MCIC September 2011 news
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No sequencing or genotyping on Friday September 23: We will be moving some of our genetic equipment to our new space on Friday, September 23, and we will not be able to process samples for sequencing and genotyping samples on that day, so please plan accordingly. We have been tight on space for quite some time, and inparticular, the genomics lab was crowded with equipment and often we have had problems with instruments overheating. Therefore we are looking forward to move some of our instruments to our new space in Selby room 014, which is adjacent to our current lab. This room was acquired thanks to the USDA/ARS lab moving to anew space. Besides housing some of our sequencing and genotyping equipment the new space will allow us to have bench space reserved for users who come and do their microscopy or genetics work at the MCIC.
Bioinformatics: CLC Bio and Partek software for Illumina sequence data analysis. By Saranga Wijeratne and Tea Meulia
Partek software for next generation sequence data analysis has been available at the MCIC for over a year, and now we are considering the purchase of the CLC Bio site license. Please contact us if you are interested.
Two commercial software packages for ‘next’ generation sequence data analysis are now available on the market. Most of the time users with little experience with Linux/Unix terminal, and no experience with the “R” statistical package find these software packages useful. MCIC users have had access to Partek® software for over year. The Partek company has pioneered the development for microarray data software analysis and has recently expanded their software for ‘next generation’ sequence data analysis. The Partek mirGenomics SuiteTM package includes software specifically designed for digital gene expression, exon/alternative splicing and RNA-Seq, copy number and association analysis, and for ChIP-chip, ChIP-seq and microRNA analysis.
Although Partek software is in its early stages of development, it provides a memory efficient analysis solution for large gnomic data. Partek works well for most of model organisms and the human genome. However its major disadvantage is that it lacks functionality to work with non-model organisms, which genomes are still not well or completely characterized and annotated. In these cases custom scripts are necessary to add functionality.
• Customers can connect to the MCIC Partek License server by sending a written request to Saranga Wijeratne <wijeratne.3@osu.edu>
• To learn more about “How to connect to the license server at: http://www.partek.com/html/updates.html/ Click on “Licence FAQ”
• The Ohio Supercomputing Center (OSC) located in Columbus OH also provides access to Partek Discovery Suite and Partek Genomic Suite under statewide licenses (current version 6.4). To access OSC Partek services, you must contact OSC help desk via e-mail (oschelp@osc.edu) and include your IP address. Any other questions regarding license server configuration and license server access issues should be also sent to the OSC help desk. More details regarding OSC Partek services are found at: http://www.osc.edu/supercomputing/statewide/partek.shtml
• If you would like to try Partek software on your computer, you can download a trial version (mac, PC or linux) at: http://www.partek.com/html/updates.html
• Instructions for installing Partek on Linux are at: http://www.partek.com/customers/Partek_Install_Linux.txt
We are now evaluating the CLC Genomic Workbench, which is also a cross-platform desktop application with a graphical user-friendly interface. CLC Genomics Workbench package uses algorithms like SIMD-accelerated assembly to rapidly analyze and visualize the huge amount of data generated by the ‘next generation’ sequencing instruments.
Some of the key Next Gen Sequencing applications of CLC Genomics Workbench include:
• Read mapping of Sanger, 454, Illumina Genome Analyzer and SOLiD sequencing data
• De novo assembly of genomes of any size (only limited by RAM available)
• Color space mapping
• Advanced visualization, scrolling, and zooming tools
• SNP detection using advanced quality filtering
• RNA-seq including support for paired data and transcript-level expression
• Small RNA analysis
• Expression profiling by tags
• EST library construction
• Gene expression analysis
• Advanced visualization, scrolling, and zooming tools
More information regarding this software can be found at: http://www.clcbio.com/index.php
We just started to evaluate the CLC Bio software. However, from discussions with other facilities, and similar to what is our experience with Partek, we expect CLC Bio to work well for well characterized and annotated model systems, but still lack functionality for some non-model systems. As it is often the case for this kind of software packages, a single software package will most likely not accommodate the many diverse needs that our customers have.
Cost
Partek: $5,000 for one year, one network user license; $3,000 for a standalone package.
CLC Bio: $14,985 for one year, one network user license; $4,995 for a standalone package.
Genotyping: Our CEQ8800 is back up and running. By Maria Elena Hernandez Gonzalez
Recently we have experienced some delays with fragment analysis on the Beckman CEQ8800, because of computer issues. These problems have been solved and we are back on track and accepting samples. The computer has been upgraded and is running on Windows XP. We are using the same CEQ 8000 series data analysis software (version 9). The company has not upgraded the software to run on Windows Vista or Windows 7.
If you would like to install the CEQ 8000 series data analysis software (version 9) on your computer, you can borrow the installation CD from MCIC. It runs on Windows XP, and will require Microsoft Office 7 installed on your computer. Microsoft Access, which is included on the Microsoft Office 7 package, allows database creation and import into Excel. Your computer will also need to have Microsoft SQL Server Desktop Engine (MSDE) installed. It is recommended to install CEQ 8000 series data analysis software on a desk computer and not on a laptop since it could make your laptop run very slow.
Regarding fragment analysis sample submission: it is important that youlet us know if you have changed your primers, have started a new multiplexing combination or have made any other changes to your protocol, because such changes affect the run and new run conditions need to be determined. In these cases, we have to test few samples to determine the best amount of sample and best ratio of sample to size marker to be loaded onto the instrument, before running the complete set.
Also, if you are a first time MCIC user, you need to register by filling out the “registration form” located on our web page http://oardc.osu.edu/mcic/. We need to have your contact information before you start a project.
Illumina GAII: Preparing libraries for the Illumina GAII sequencing is getting easier and also more cost effective. By Asela Wijeratne.
Automation and throughput
Illumina has recently developed new protocols and released new kits for genomic, mRNA and smallRNA library preparation called TrueSeq protocols. The TruSeq protocols are easier to implement with lesser number of pipetting steps. These improvements reduce the mistakes and increase the throughput. In addition, these protocols allow automation of sample preparation. The TruSeq mRNA sample preparation protocol is completely gel free and can be fully automated with a suitable liquid handler. The TruSeq genomic DNA protocol coupled with Covaris nebulizer allows gel free automation of DNA-seq procedure for some applications. With these procedures, one can easily make 96 libraries within 2-3 days. The TruSeq Small RNA kit still requires gel-based size fractionation, making it a little harder to fully automate. However, there are several companies that have designed gel-free size selection equipment and currently, we are looking in to these options.
Indices
Each TrueSeq genomic and mRNA library preparation contains enough reagents for 40-44 libraries (even though Illumina advertises for 48 reactions) and includes index oligonuceoltides for multiplexing, combining samples and sequencing them in one flow-cell lane. In cases where the sequencing coverage and depth are not an issue, indexing and multiplexing samples reduces the cost of sequencing. Illumina makes 12 indices, however one kit contains just six different indices and to get all the 12 indices one needs to purchase two kits. TruSeq Small RNA kits contain a total of 48 indices that are divided into 4 kits. Indexing allows combining of different samples together.
Alternative protocols
There are number of different kits and protocols available for making libraries for different applications. While it is impossible to review all these in a short note like this, there are two protocols worth mentioning.
Even though, the improved Truseq protocol allows using as low as 100ng of total RNA, it is still well above the amount of total RNA that can be extracted from a few cells (it is believed that a single mammalian cell contains about 20-30 pg of total RNA). In cases like this, SMARTer™ Ultra Low RNA Kit for Illumina® Sequencing (Clontech), which has been shown to work with total RNA amounts as little as 10pg, can be used with the Illumina protocol.
For the DNA sequencing, one of the problematic steps is breaking DNA and one of the successful methods involves using sonication with Covaris. However, the instrument costs about $45,000, making it cost-prohibitive for most case. An alternative method developed by Epicenter uses simultaneous fragmentation and adaptor tagging by in vitro transposition. This method has the added advantage of having several steps combined in the library preparation protocol, allowing completion of the sample preparation within a day. We would hope this method would be integrated into Illumina standard protocols as recently Illumina has acquired Epicenter.
Cost
Truseq kits reduce the sample preparation cost significantly:
DNA-seq sample preparation kit: $2600 for 48 samples
RNA-seq sample preparation kit: $3,350.00 for 48 samples
Small RNA – seq sample preparation kit: $2,600 for 24 samples
Note: Illumina charges 10% of the above price for shipping and the kits also do not contain all of the reagents necessary for the completion of the libraries.
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