BioMicroCenter:News

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== Welcome to the MIT BIOMICRO CENTER ==
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== BioMicro Center News ==
 +
=== APRIL 20, 2013 ===
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=== May 2009 - HIGHLIGHTS ===
+
We have noticed a number of technical issues with some Illumina runs. We want to share with you to make sure you are aware of some changes and newly identified technical issues with the platform and what we are doing to correct them where we can. All of these changes are from the Illumina side and none were especially well documented (some not at all). These issues are unlikely to be limited to the BMC, so samples from elsewhere on campus or around the country may also have these issues. Please read this as it may have some impact on your analyses.  <BR><BR>
-
* Improvements in Microarray Processing
+
-
* Improvements in Illumina Sequencing
+
-
* Pilot project for large scale data storage
+
-
* BioMicro Center renovation ongoing.  
+
-
It has been a few months since our last update and I would like to share with everyone several improvements we have made in some of our core technologies.  
+
Just to begin, all of these changes are subtle and not obvious in most cases directly from the sequencers. It was the rare cases that had dramatic effects that caused us to notice them. If you decide you need to have samples rerun, we will work with you to try to get Illumina to replace the reagents and to get the samples rerun. Unfortunately, there is no way we can possibly do bulk reruns of several months’ worth of studies. <BR><BR>
-
One large change has been in our handling of microarrays. Affymetrix will be retiring their old labeling kits later this year and we have been evaluating different technologies to replace the old kits. We have tried out new kits for both eukaryotes and prokaryotes and have been pleased with the results. Both new kits require significantly less sample input (<100 ng total RNA) and are easier to handle larger numbers of samples. We are also looking at other labeling techniques that would require even less input RNA (<1ng or even from single cells). More information can be found on our website. If you are interested in some of the newer technologies, please contact Manlin or myself.
+
The most concerning issue is a dropout of GC rich regions in clustering. This has been an on-again off-again issue with Illumina that we have addressed over a year ago by improvements in amplification cycling conditions and enzyme selection. Some time, several months ago (we do not have a precise window), Illumina appears to have changed the chemistry of one of their clustering components and that caused a major change in performance on GC rich areas. This can be seen as an absence of reads from very GC rich areas but, because these areas are rare in most genomes, they cannot be seen on the flowcell wide metrics. This issue is found on current HiSeq and MiSeqV2 kits but not on MiSeqV1 kits nor, we suspect, on the GAII. We have been able to address this problem by adding a brief boiling step during NaOH denaturation of the samples and have implemented this as SOP starting about two weeks ago. This drop out of regions can cause significant issues for several studies – most notably ChIP analyses – when you are comparing data from different chemistries. <BR><BR>
-
In addition to microarrays, we have also upgraded to the latest kits from Illumina for next-generation sequencing. These new kits replace the old cleavage buffer with a new formulation that allows for much longer reads. We have preformed some tests with these new kits and have been able to achieve read lengths of 144nt (72nt each from the front and back) with over 90% of the reads showing 1 error or less at densities of typically >5 million reads per lane. We have placed some of the preliminary analyses on our website under the genomic sequencing section.
+
A second concern is one that has been reported in the community but we have not identified on our machines – yet – where samples from a run are being observed in the following run as minor contaminants. This issue is limited to the MiSeq and HiSeq2500 (we do not have the latter) where the tubes that add sample to the flowcell are not changed. This contamination is reported to be <1% and so would not show up on our quality metrics. However, if your MiSeq analyses are being based on finding a few reads in a large pool of discarded data or you are doing a number of sequential runs, you may wish to validate your data more carefully using an alternative technique such as qPCR or sanger sequencing. There is currently no technical fix to this problem.  <BR><BR>
-
We have also started a pilot program to help labs handle the large amounts of data produced by these high-throughput methodologies in response to requests from several labs. We have purchased a new storage server and are providing space on the server at $500 per terabyte per year. The server is set up to be able to be accessed from all three major operating systems (Linux, Windows and Mac) and the data is backed up using MIT’s TSM (normally $65/mo).   Space on this pilot server is limited and will be allocated on a first come / first served basis. Please contact Stephen Goldman or myself if you have questions. More information about this program can be found in the computing section of our website.
+
A third issue has been around for a while though we had not appreciated the implications. Illumina’s newer versions of basecalling software have become less capable of handling uniform sequence (all A’s for example). In earlier versions, only 5 basepairs of variability were needed and intensities could be determined by the control lane we run on all HiSeq flowcells. Now, it appears that nt 1-25 all must have representation of all 4 bases at all positions, even with a control lane. This has always been an issue on the MISeq and we have solved it by spiking in 30%PhiX in the lane (as opposed to our normal 0.1% spike in). Similar solutions can be used on the HiSeq. Given this change, we are re-evaluating whether there is value in using the 8th lane as a control. The latest version of MiSeq software (only a couple days old) supposedly allows us to lower the fraction to 5%, but how successful this is remains to be seen. Base rearrangement with the GAII allows the GAII to avoid this issue. <BR><BR>
-
Lastly, we are still in the process of remodeling the BioMicro Center lab space. Thank you all for your patience as we have been in transition and we hope by the end of this month to have the process completed. The final layout is also on our website along with all of our monthly updates.
+
Finally, it appears that custom priming on the MiSeq is not the same as custom priming on the HiSeq and GAII. It can still be done, but the Tm requirement is much higher. Primers that work on the HiSeq may fail on the MiSeq. As long as your Tm matches or exceeds the Tm used for Illumina primers, the MiSeq should work, but the MiSeq’s different chemistry (formamide instead of heat denaturation) is less forgiving. <BR><BR>
 +
In summary, we have a number of technical challenges that may (or may not) effect you and we want to make sure you have all the information we can give you. I want to thank the researchers and labs that have been very patient as we have struggled running their samples which led us to identify these problems. If you believe these issues have effected your data, please do not hesitate to contact me and we can discuss how to move forward.  <BR><BR>
-
|-
 
-
|valign="top" width="45%"|
 
-
===RECENT & UPCOMING CHANGES===
+
=== MARCH 11, 2013 ===
 +
Quick update from BioMicro: <BR><BR>
 +
The [[BioMicroCenter:Wafergen|Wafergen qPCR system]] is now operational. We have done a couple pilot experiments so far and it does seem to work, if there are a few more limitations than we anticipated. We are working with Wafergen to see how many of these can be alleviated but you are more than welcome to try it out and see if it would be useful to you. They have given us quite competitive pricing that is a lot lower than the cost for the [[BioMicroCenter:Fluidigm|Fluidigm BioMark]] . Please email us if you are interested in training.
-
{{BioMicroCenter:News/Changes/Content}}
 
-
<B><BIG>PREVIOUS NEWSLETTERS </BIG></B>
+
=== JANUARY 9, 2013 ===
 +
Happy new years to everyone. A couple new things happening in BioMicro that we want to make everyone aware of. <BR><BR>
 +
First, this month begins a year long experiment in joining the BioMicro Center Informatics team and the KI Bioinformatics and Computing Core in to a single team. Our two teams have been collaborating for several years, sharing computational infrastructure, etc. but this year we will be formalizing and expanding the relationship with the goal of creating a more efficient unified core. Informatics analysis requests should still be sent to Charlie Whittaker or to myself as usual, but will be spread across the joint team based on expertise and on availability. You are also, as always, welcome to contact any of the informatics scientists directly. We hope this will allow us to reduce waiting times and to keep costs under control.  <BR><BR>
 +
During the trial period (and hopefully going forward), pricing for informatics will be available in two flavors. First, for projects needing routine work, the subsidized rate will be $70/h for all CORE members (Biology, BE, KI, CEHS). For more involved projects, we have second option to purchase a “share” of the informatics team. This is an annual commitment for a fraction of an informaticist and will cost $960/mo for an average of 4h/week of informatics support. The monthly usage levels do not have to be exact and can be used in large blocks. The hours in the share can be used with any member of the team and the informaticist can vary from project to project.  <BR><BR>
 +
Finally, and importantly, we will be changing the way we are storing Illumina sequencing data long term. In the past, we have saved the fastq, sam and bam files, along with the quality control data, in a zipped file. These zipped files now occupy over 50TB of storage which is limiting  how we are able to handle new sequencing runs. To address this, we will be deleting the fastq and sam files from the archive and storing only the binary bam and quality control files. The fastq and sam files can be regenerated rapidly from the bam files using Picard and SamTools (though reads may not be in the same order). As always, we strongly encourage you to keep your own copy of the Illumina data and use our version only as a backup. We will begin this conversion next week.
 +
If you have any concerns, please do not hesitate to contact me.
-
{|
 
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|'''[[BioMicroCenter:News/2009|2009]]'''
 
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|-
 
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|{{BioMicroCenter:News/2009/Content}}
 
-
|-
 
-
|'''[[BioMicroCenter:News/2008|2008]]'''
 
-
|-
 
-
|{{BioMicroCenter:News/2008/Content}}
 
-
|}
 
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 +
 +
|valign="top"|
 +
 +
== ABOUT THE BIOMICRO CENTER ==
 +
 +
The MIT BioMicro Center was founded in 2000 as the core bio-fabrication and microarray processing facility at MIT. The Center is a joint endeavor between the [http://biology.mit.edu Department of Biology], the [http://ki.mit.edu Koch Institute for Integrative Cancer Research], the [http://be.mit.edu Department of Biological Engineering] and the [http://cehs.mit.edu MIT Center for Environmental Health Sciences.] The BioMicro Center offers a wide range of genomic services to researchers at MIT. The majority of services rendered pertain to massively parallel sequencing using the Illumina Genome Analyzer (both library preparation and sequencing). Commercial array processing and include both the Affymetrix Gene Chip and Agilent DNA array platforms continues to be a significant portion of our portfolio. Real-time PCR and Agilent BioAnalyzer services are available in the facility both as services available to researchers, as well as for quality control of microarray and sequencing samples. In addition, the Center has a presence in high-throughput screening with robotics and plate reading as well as informatics and computational support. The BioMicro Center serves the [http://ki.mit.edu Koch Institute] as the [http://ki.mit.edu/sbc/microarray MicroArray Technologies Core] and as part of the [http://ki.mit.edu/sbc/bioinformatics Bioinformatics and Computing Core] and the [http://cehs.mit.edu MIT Center for Environmental Health Sciences] as part of the [http://cehs.mit.edu/facilities.html#Genomics_and_Bioinformatics_Core Genomics and Imaging Core]<BR><BR>
 +
 +
Experimental and analytical work done in the BioMicro Center is funded by the NIH and must be made available through the NIH's open access policy. All Koch Institute and CEHS labs '''must''' acknowledge their core grants for work done in the core with the following language.
 +
* KI ''"This work was funded by the National Cancer Institute of the NIH under award P30-CA14051"''
 +
* CEHS ''"This work was funded by the National Institute of Environmental Health Sciences of the NIH under award P30-ES002109"''
 +
 +
== PUBLICATIONS ==
 +
'''2013'''<BR><BR>
 +
'''2012'''<BR><BR>
 +
<biblio>
 +
#Paper1 pmid=22981692 <!-SL Boyer: Heart->
 +
#Paper2 pmid=22847430 <!-SL Saeij->
 +
#Paper3 pmid=22102570 <!-HD Chisholm->
 +
</biblio>
 +
'''2011'''<BR><BR>
 +
<biblio>
 +
#Paper1 pmid=21892155 <!-SL Sur->
 +
</biblio>
 +
'''2010'''<BR><BR>
 +
<biblio>
 +
#Paper1 pmid=20720539 <!-SL Young->
 +
#Paper2 pmid=20581084 <!-SL Zwaka->
 +
</biblio>
 +
'''2009'''<BR><BR>
 +
<biblio>
 +
#Paper1 pmid=19531355 <!-SL Amon->
 +
</biblio>
 +
 +
== PREVIOUS NEWSLETTERS ==
 +
 +
'''[[BioMicroCenter:News2012|2012]]'''<BR>
 +
'''[[BioMicroCenter:News2011|2011]]'''<BR>
 +
'''[[BioMicroCenter:News2010|2010]]'''
<br>
<br>
-
<B><BIG>RECENT CHANGES TO THE WEBSITE <\BIG><\B>
+
 
 +
== RECENT CHANGES TO THE WEBSITE ==
{{BioMicroChanges}}
{{BioMicroChanges}}
|}
|}

Revision as of 11:08, 29 April 2013

Image:BioMicroCenter-header6.jpg

.

Contents

Welcome to the MIT BIOMICRO CENTER

BioMicro Center News

APRIL 20, 2013

We have noticed a number of technical issues with some Illumina runs. We want to share with you to make sure you are aware of some changes and newly identified technical issues with the platform and what we are doing to correct them where we can. All of these changes are from the Illumina side and none were especially well documented (some not at all). These issues are unlikely to be limited to the BMC, so samples from elsewhere on campus or around the country may also have these issues. Please read this as it may have some impact on your analyses.

Just to begin, all of these changes are subtle and not obvious in most cases directly from the sequencers. It was the rare cases that had dramatic effects that caused us to notice them. If you decide you need to have samples rerun, we will work with you to try to get Illumina to replace the reagents and to get the samples rerun. Unfortunately, there is no way we can possibly do bulk reruns of several months’ worth of studies.

The most concerning issue is a dropout of GC rich regions in clustering. This has been an on-again off-again issue with Illumina that we have addressed over a year ago by improvements in amplification cycling conditions and enzyme selection. Some time, several months ago (we do not have a precise window), Illumina appears to have changed the chemistry of one of their clustering components and that caused a major change in performance on GC rich areas. This can be seen as an absence of reads from very GC rich areas but, because these areas are rare in most genomes, they cannot be seen on the flowcell wide metrics. This issue is found on current HiSeq and MiSeqV2 kits but not on MiSeqV1 kits nor, we suspect, on the GAII. We have been able to address this problem by adding a brief boiling step during NaOH denaturation of the samples and have implemented this as SOP starting about two weeks ago. This drop out of regions can cause significant issues for several studies – most notably ChIP analyses – when you are comparing data from different chemistries.

A second concern is one that has been reported in the community but we have not identified on our machines – yet – where samples from a run are being observed in the following run as minor contaminants. This issue is limited to the MiSeq and HiSeq2500 (we do not have the latter) where the tubes that add sample to the flowcell are not changed. This contamination is reported to be <1% and so would not show up on our quality metrics. However, if your MiSeq analyses are being based on finding a few reads in a large pool of discarded data or you are doing a number of sequential runs, you may wish to validate your data more carefully using an alternative technique such as qPCR or sanger sequencing. There is currently no technical fix to this problem.

A third issue has been around for a while though we had not appreciated the implications. Illumina’s newer versions of basecalling software have become less capable of handling uniform sequence (all A’s for example). In earlier versions, only 5 basepairs of variability were needed and intensities could be determined by the control lane we run on all HiSeq flowcells. Now, it appears that nt 1-25 all must have representation of all 4 bases at all positions, even with a control lane. This has always been an issue on the MISeq and we have solved it by spiking in 30%PhiX in the lane (as opposed to our normal 0.1% spike in). Similar solutions can be used on the HiSeq. Given this change, we are re-evaluating whether there is value in using the 8th lane as a control. The latest version of MiSeq software (only a couple days old) supposedly allows us to lower the fraction to 5%, but how successful this is remains to be seen. Base rearrangement with the GAII allows the GAII to avoid this issue.

Finally, it appears that custom priming on the MiSeq is not the same as custom priming on the HiSeq and GAII. It can still be done, but the Tm requirement is much higher. Primers that work on the HiSeq may fail on the MiSeq. As long as your Tm matches or exceeds the Tm used for Illumina primers, the MiSeq should work, but the MiSeq’s different chemistry (formamide instead of heat denaturation) is less forgiving.

In summary, we have a number of technical challenges that may (or may not) effect you and we want to make sure you have all the information we can give you. I want to thank the researchers and labs that have been very patient as we have struggled running their samples which led us to identify these problems. If you believe these issues have effected your data, please do not hesitate to contact me and we can discuss how to move forward.


MARCH 11, 2013

Quick update from BioMicro:

The Wafergen qPCR system is now operational. We have done a couple pilot experiments so far and it does seem to work, if there are a few more limitations than we anticipated. We are working with Wafergen to see how many of these can be alleviated but you are more than welcome to try it out and see if it would be useful to you. They have given us quite competitive pricing that is a lot lower than the cost for the Fluidigm BioMark . Please email us if you are interested in training.


JANUARY 9, 2013

Happy new years to everyone. A couple new things happening in BioMicro that we want to make everyone aware of.

First, this month begins a year long experiment in joining the BioMicro Center Informatics team and the KI Bioinformatics and Computing Core in to a single team. Our two teams have been collaborating for several years, sharing computational infrastructure, etc. but this year we will be formalizing and expanding the relationship with the goal of creating a more efficient unified core. Informatics analysis requests should still be sent to Charlie Whittaker or to myself as usual, but will be spread across the joint team based on expertise and on availability. You are also, as always, welcome to contact any of the informatics scientists directly. We hope this will allow us to reduce waiting times and to keep costs under control.

During the trial period (and hopefully going forward), pricing for informatics will be available in two flavors. First, for projects needing routine work, the subsidized rate will be $70/h for all CORE members (Biology, BE, KI, CEHS). For more involved projects, we have second option to purchase a “share” of the informatics team. This is an annual commitment for a fraction of an informaticist and will cost $960/mo for an average of 4h/week of informatics support. The monthly usage levels do not have to be exact and can be used in large blocks. The hours in the share can be used with any member of the team and the informaticist can vary from project to project.

Finally, and importantly, we will be changing the way we are storing Illumina sequencing data long term. In the past, we have saved the fastq, sam and bam files, along with the quality control data, in a zipped file. These zipped files now occupy over 50TB of storage which is limiting how we are able to handle new sequencing runs. To address this, we will be deleting the fastq and sam files from the archive and storing only the binary bam and quality control files. The fastq and sam files can be regenerated rapidly from the bam files using Picard and SamTools (though reads may not be in the same order). As always, we strongly encourage you to keep your own copy of the Illumina data and use our version only as a backup. We will begin this conversion next week. If you have any concerns, please do not hesitate to contact me.



ABOUT THE BIOMICRO CENTER

The MIT BioMicro Center was founded in 2000 as the core bio-fabrication and microarray processing facility at MIT. The Center is a joint endeavor between the Department of Biology, the Koch Institute for Integrative Cancer Research, the Department of Biological Engineering and the MIT Center for Environmental Health Sciences. The BioMicro Center offers a wide range of genomic services to researchers at MIT. The majority of services rendered pertain to massively parallel sequencing using the Illumina Genome Analyzer (both library preparation and sequencing). Commercial array processing and include both the Affymetrix Gene Chip and Agilent DNA array platforms continues to be a significant portion of our portfolio. Real-time PCR and Agilent BioAnalyzer services are available in the facility both as services available to researchers, as well as for quality control of microarray and sequencing samples. In addition, the Center has a presence in high-throughput screening with robotics and plate reading as well as informatics and computational support. The BioMicro Center serves the Koch Institute as the MicroArray Technologies Core and as part of the Bioinformatics and Computing Core and the MIT Center for Environmental Health Sciences as part of the Genomics and Imaging Core

Experimental and analytical work done in the BioMicro Center is funded by the NIH and must be made available through the NIH's open access policy. All Koch Institute and CEHS labs must acknowledge their core grants for work done in the core with the following language.

  • KI "This work was funded by the National Cancer Institute of the NIH under award P30-CA14051"
  • CEHS "This work was funded by the National Institute of Environmental Health Sciences of the NIH under award P30-ES002109"

PUBLICATIONS

2013

2012

  1. Wamstad JA, Alexander JM, Truty RM, Shrikumar A, Li F, Eilertson KE, Ding H, Wylie JN, Pico AR, Capra JA, Erwin G, Kattman SJ, Keller GM, Srivastava D, Levine SS, Pollard KS, Holloway AK, Boyer LA, and Bruneau BG. . pmid:22981692. PubMed HubMed [Paper1]
  2. Minot S, Melo MB, Li F, Lu D, Niedelman W, Levine SS, and Saeij JP. . pmid:22847430. PubMed HubMed [Paper2]
  3. Kelly L, Huang KH, Ding H, and Chisholm SW. . pmid:22102570. PubMed HubMed [Paper3]
All Medline abstracts: PubMed HubMed

2011

  1. Mellios N, Sugihara H, Castro J, Banerjee A, Le C, Kumar A, Crawford B, Strathmann J, Tropea D, Levine SS, Edbauer D, and Sur M. . pmid:21892155. PubMed HubMed [Paper1]

2010

  1. Kagey MH, Newman JJ, Bilodeau S, Zhan Y, Orlando DA, van Berkum NL, Ebmeier CC, Goossens J, Rahl PB, Levine SS, Taatjes DJ, Dekker J, and Young RA. . pmid:20720539. PubMed HubMed [Paper1]
  2. Dejosez M, Levine SS, Frampton GM, Whyte WA, Stratton SA, Barton MC, Gunaratne PH, Young RA, and Zwaka TP. . pmid:20581084. PubMed HubMed [Paper2]
All Medline abstracts: PubMed HubMed

2009

  1. Boselli M, Rock J, Unal E, Levine SS, and Amon A. . pmid:19531355. PubMed HubMed [Paper1]

PREVIOUS NEWSLETTERS

2012
2011
2010

RECENT CHANGES TO THE WEBSITE

22 July 2014

+      15:16 BioMicroCenter:Software‎ (3 changes) . . (+744) . . (Page history) [Jingzhi Zhu‎ (3×)]
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