drosophila Y chromosome appears to be the result of millions of years of gene gains. The team demonstrates that the new gene they detected, named FDY for flagrante delicto Y, was formed about 2 million years ago in a single duplication event of the gene vig2 and its flanking sequence from chromosome 3R. That flanking sequence originally included four other genes, “but they became pseudogenes through the accumulation of deletions and transposable element insertions, whereas FDY remained functional, acquired testis-specific expression, and now accounts for ~20% of the vig2-like mRNA in testis,” the scientists report. Today, FDY shares 98% sequence identity with its vig2 parent.
The paper details the team’s effort to sequence the FDY region, using RT-PCR, clonal sequencing, and publicly available genome assemblies. Most existing assemblies did not fully cover the region. “Fortunately … the PacBio [MHAP] assemblies covered not only FDY, but also substantial flanking regions,” the scientists write. With that resource, they had their first view of the full sequence of the region. By comparing it to Sanger and Illumina sequence data, they concluded that the PacBio assembly is complete and accurate.
Carvalho et al. went on to figure out when FDY likely appeared in the genome. Their sequence divergence analysis suggests that the duplication occurred once, about 2 million years ago. The gene was found in samples of D. melanogaster from around the world, but does not appear in the fly’s closest relatives.
“Hence a female-biased gene (vig2) gave rise to a testis-biased gene (FDY),” the authors write. “This seems to be a case of gene duplication followed by neofunctionalization, the first reported, to our knowledge, for the Drosophila Y.”
http://www.pacb.com/blog/scientists-reveal-recent-autosome-to-y-duplication-event-in-drosophila/
Stepwise evolution of pandrug-resistance in Klebsiella pneumonia-- Published online:19 October 2015---("Single Molecule Real Time (SMRT) sequencing(PACBIO) we determined the complete genome of a pandrug-resistant Klebsiella pneumoniae isolate, representing the first complete genome sequence of CRE resistant to all commercially available antibiotics.") --
Abstract
Carbapenem resistant Enterobacteriaceae (CRE) pose an urgent risk to global human health. CRE that are non-susceptible to all commercially available antibiotics threaten to return us to the pre-antibiotic era. Using Single Molecule Real Time (SMRT) sequencing we determined the complete genome of a pandrug-resistant Klebsiella pneumoniae isolate, representing the first complete genome sequence of CRE resistant to all commercially available antibiotics. The precise location of acquired antibiotic resistance elements, including mobile elements carrying genes for the OXA-181 carbapenemase, were defined. Intriguingly, we identified three chromosomal copies of an ISEcp1-blaOXA-181 mobile element, one of which has disrupted the mgrB regulatory gene, accounting for resistance to colistin. Our findings provide the first description of pandrug-resistant CRE at the genomic level, and reveal the critical role of mobile resistance elements in accelerating the emergence of resistance to other last resort antibiotics.
Introduction
The “golden era” when modern medicine saved lives through antibiotic treatment is under serious threat1. In 2013, the Centers for Disease Control and Prevention (CDC) released a landmark report on “Antibiotic Resistance Threats2”. Three microorganisms were tagged as posing a threat level of urgent – Clostridium difficile, carbapenem-resistant Enterobacteriaceae (CRE) and drug-resistant Neisseria gonorrhoeae2. CRE, which include organisms such as Klebsiella pneumoniae and Escherichia coli, are resistant to almost all currently available antibiotics. Almost 50% of patients who develop bloodstream infections with these organisms die from the infection2. In healthcare settings, carbapenem resistant Enterobacteriaceae have increased sharply over the past decade3. Carbapenem resistance is typically mediated by the production of beta-lactamases4, and patients with CRE infections are treated with last-resort antibiotics such as colistin5.
The CDC and the European Centre for Disease Prevention and Control (ECDC) have jointly developed definitions for multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria6. Pandrug-resistance implies non-susceptibility to all commercially available antibiotics relevant to the treatment of a particular bacterial infection. Although there has been an anecdotal report of probable pandrug-resistance in K. pneumoniae7, no such isolates have been comprehensively analyzed.
In this manuscript, we describe the genetic basis of pandrug-resistance in a K. pneumoniae isolate using single molecule real-time (SMRT) sequencing. We show that a genetic element conferring resistance to carbapenem antibiotics has been acquired and mobilized, leading to insertional inactivation of a gene that results in resistance to colistin. Overall, our analysis provides a comprehensive description of a pandrug-resistant K. pneumoniae isolate at the whole genome level.
Results
Case Record
An 87 year old man, hospitalized in the United Arab Emirates in April 2014, was found to be colonized with multidrug-resistant Klebsiella pneumoniae. The isolate grew from urine and a pre-sacral pressure area but blood cultures were sterile. Susceptibility testing by way of a commercial semi-automated method (Vitek, bioMérieux) showed resistance to all antibiotics tested. The urinary isolate (strain MS6671) was therefore sent to a reference laboratory for further testing. Other K. pneumoniae isolates with this antibiotic resistance phenotype were not detected at the index patient’s hospital.
Pandrug-resistant phenotype of K. pneumoniae MS6671
MS6671 was found to be non-susceptible to all antibiotics tested, which includes cephalosporins, penicillins, carbapenems, aztreonam, aminoglycosides, ciprofloxacin, colistin, tetracyclines, tigecycline, chloramphenicol, trimethoprim-sulfamethoxazole and fosfomycin (Table 1). Thus, the isolate can truly be described as pandrug-resistant6.
Table 1: Antibiotic resistance in K. pneumoniae MS6671.
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K. pneumoniae MS6671 general genome features
The complete genome of K. pneumoniae MS6671 consists of a circular chromosome 5,402,900 base-pairs in length with an average G-C content of 57%, five circular plasmids and a linear plasmid prophage (Supplementary Table S1). The sequence type of the isolate was ST147. The chromosome of MS6671 is highly similar to K. pneumoniae NTUH-k20448; a hypervirulent strain associated with liver abscess and meningitis, with most variation attributable to differences in their mobile genetic element (MGE) content (Supplementary Figure 1). Further details of the complete genome are provided in the Supplementary Results.
Genetic determinants of pandrug-resistance
In order to determine the genetic basis of pandrug-resistance, we interrogated the genome to identify acquired and intrinsic resistance genes. The majority of acquired antibiotic resistance genes were located on the chromosome, with most beta-lactamase and aminoglycoside resistance genes carried within two copies of a class 1 integron or as part of mobile elements that incorporate the ISEcp1 insertion sequence (Table 1, Supplementary Table S2 and Supplementary Results). Mutations in gyrA and parC that have previously been linked with fluoroquinolone resistance (GyrA Ser83Ile and ParC Ser80Ile) were identified9,10. Fosfomycin resistance was mediated by a chromosomally encoded copy of fosA11. Mutations in chromosomal genes encoding major outer membrane porins (OmpK35 and OmpK36) were also identified. A novel variant of the ompK36 gene was encoded on the chromosome. The amino acid sequence change is located in loop 3 (L3) of the porin, which constitutes the porin channel eyelet12. L3 mutations have previously been associated with increased resistance to carbapenems13,14,15. Additionally, ompK35 has been disrupted by IS insertion. Inactivation of ompK35 has been associated with increased resistance to a number of different classes of antibiotics, including quinolones and cephalosporins16,17. Genes encoding three beta-lactamases, including an extended-spectrum beta-lactamase (ESBL) and a carbapenemase, were detected at different genomic locations – blaSHV-36, blaCTX-M-15, blaOXA-181.
Insertional inactivation of mgrB by a carbapenem-resistance element and colistin resistance
OXA-181 is an oxacillinase capable of hydrolysing carbapenems18. Three copies of an ISEcp1-blaOXA-181 transposon were identified throughout the chromosome (Fig. 1). One of these insertions has resulted in the inactivation of the mgrB gene, a negative regulator of phoPQ. Insertions in mgrB have previously been shown to cause colistin resistance in K. pneumoniae clinical isolates19,20,21. Examination of the DNA flanking the ISEcp1-blaOXA-181 transposons shows that the primary insertion site is within MS6671_10430, followed by intra-chromosomal transposition of ISEcp1-blaOXA-181 and a 37?bp fragment of MS6671_10430 to two other locations in the genome (Fig. 2). The three transposons are bracketed by imperfect 14?bp inverted repeats and flanking 5 bp direct repeats (TATCT, TGAAA and TATAA), providing direct evidence for their transposition activity (Supplementary Table S3).
Figure 1: Diagram of the pandrug-resistant K. pneumoniae MS6671 chromosome highlighting the position and context of mobile genetic elements that harbor antimicrobial resistance genes.
Figure 1
The chromosome of MS6671 is represented to scale by the black bar with ISEcp1 and integron insertion points indicated with red rectangles. Pop-outs display schematic representations of the four ISEcp1 elements which harbor beta-lactamase genes (three copies of blaOXA-181 and one copy of blaCTX-M-15) and a class 1 integron located on the chromosome of MS6671. Insertion elements are highlighted in yellow. The coordinates of each element are indicated above and below the genome bar.
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Figure 2: Comparison of ISEcp1-blaOXA-181 transposons from MS6671.
Figure 2
Pairwise nucleotide comparison of ISEcp1-blaOXA-181 (OXA-181) transposons and flanking genomic regions from K. pneumonaie MS6671. ISEcp1 elements are represented by blue rectangles. Protein-coding genes are represented by coloured arrows: blaOXA-181 (green); MS6671_10430 encoding a hypothetical protein (red); other (brown). Left and right flanking inverted repeats (IRL, IRR, IRRalt1, IRRalt2) are represented by yellow bars and 5 bp direct repeat sequences created by duplication of the target sequence during transposition are given (TGAAA, TATCT or TATAA). In the primary insertion site, a single 2,855?bp transposon carrying blaOXA-181 has inserted at TGAAA (position 1152428..1155282) within MS6671_10430. This transposon, similar to Tn2013 previously described in K. pneumoniae Kp331, is flanked by 14-bp inverted repeat sequences, namely IRL and IRRalt1. IRRalt2 lies 23?bp downstream of IRRalt1 within the MS6671_10430 sequence. Mobilisation of the ISEcp1-blaOXA-181 transposon using IRRalt2 instead of IRRalt1 has resulted in a 37?bp fragment of MS6671_10430 (indicated by a small red rectangle) being packaged at the 3’ end of the other two ISEcp1-blaOXA-181 transposons inserted at TATCT and TATAA (position 126108..128999 and position 3345804..3348695, respectively). Grey shading indicates regions of homology (100% nucleotide sequence identity) between sequences.
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In a similar fashion, a single copy of ISEcp1-blaCTX-M-15 has inserted into ompK35, leading to inactivation this gene (Fig. 1). The ISEcp1-mediated mobilisation and transposition of blaCTX-M-15, blaOXA-181 and other clinical relevant beta-lactamase resistance genes, including blaCMY and blaACC, has been reported previously22,23,24,25,26.
K. pneumoniae MS6671 contains two copies of a class 1 integron
A class 1 integron was identified on the chromosome encoding multiple antibiotic resistance genes (arr-3, aac(6')-Ib-cr, rmtF, catB1) (Fig. 1). These genes result in resistance to rifampin, all aminoglycosides and chloramphenicol. A near-identical copy of this integron was also found on one of the six plasmids (Supplementary Figure S2).
Discussion
This is the first genomic analysis of a pandrug-resistant CRE isolate, as defined by the rigorous CDC/ECDC assessment criteria6. With the advantage of long-reads provided by SMRT sequencing we were able to identify the genomic context of multiple resistance elements. In contrast to short-read technologies, SMRT sequencing allows complex resistance elements to be properly characterized27. This technology platform was used to investigate the German E. coli O104:H11 outbreak28 and more recently to identify plasmid-borne resistance in a large-scale study of CRE following an outbreak at the National Institute for Health Clinical Center29,30. Critically, elucidation of the complete K. pneumoniae MS6671 genome using long-read sequencing enabled the context of multiple, identical carbapenem resistance elements to be determined. Based on this analysis we propose a model for the development of pandrug-resistance in this K. pneumoniae isolate, whereby mobile resistance determinants are responsible for driving additional resistance. In this example, ISEcp1 carrying the blaOXA-181 carbapenem resistance gene has inserted three times in the chromosome, with one event causing colistin resistance by insertional inactivation of mgrB.
ISEcp1-like insertion sequences are the most common genetic element associated with blaCTX-M, blaCMY and blaACC genes and have more recently been associated with blaOXA-18122,23,24,25,26,31. By recognizing a variety of DNA sequences as right inverted repeats (IRR), ISEcp1s are capable of mobilising adjacent genes and inserting at new location32,33. Similar to previous reports on the hydrolytic activities of OXA-18118,34,35, elevated MICs for ertapenem, imipenem, meropenem and doripenem were observed for MS6671, indicating hydrolytic activity of OXA-181 towards these carbapenems and a possible blaOXA-181 copy number effect (Table 1). Notably, doripenem resistance was higher than previously reported18. The ompK36 variant encoded by MS6671 has previously been associated with increased resistance to doripenem and doripenem-colistin13, and may contribute to the elevated MIC for doripenem observed in MS6671. Porin deficient E. coli expressing OXA-48-like beta-lactamases have also been shown to have elevated MICs towards carbapenems18.
Inactivation of mgrB has recently been associated with resistance to colistin, and appears to be the most common mechanism for polymyxin resistance in K. pneumoniae19,20. Specifically, disruption of mgrB results in over expression of the phoPQ signaling system and of the pmrHFIJKLM operon which controls modification of LPS, the target of polymyxin antibiotics36. Insertional inactivation of mgrB with IS5-like or IS1 elements has been previously reported21,37, however, the present study is the first to show colistin resistance caused by insertion of a carbapenem resistance element itself. While we cannot rule out the possibility that this mechanism may have occurred in other colistin-resistant K. pneumoniae carrying ISEcp1-blaOXA-18138, the generation of a complete genome sequence of MS6671 provides unequivocal evidence for this novel insertion event.
We also found a fourth ISEcp1 element encoding an ESBL (CTX-M-15), which was inserted within the outer membrane porin gene ompK35. Disruption of ompK35 reduces the permeability of the outer membrane and mutants lacking this porin have increased resistance to quinolones, tetracyclines, beta-lactams and chloramphenicol39. Pan-aminoglycoside resistance was mediated by the rRNA methyltransferase RmtF, which was encoded on both chromosomal and plasmid copies of a class 1 integron. We did not have access to investigational antibiotics, such as plazomicin, eravacycline, ceftazidime/avibactam or ceftolozane/tazobactam, to assess their potency against MS6671.
MS6671 was defined by multi-locus sequence typing (MLST) as being ST147. K. pneumoniae ST147 was first described in Hungary in 200840. Subsequently, it has been frequently associated with carbapenem resistance, with ST147 producing KPC well described in Greece and Italy41,42. For example, epidemics of VIM-producing carbapenem resistant K. pneumoniae ST14743, and KPC-2-producing carbapenem resistant K. pneumoniae44,45 have been reported in Greece. Notably, ST147 isolates carrying both blaVIM and blaKPC-2 genes were identified41,46. Carbapenem resistant ST147 carrying the blaNDM-1 gene have been isolated in Iraq, Switzerland, Canada and the United Kingdom47,48,49. In MS6671, carbapenem resistance was most likely mediated by the beta-lactamase OXA-181, possibly in combination with permeability defects as has been reported previously in other strains18,31. ST147 K. pneumoniae producing OXA-181 have been previously reported from the Indian sub-continent18,31,50. Clearly, in addition to the KPC-producing, carbapenem-resistant K. pneumoniae ST258 clone51, ST147 also represents a clone of K. pneumoniae with a potential for global significance.
Fortunately, in six months there have been no further isolates with this resistance phenotype at the index patient’s hospital. However, the occurrence of this strain in the Arabian Gulf is of great significance. OXA-48-like-producing K. pneumoniae are frequent in this region52. It is unknown if this strain originated in the index patient, in another patient at the same hospital or was imported from another hospital, perhaps in another country. There are a large number of expatriates in the Gulf region, and travel to the Indian sub-continent, Europe and the United States is frequent53. The potential for international transfer of multidrug-resistant bacteria54,55 emphasizes the need for global surveillance efforts as one part of a strategy to control antibiotic resistance3.
In summary, we have provided the first report of a pandrug-resistant isolate of CRE using high-resolution genome data. The CDC has denoted CRE as an urgent threat. The emergence of this highly resistant strain, in a clone that has proven capable of causing outbreaks, raises this threat level even higher.
Methods
Antibiotic Resistance Phenotypic Testing
The K. pneumoniae isolate (hereafter referred to as MS6671) was sent to a reference laboratory (University of Queensland, Centre for Clinical Research) where confirmatory susceptibility testing was performed in order to determine the minimal inhibitory concentrations of all antibiotics used by the Centers for Disease Control and Prevention (CDC) and European Centre for Disease Prevention and Control (ECDC) in defining a pandrug-resistant isolate6. Susceptibility of most tested antibiotics was determined using Etests and following the breakpoints of the European Committee for Antibiotic Susceptibility Testing (EUCAST)56, except for cefazolin, cefoxitin, cefotetan, tetracycline, doxycycline, and minocycline which were determined using the breakpoints of the Clinical and Laboratory Standards Institute (CLSI)57. The minimum inhibitory concentration of colistin (sulfate, Sigma-Aldrich) was determined by broth microdilution in cation-adjusted Mueller-Hinton broth (Oxoid).
Genome sequencing
Pacific Biosciences (PacBio) RS II Single-Molecule Real Time (SMRT) sequencing of K. pneumoniae MS6671 was performed using ~4?µg of the genomic DNA sheared using g-TUBETM (Covaris®) into fragments size targeted at 10?kb. Purification of the sheared DNA was then carried out using 0.45-fold volume of washed Agencourt AMPure XP magnetic beads (Beckman Coulter Inc.). SMRTbell template libraries were subsequently prepared using the commercial Template Preparation Kit from Pacific Biosciences Inc. that involved steps of DNA end repair, adapters ligation followed by exonuclease digestion of incompletely ligated products. Next, 0.83?nM of the libraries were then annealed with sequencing primers followed by binding to 50?nM of P4 DNA polymerase, as provided in the Template Binding Kit from Pacific Biosciences Inc. For enhanced loading efficiency, 15 pM of the bound complexes were immobilized into Magbeads (Pacific Biosciences Inc.) prior to loading into the sequencing zero-mode waveguides (ZMWs). Duration for the sequence collection was set at 180?minutes with stage start option. Reads with length that were less than 50?bp were filtered off upon acquisition of the sequencing data and minimum polymerase read quality was set at 0.75.
Genome assembly
De novo genome assembly of PacBio SMRT reads from the K. pneumoniae MS6671 genome was performed using the hierarchical genome assembly process (HGAP)58 from the PacBio SMRT analysis software suit (version 2.2.0), with default parameters and a seed read length cut-off of 5?kb. Following assembly, all contigs were screened for duplicate sequences at their 3' and 5' ends. Overlapping sequences were manually trimmed and joined based on sequence similarity. Individual contigs with duplicate sequences on their 5' and 3' ends were manually trimmed and circularised. Following circularisation the chromosome and plasmid sequences were polished using quiver58 whereby the raw reads were mapped back to the chromosome and plasmid sequences to validate the assembly and resolve any remaining sequence errors. Non-circularised chromosomal and plasmid contigs were closed using primers designed on their 5' and 3' ends. The amplified PCR products were sequenced by the Australian Genome Research Facility and their sequences were manually integrated into the assembly.
Genome annotation
Gene calling and automatic functional annotation of the complete MS6671 chromosome and plasmids was performed using Prokka (Prokka: Prokaryotic Genome Annotation System - http://vicbioinformatics.com/) identifying 5,054 putative coding regions on the chromosome with an additional 644 putative coding regions distributed amongst the 5 plasmids and linear plasmid prophage. The complete annotated genome sequence has been deposited at the European Nucleotide Archive (Bioproject: PRJEB7538, Accessions: LN824133-LN824139).
Identification of antibiotic resistance genes
Initial identification of antimicrobial resistance genes from the complete PacBio assembly was performed using ResFinder (version 2.0)59. Additional screening for antimicrobial resistance genes was performed by comparison (BLASTp; sequence identity?>?=40%; E-value?<?=0.0001) of all predicted coding regions against the Antibiotic Resistance Genes Database (ARDB)60 and the Comprehensive Antimicrobial Resistance Database (CARD)61. Antimicrobial resistance genes were then subject to manual inspection to improve their functional annotation, correct start sites and identify point mutations, which may contribute to a resistant phenotype. Finally, resistance gene loci were screened for known insertion sequences and integrons by comparison against the ISFinder database62 and Integrall63, respectively.
http://www.nature.com/articles/srep15082