Articles by james

We have developed an new flow for processing Thermo RAW files that works both with the most recent XCalibur V2.1, as well as with earlier versions. This flow has been giving good results in internal testing, and we are now releasing it for beta testing to any interested, actively supported Sorcerer customer.

Thermo LTQ Velos users will have noticed the major changes to the XCalibur software that were introduced at version 2.1. The installation process is different, and requires a new component called Thermo Foundation, and some of the file names and locations have changed. All of these changes are no longer compatible with the ReAdW program that is used within the CrossOver environment by Sorcerer. One workaround which has been commonly suggested in the Thermo field is to down-rev the XCalibur used on the instrument to V2.0 and to continue using the old software for analysis. This remains a viable option, but with our newly developed solution, it is now also possible to use 2.1 RAW files on Sorcerer.

We are moving to a new spectrum extractor called msconvert (part of the ProteoWizard suite)  which works with a different version of the Thermo libraries, and for which we have developed a new integration in the CrossOver environment. We are offering this as a beta release to our in-warranty customers. This solution  entails a few Linux operations to reinstall CrossOver with the latest release, to configure the required libraries and to install a new Sorcerer workflow script; it is fairly straightforward for people comfortable with the Linux environment, or alternatively, we can do it for you if you give us remote access to your system. Please contact us at support@sagenresearch.com for more information.

Our R&D team is busy working on the next major version of the Sorcerer-PE software, and expects to release it to then-in-warranty customers in the next few weeks.  Early previews and beta tests of some of the components will be made available by arrangement to qualified customer sites.

Highlights of the upcoming release include:

• ETD fragmentation support and analysis
• MUSE scripting modules for rescoring peptide matches with Olsen-Mann and Sadygov-Coon scores
• Interoperation with major components of the Yates lab Sequest suite, including the DTASelect filtering and statistical analysis tool, and the Census quantitation application
• Enhancements to the SEQUEST engine which provide first-pass cross-correlation scoring and E-values for greater accuracy and sensitivity

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We’ve developed a new Muse workflow for target-decoy analysis and false discovery rate estimation, based on our integration of DTASelect from the Yates lab. DTASelect can now use target-decoy FASTA files that are installed on Sorcerer to support its statistical analysis. It provides an easy-to-interpret results report complete with match statistics and estimated false discovery rates.

Our DTASelect on Sorcerer page on this blog has been updated to describe the target-decoy workflow, in addition to the existing material on installing, configuring and running DTASelect and the Muse script. Please visit it to get links to the latest scripts and for a detailed How-To.

Three of the world’s leading experts on MS-MS protein identification came together recently at Sage-N Research’s annual user group meeting, and presented methods and results for the techniques and tools with which they are associated:

• Jimmy Eng, co-inventor of Sequest and developer of many proteomics tools, presented tips for Sequest analysis
• Josh Elias, who pioneered the systematic use of decoy databases for FDR estimation, gave a talk on how to use that technique to address Peptide ID signal-to-noise.
• Alexey Nesvizhskii spoke about the tools he co-authored, in “Peptide identification and protein inference using PeptideProphet and ProteinProphet”

Their talks were very wide-ranging and full of practical insights for the proteomics user community, and they explored the different research interests, data sets, analysis methods and workflows in the individual labs.  However, they all had this in common: they had kept a careful eye on their search settings, monitored sensitivity and error rates, and come to a common, if perhaps not entirely intuitive, conclusion: the most sensitive search and the lowest error rates for shotgun proteomics are achieved when using semi-enzymatic searches — that is, when one end, but not both, of the peptide is allowed to diverge from the expected cleavage site.

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Many of our customers have found DTASelect to be a very useful postprocessing tool for Sequest results, and have reported success using it with Sorcerer output. Up until now, however, these customers have generally run the tool manually on a separate desktop computer. Now we have developed a Muse script to make it easy to do this automatically, on Sorcerer itself.

See our DTASelect on Sorcerer page on this blog for a detailed How-to on installing, configuring and running DTASelect and the Muse script.

If you are interested in using Ascore as described in the application note on the blog, please contact us for new Muse scripts for your Sorcerer. We’ve just updated them, and they are needed to work with the recent v4.0 release of TPP, which is what’s in the current Sorcerer release.

Here’s a how-to for technically advanced users who need to update the Java platform on Sorcerer. It’s not required for the base Sorcerer software, including ScaffoldBatch, but it may be necessary for Phenyx installation. Please consult our technical support staff before deciding to do the update.

These instructions assume that you have a recent 64-bit Sorcerer operating platform (either RHEL 5.2 or Centos 5-based), and that your Sorcerer software is at V3.5.

Here are the steps:

1. Get the latest Java Development Kit (JDK)  (currently v6 update 18) from http://java.sun.com/javase/downloads/index.jsp. Click on the ‘Download JDK’ button. Get the Linux x64 platform, and download the non-rpm file which has a name like jdk-6u18-linux-x64.bin
2. Log in as root in a terminal window and type: cd /opt
3. Copy the file you downloaded to /opt, and unpack it:  /bin/sh jdk-6u18-linux-x64.bin
4. Note the name of the pathname to java in the unpacked directory for use in the next step, e.g. /opt/jdk1.6.0_18/bin/java
5. Type:  /usr/sbin/alternatives --install /usr/bin/java java /opt/jdk1.6.0_18/bin/java 2
   • This sets up a system of links from /usr/bin/java to the new installation
6. Type: /usr/sbin/alternatives --config java
   • Enter ‘2′ at the prompt to select the newly installed alternative
7. Check you have the latest java by typing:  java -version

(Optional) Update Firefox Java plugin:

1. Create a plugins directory in the Firefox installation directory if the plugins directory does not exist. Please check your version of Firefox to determine the correct path to use: mkdir /usr/lib64/firefox-3.x.x/plugins
2. Create a symbolic link to the new Java plugin. Again please check your Firefox and JRE version for the correct paths: ln -s /opt/jdk1.6.0_18/jre/lib/amd64/libnpjp2.so /usr/lib64/firefox-3.0.5/plugins/

Sage-N Research is hosting its annual users’ meeting on the afternoon of Sunday, 31st May, immediately before the ASMS meeting in Philadelphia. We are proud to announce a compelling  agenda with talks from the principal developers of several of the key proteomics data analysis methods that are used as standard in the community, including SEQUEST, target-decoy search strategies, and Peptide/Protein Prophet.  The insights our clients will take away from this meeting will be very relevant to their use of Sorcerer, and promise to enhance their proteomics analysis productivity greatly.

2009 Users’ Meeting Arrangements

The meeting is open to in-warranty Sorcerer customers and by invitation only. Pre-registration is required. A light buffet and refreshments are being provided, and there will be a drawing for customer door prizes. Attending this meeting is your chance to win one of three Acer Aspire One netbook computers that we are giving away to our customers (must pre-register and be present to win)!

Date: Sunday 31st May 2009
Time: 1:30 PM to 5:00 PM
Address: Courtyard Marriott Hotel, 21 N. Juniper St, Philadelphia.
Room: Ballroom Level 1

Agenda

1:30 PM   Welcome and introductory remarks

1:45 PM    “What’s new in Sorcerer”
James Candlin, Sage-N Research, Inc.

2:05 PM    “Sequest analysis tips”
Jimmy Eng, University of Washington

2:45 PM    “Using target-decoy searching to visualize peptide identification signal-to-noise”
Dr. Joshua Elias, Stanford University School of Medicine

3:15 PM    Break and refreshments

3:50 PM   “Peptide identification and protein inference using PeptideProphet and ProteinProphet”
Dr. Alexey Nesvizhskii, University of Michigan

4:30 PM   Panel Discussion: “Putting it together: strategies for a productive proteomics analysis workflow”

4:50 PM   Concluding remarks

5:00 PM   End of meeting