FAQs

General Core FAQs


Between what times can samples be dropped off at the facility?
The Molecular Genetics Core Facility is open from 9 am – 5 pm Monday through Friday. Samples can be dropped off at anytime during open hours. The facility is locked during all other times.


How do I cite the Molecular Genetics Core Facility in a paper?
When submitting a paper to a publication, if any of the services within the MGCF were utilized, it is important that you acknowledge our facility, the individual core, and the work we performed. For all projects through the Sequencing Facility and/or Genomic Mapping Facility, please include the grant we receive from the Intellectual and Developmental Disabilities Research Center (IDDRC) which is 1U54HD090255. For Microarray Facility work, please include the IDDRC grant number as well as the grant number for the Neuromuscular Disease Project which is NIH-P50-NS40828. An example acknowledgment would be, “Microarray expression experiments were performed by the Microarray Core Facility of the Molecular Genetics Core Facility at Children’s Hospital Boston supported by NIH-P50-NS40828 and 1U54HD090255.”


What forms of payment does the Molecular Genetics Core Facility accept?
All Children’s Hospital Boston researchers using Core services must provide an active cost center number (internal fund number) when registering. All researchers from outside institutions will be sent invoices that must be paid by check. For those researchers using the Microarray Core Facility, purchase order numbers must be provided in advance. Credit cards are not an acceptable form of payment.



DNA Sequencing Facility FAQs


Where do I bring my sequence samples and sample sheets?
The Molecular Genetics Core Facility is located on the 16th floor of the Center for Life Sciences Building located at 3 Blackfan Street across from the main Children’s Hospital entrance. Once on the 16th floor, follow the signs for the Molecular Genetics Core. The sample drop-off location and refrigerator is located towards the end of the lab suite as marked appropriately. Place your sequence samples in the provided lettered box corresponding to the first letter of your last name and then into the refrigerator. Be sure to clearly label your samples with tape, including your name and the date submitted. Place your corresponding sample sheet in the tray associated with the type of DNA template you are submitting for sequencing.


Do you have additional drop-off locations for sequencing?
We do not have any additional drop-off locations for submitting samples. All samples must be sent to or brought to the 16th floor of the Center for Life Sciences Building.


Can we set up one single account for our lab or should each lab member create one individually?
It is better if each individual lab member creates a separate account. If the facility needs to contact users about sequencing data or samples, it is easier and better for everyone for the facility to have direct contact information to the person submitting the samples.


How long does it take to process sample submissions?
It takes generally 2 days to process the samples. However, if we receive a very low amount of samples during a day, it can sometimes take up to 3 days because we run no less than a full plate of 96 samples at a time.


Is there any way to edit my sample submission?
Once a sample request has been generated and accepted it cannot be edited. If there are changes that need to be made, post a note on the incorrect sample request informing the facility of the error and then submit a new, correct form. If there are simply samples missing from the first request, create another one that contains only the missing samples.


What type of tubes or plates do you recommend we use to submit samples?
We ask that all our customers submitting individual samples use 200 ul striptubes with unattached stripcaps. These are the ONLY types of tubes that work with our current protocol. For full plate sequence submissions, we ask that customers submit samples in 96-well, flat-surfaced, skirtless plates enclosed with an adhesive film. For a more detailed description, please see the “Submitting Samples” section of the DNA Sequencing Core Facility page on this site.


Do you supply any sequencing primers?
We do NOT supply any primers for sequencing.


Do I add my reverse primers and forward primers in the same tube?
No. You must have a separate reaction for each primer to sequence your template.


Do I add the primer to my template or supply the primer separately?
Please combine the primer and template in the same tube according to the details outlined under the “Submitting Samples” section of the DNA Sequencing Core Facility page on this site. The template and primer will remain stable in the refrigerator for over a week.


How do I sequence through a G-C rich area or a hairpin in my template?
We suggest suspending your sample in 10% DMSO. This should be submitted in the same tube with your template and primer. If this does not work, please contact us to discuss other possible solutions.


How do I sequence through a poly-N in my template?
Often, large poly N sequence within a template uses up that specific di-deoxy nucleotide, resulting in noisy sequence after it. It is easiest to design primers right after the poly N sequence.


What length reads can I expect?
Our sequencers can produce quality read lengths up to 1000 bases. Remember that the length of the sequence relies heavily on the quality of the template submitted.


What software can I use to view my chromatogram results?
There are many sequence viewing software programs on the market, and almost all of them can be used to view your results. There is a list of some very basic, free programs with links to websites for downloading under the “Software” section of the DNA Sequencing Core Facility page on this site.


My sequence is very strong at first, but then trails off quickly – how do I fix this?
There are two major causes for this result. If not enough template is submitted for the reaction, the sequence will be weak and trail off. In these cases we recommend submitting a larger amount of template. However, due to the nature of dye terminator cycle sequencing, a large amount of template DNA results in a large amount of starting points for your primers. This results in most of the fluorescently dyed nucleotides to be incorporated at the beginning of the template. In these cases, we recommend customers to submit their template DNA at a lower concentration. If you are unsure what to do, please contact sequencing@iddrc.org and our technicians will help you with the proper correction.



Genomic Mapping Facility FAQs


Which fluorescent dyes can I use when designing labeled primers?
Our Applied Biosystems 3730 DNA Analyzer uses the G5 filter set for genotyping analysis. This filter can read 6-FAM (blue), VIC (green), NED (yellow), and PET (red) and these should be used to label your primers. Do not use LIZ as this is the dye label on our size standard.


What is the size range of products that can be read for genotyping?
All products should be at least 75 bp in length and no longer than 490 bp. The largest fragment on our size standard is 500 bp, so any products longer than this will not be called properly.


What size standard is used?
We use the Appleid Biosystems GeneScan LIZ-500 Size Standard.


How do I determine the proper dilution for my pre-run samples when submitting?
Not all PCR reactions work equally efficient. To determine the appropriate dilutions for custom markers, we suggest submitting test samples for each marker at various dilutions. We recommend testing dilutions of 1:1, 1:10, and 1:20 to assess the appropriate future submission amounts that produce the best results.


What type of tubes or plates do you recommend we use to submit samples?
The plate type depends on whether we are running the reactions or whether the reactions are pre-run. For a detailed description, please read the “Submitting Samples” section of the Genomic Mapping Facility page on this site.



Microarray Core Facility FAQs


How long will it take to receive my data?
The turn-around time for microarray projects varies based on our current queue of projects and our stock of the specific microarrays needed for each project. Depending on the size of the project (number of samples), it usually takes only about a week to process the samples. However, if we do not have the specific arrays in stock for your project, there can be an extra delay for shipping time from the manufacturer. Also, if there is a back-log of projects to process, this can cause delays as well. To determine our current assessment on project processing time, please contact microarray@iddrc.org and one of our microarray technicians will promptly respond.


How do I decide between using the Affymetrix platform versus the Illumina platform for gene expression?
There are a few factors to consider when deciding on the correct platform for a microarray gene expression project. Both platforms demonstrate good consistency from chip to chip or sample to sample with excellent CVs. Cost is often a factor for researchers and the Illumina platform has a lower per-sample cost than the Affymetrix platform. Sample number is also a consideration as the Illumina beadchip design requires that samples be submitted in groups of 6, 8, or 12 at a time whereas the Affymetrix platform uses one chip per sample. Before beginning a project, please consider these factors. If you need further assistance with project consultation, please feel free to contact us at microarray@iddrc.org.


What is the difference between the Mouse WG6 and Ref8 Illumina beadchips?
Both of these Illumina beadchips are genome-wide expression arrays. The key difference is the depth of coverage per sample. On the WG6 chips there are ~48,000 transcripts per sample, whereas there are only ~24,000 per sample on the Ref8. Due to the variations in probe density per sample on the arrays, Illumina is able to fit 8 sample spaces on each Ref8 chip, but only 6 on each WG6 chip. For more details on these chips, please consult the Illumina website.


Are there gene expression arrays able to process FFPE or degraded RNA samples?
In 2008, Illumina released an array protocol for paraffin-embedded or degraded RNA samples called the WG-DASL Expression Arrays. These arrays fit 12 samples to a slide and incorporate a modified amplification protocol that can be used with degraded template. There are also other FFPE sample preparation protocols and reagents that can be used and then loaded onto either Affymetrix or Illumina microarrays. For more information about these products, please email microarray@iddrc.org


 

Does the MGCF offer microarray informatics support?
For association or linkage study analysis, collaborations can be arranged with staff from the Program in Genomics at Children’s Hospital Boston. The Core does not internally have staff to support microarray gene expression analysis, but collaborations with staff from the Children’s Hospital Informatics Program can be coordinated. Also, links to some recommended software packages can be found under the “Software” section of the Microarray Core Facility page on this site.



Next-Gen Sequencing Core Facility FAQs

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How do I submit a project in the Next-Gen Sequencing Facility?
The first steps to submitting a project are to register on our Next-Gen Sample Submission website and contact us at nextgen@iddrc.org to set up your project consultation. Once we have determined which next-gen services are needed for your project and discussed the experimental design, you will complete the appropriate on-line order form. Once your order is submitted into the system, download and print the pdf of your order and submit your samples with the printout to our core facility.


Do I need a project consultation?
We require a project consultation for all next-gen users for many reasons: to ensure that the services we offer will provide the type of data in which the user is interested; to ensure that the experimental design is compatible with our current setup; to discuss sample multiplexing; etc. Please be sure to have a list of your questions ready. We are happy to do your consultation in person or as a phone conference.


Can a quote be prepared for my next-gen sequencing project?
Unfortunately, we are not able to give specific quotes for next-gen sequencing projects. This is because there are steps in the preparation, such as templated bead preparation, that have to be optimized and are charged based on the number of replicates run for each reaction set. However, using our pricing guidelines available on the next-gen facility page, you can generate a good estimate of your project cost.


Can more than one sample be combined in a single run?
Yes. With the SOLiD system, we are able to multiplex up to 96 customer samples in one reaction set. Each reaction set can then be loaded onto a full slide, a quarter (i.e. quad), or eighth (i.e. octet) of a slide. Note that Applied Biosystems/Life Technologies currently has 96 biological bar-codes available for use, but we reserve bar-code #1 for our internal spike-in control, and use bar-codes #2-96 for customer samples. If you are preparing your own sequencing libraries we will discuss which bar-codes are optimal for your samples during your project consultation.


Can I share my slide with other users?
Yes. We do allow researchers to share sequencing slides. In the case of a shared slide, we distribute the sequencing charges accordingly, based on the amount of the slide each researcher uses. Please discuss this with us during your project consultation.


What is the turn-around time for receiving data?
The turn-around time for next-gen sequencing varies greatly based on the number of projects on-going and whether the core facility will be preparing the libraries. During your project consultation, we will determine exactly which services the core will provide for your project and discuss the time considerations for those services.


How will I receive my data?
The next-gen order submission website also acts as a repository for your primary data files. This data will permanently be accessible on the Sample Submission and Tracking site.


How do I analyze my data?
There are innumerable ways to analyze your data. We suggest that you consider your data analysis before submitting your project. Some basic analysis options are available through your 3 month subscription to the Geospiza GeneSifter Analysis website that is included in your run. Information about other analysis options can be found on the Applied Biosystems SOLiD Software Community website.


How do I extend a subscription to the Geospiza GeneSifter software?
To extend a subscription, please contact Geospiza at support@geospiza.com and be sure to identify yourself as a customer of the Molecular Genetics Core Facility at Children’s Hospital Boston. An account manager will get in touch with you to discuss pricing of the full GeneSifter Edition. However, if you do another SOLiD sequencing run in our facility before the end of your initial subscription, we will add your next 3 free month subscription to the end of your current one and notify you of the new expiration date for your account.


What is the data storage charge?
Some researchers choose to save the intermediary data files (.spch files) from their run. Due to the large size of these files, we do not automatically save these files by default, so you must request this before a run is started. The charge associated with this is the cost for the server space and data backup. Upon completion of every run, we automatically save and upload all of the primary data including the color-space sequence data (.csfasta files), the sequence quality data file (.qual files) and the run statistics file (.stat files) for each sample to your personal profile on the website at no additional cost.