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from GenomeWeb

NEW YORK (GenomeWeb News) – Pacific Biosciences reported after the close of the market Tuesday that its fourth-quarter revenues fell 52 percent year over year but were up sequentially as product revenue jumped from the third quarter.

The Menlo Park, Calif.-based single molecule sequencing technologies firm reported total revenues of $5.9 million for the three months ended Dec. 31, compared to $12.4 million for the fourth quarter of 2011. The firm beat analysts’ consensus estimate for revenues of $4.4 million, and though its sales were down sharply year over year, its revenues more than doubled from $2.8 million in Q3 2012. Read more

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MENLO PARK, Calif., Jan. 29, 2013 (GLOBE NEWSWIRE) — Pacific Biosciences of California, Inc. (Nasdaq:PACB) provider of the PacBio^® RS High Resolution Genetic Analyzer, today announced it is releasing a new software upgrade that provides higher quality genome assemblies with near perfect base level accuracy in addition to other key features. The software upgrade extends the range of projects that uniquely benefit from the company’s Single Molecule, Real-Time (SMRT^®) DNA Sequencing method, and will be available to customers for download on January 31^st. Read more

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Average Read Lengths of 5,000 Bases and Reads as Long as 20,000; PacBio Hosting Workshop at ASHG Annual Meeting

MENLO PARK, Calif., Nov. 6, 2012 (GLOBE NEWSWIRE) — Pacific Biosciences of California, Inc. (Nasdaq:PACB) provider of the PacBio^® RS High Resolution Genetic Analyzer, today announced the latest enhancements to its DNA sequencing system, the XL release featuring new chemistry and software for extraordinarily long read lengths that average 5,000 bases.

Pacific Biosciences’ Single Molecule, Real-Time (SMRT^®) sequencing generates reads an order of magnitude longer than other leading DNA sequencing technologies. With the latest advance, the average read length increases 67 percent from 3,000 to 5,000 bases, with some reads as long as 20,000 bases. Long reads are critical for resolving genetic complexity in applications such as the assembly and finishing of genomes, phasing genomic variation over long distances, understanding human nucleotide repeat disorders, and resolving the structure of alternatively spliced transcripts. Read more

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By Monica Heger from GenomeWeb News

Pacific Biosciences booked four new systems in the third quarter of 2012, an increase from just one in the previous quarter, but down from six in the year-ago quarter. The Q3 orders bring its backlog to five systems.

Additionally, the company is planning to launch new chemistry in the fourth quarter that it says will increase average read lengths to 5,000 bases with five percent of reads above 13,000 bases. PacBio’s current C2 chemistry yields average reads of 3,000 bases with five percent above 8,000 bases.

“Long reads are especially valuable for de novo assembly applications and targeted sequencing of large repeat regions and regions with complex structural variation,” CEO Mike Hunkapiller said during a conference call discussing the company’s 2012 third-quarter earnings. Read more

From GenomeWeb News…

One factor in the continued struggles for life science tools stocks is concern over funding from government sources both in the US and abroad.

Leading the decliners for the second month in a row was Pacific Biosciences, which followed its 36% decline in stock price in August with a 55% fall in September. The firm’s stock has tumbled from $11.01 at the end of July to $2.89 in afternoon trade today on the Nasdaq.

A primary catalyst for the drop in September was PacBio’s disclosure that it is laying off 130 employees as a result of “uncertainties associated with the economic environment” and to put the company in a position for success in the long term.

Other sequencing technology firms hit hard include Complete Genomics, seeing its stock fall 35% for the month, and Illumina whose stock fell more than 20%.

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On September 20, 2011, Pacific Biosciences of California, Inc. (the “Company”) implemented a reduction in its workforce of approximately 130 employees, or approximately 28% of its total workforce. The actions taken were in consideration of uncertainties associated with the economic environment and to position the Company for long-term success. The Company’s current infrastructure was staffed to support a faster adoption rate for its products. The reduction implemented will allow the Company to continue support of its growing customer base with improved service and continued product enhancements, while at the same time conserving cash.

The company reported the layoffs in a document filed with the US Securities and Exchange Commission.

(read the document…)

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from GenomeWeb

NEW YORK (GenomeWeb News) – Pacific Biosciences reported after the close of the market Tuesday an increase in its grant revenues for the fourth quarter and full-year 2010, and a $140.2 million loss for the year as it prepares for the commercial launch of its PacBio RS system.

The Menlo Park, Calif.-based firm reported fourth-quarter revenues of $280,000, up from $220,000 for the fourth quarter of 2009. All of its Q4 and FY 2010 revenues are from government grants.

PacBio anticipates commercial launch of its first product — the real-time, single-molecule PacBio RS sequencing system — in the second quarter of this year.

(read the entire story… )

from Genetic Engineering News- Third-Generation Sequencing Debuts
by Vicki Glaser

Helicos BioSciences

In Helicos’ tSMS technology, labeled nucleotides are mixed with nucleic-acid templates immobilized on a flow cell. Detection of the fluorescent signals emitted as a result of each base addition is performed in the HeliScope™ Genetic Analysis System. The Helicos system can sequence 105–180 megabases/hour with average read lengths of 33–36 bases from templates ranging in length from 25 to 5,000 bases.

It will enable a range of applications including chromatin profiling by direct sequencing of immunoprecipitated DNA, direct RNA sequencing, small RNA quantitation, digital gene expression, copy number variation assessment, and epigenetic analysis.

For direct RNA sequencing, the system can produce 300–400 million aligned reads/run with an average read length of 34 nucleotides (range 25–55) and a <5% per nucleotide error rate. Dr. Milos presented qualitative and quantitative data from RNA studies using tSMS to map the 3´ ends of RNA transcripts from yeast and human liver cells, producing a high-resolution map of 3´ polyadenylation sites. Another project under way is using direct RNA sequencing to study a pool of micro-RNAs and generate miRNA count distribution. Early results suggest that the technique yields greater quantitative accuracy than conventional cDNA-based methods.

Pacific BioSciences

In Pacific BioSciences SMRT™ sequencing technology, sequencing takes place on SMRT cells, each of which contains thousands of zero-mode waveguides (ZMWs). Each ZMW represents a hole tens of nanometers in diameter in a metal film that has been deposited on a silicon dioxide substrate.

The PacBio system generates both DNA sequence and epigenomic information directly from the real-time sequencing of genomic DNA. Single-molecule sensitivity enables faster results and longer read lengths.

Within about two years, the company plans to offer an application that will enable direct RNA sequencing in real time on the SMRT system without the need to convert RNA to cDNA. This application will provide insights into the epigenetics of RNA. An example was presented at the AGBT conference in which RNA sequencing using SMRT technology could distinguish pseudo-uridine from its native analog.

(read the full article…)

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Which sequencing platform/technology is best suited for RNA-Seq applications?

Platform                                  Votes

Roche/454 – GS FLX                        4
Illumina – Genome Analyzer            12
Illumina – HiSeq                             16
Applied Biosystems – SOLiD            6
Helicos – HeliScope                         2
Pacific Biosciences                          2

Total Votes                                   42

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From GenomeWeb

Illumina’s Q3 Revenues Jump 50 Percent on Strength of HiSeq
Illumina reported after the close of the market Tuesday that its third-quarter revenues jumped 50 percent, easily beating analysts’ consensus estimate.

Life Technologies’ Q3 Revenues Rise 8 Percent on Record SOLiD 4 Sales
The company’s CE and next-generation sequencing business saw strong results, while its PCR business suffered from difficult year-ago comparison figures, it said.

Roche’s Sequencing Business Grows 12 Percent YTD as Applied Science Sales Shrink in Q3
Roche said it has seen “robust uptake” in the European Union and Asia Pacific for the 454 GS Junior sequencer, a benchtop sequencer launched in May that is geared at small laboratories.

Helicos BioSciences Cuts 14 More Positions in Q3
The firm also cut 40 jobs in May amid its plans to restructure and focus on the molecular diagnostics market. Part of its restructuring includes the departure of J. William Efcavitch as chief technology officer.

The firm will cut another 14 positions, on top of the previously announced 40 jobs, as it aims to reduce its operating costs. Additionally, Lapidus steps down as Chairman.

Pacific Biosciences Files for IPO in Q3
The firm has raised around $370 million since its inception, and announced plans to go public in an offering that could potentially bring in $200 million.

The firm went on to sell 12.5 million shares at $16 per share, the mid-point of its estimated offering range, at the initial public offering in October.

Helicos BioSciences Corporation has now named all three major competitors in a lawsuit claiming willfull infringement on their patents which broadly cover sequencing-by-synthesis technology.  They claim that Pacific Biosciences, Life Technologies, and Illumina have all incorporated Helicos’ patented technology “at the heart of” their respective sequencing systems and products.

The patents cover sequencing-by-synthesis methods using labeled nucleotides. The nucleotides are labeled with detectable markers, such as fluorescent markers, that enable determination of each nucleotide incorporated into the DNA strand being extended by the polymerase. The patents describe processes that involve, for example, identifying each new nucleotide by observing its detectable label and neutralizing or removing the label before addition of the next nucleotide. The specific claims apply to both “real time” and “step and repeat” approaches.

“After a careful examination of the sequencing products and technologies offered by Illumina and Life Technologies, we are convinced that they, in addition to Pacific Biosciences, infringe the Helicos patents, which are in full force and effect up through 2028,” stated Dr. Ivan Trifunovich, President and CEO of Helicos.

Recently, Helicos had stated a strategic initiative to vigorously protect their next-generation sequencing IP.

(Read the press release… )

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• SEQanswers – RNA Sequencing

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• Biostar – RNA-Seq

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    I'm using edgeR in order to perform differential expression analysis from RNA-seq experiment. I have 6 samples of tumor cell, same tumor and same treatment: 3 patient with good prognosis and 3 patient with bad prognosis. I want to compare the gene expression among the two groups. I ran the edgeR pakage like follow: x […]
  • Normalising tag count to RPKM
    Hi! I was wondering if their is a way to normalise the number of reads in a region and the RPKM of the nearest gene to that region, so that a correlation could be computed. Like the following data shows number of tags in first column and RPKM in second column Tags RPKM 15 0.14619 11 0 203 0.2259 129 10.701 300 7.0772 122 2.3234 346 10.666 77 3.117 201 16.749 […]
  • a simple question on RNA-Seq terminology
    This question may be very simple and basic, but I just need to confirm that I understand the differences among those terminologies in the RNA-Seq context. Suppose I have a sample called SLR, and it is sequenced on 5 lanes, so I have (among other output files) BAM files like L1_SLR, L2_SLR, L3_SLR, L5_SLR and L7_SLR.bam. Here, the letter "L" denotes […]
  • FInding regions of interest with minimum coverage
    Hi, I have a bam file of all my accepted hits (tophat output) and an gtf file with my genes of interest for which I am trying to find potential antisense transcripts. I would like to create a list - preferably one that can be visualized in a genome browser - that shows all genes that have antisense reads in the accepted hits.bam file provided that there are […]
  • How to remove the intronic reads before counting
    I got RNASeq data in several samples. I checked the FastQC, seems the read quality are good (Hiseq 2000). But the problem is many reads are mapped to intronic region, and the regions have no any reference exons there (Refseq, ensembl, gencode). We don't know what they are. We guess the problem happend in library preparation, the concentration was low. N […]
  • Which strand of the mRNA molecule does the sequencer output as a "read"?
    In Illumina Stranded RNA-Seq (using the dUTP method), do the final reads in the fastq files correspond to the initial molecule (that was transcribed), or to the reverse complement of the molecule? C […]