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=== Custom Primers ===
=== Custom Primers ===
When submitting [[BioMicroCenter:Sequencing
When submitting [[BioMicroCenter:Sequencing|'''custom primers''']], be sure to verify their compatibility with the standard Illumina Primers used. If you'd like a second opinion on the compatibility of your primers, we'd be more than happy to sit down with you and verify the primer design based on your constructs
== HOW LONG WILL IT TAKE FOR MY HISEQ SAMPLE TO BE SEQUENCED ==
== HOW LONG WILL IT TAKE FOR MY HISEQ SAMPLE TO BE SEQUENCED ==
STOP BY: The BioMicro Center is located at 68-316 on the MIT campus.
EMAIL: The BioMicro staff can be emailed at email@example.com
PHONE: The main lab number is 617-715-4533
Our director, Stuart Levine, can be reached at 617-452-2949.
The volume and concentration needed for submission will depend on the type of sample prep. However, if you are submitting prepped, sequence-ready samples, we require a minimum of 10uL at a concentration of at least 1.0nM. This allows us to perform quality control and have enough of the library for clustering. Any samples less than 1.0nM cause significant difficulties with clustering the flowcell, which is why we cannot guarantee optimal read count and quality should you like us to proceed with sequencing a low concentration sample.
When submitting custom primers, be sure to verify their compatibility with the standard Illumina Primers used. If you'd like a second opinion on the compatibility of your primers, we'd be more than happy to sit down with you and verify the primer design based on your constructs.
Another important aspect to consider is the fact that clustering conditions differ when comparing the HiSeq to the MiSeq. The MiSeq clusters at about 5 degrees higher than the cBot, which is used to prepare flowcells for the HiSeq. This means that all custom primers must be designed appropriately so Tm's are compatible with whatever sequencing route you choose.
HOW LONG WILL IT TAKE FOR MY HISEQ SAMPLE TO BE SEQUENCED
This is a very hard question to answer because it involves a number of variables. The simplest answer is, samples preparation typically takes ~1 weeks for standard samples with all reagents on hand (QC, prep, QC). For newer or rarely used methods, the number can extend to a month if we need to gather reagents. Once the sample is ready, it goes in to the sequencing queue. This queue is often the longest part of the process. Once the full flowcell is ready and put on a sequencer, it takes ~1.3h per base to sequence (this used to be 1h but upgrades to the machines have slowed them down some). A 40nt read takes just over 2 days. Turn around (for PE samples) functionally takes about a 1/2 day. So a 100+100PE sample will take about 11 days once it is on the sequencer - if nothing goes wrong. Overall, when the queue is flowing at full speed, a 40nt SE submission with sample preparation can be returned with data in right around 3 weeks. 40+40PE samples more typically take ~4-6 weeks and 80+80PE runs typically average ~6-8 weeks for CORE lab members with the extra time coming from delays in filling out flowcells.
GAII and MISEQ are much simpler. Once the sample is prepped and the machine is working, it will be loaded immediately. Total times should be less than 3 weeks.
Illumina Queue: Full Flowcells
The primary requirement for a run is a full flowcell. Each flowcell is composed of 7 lanes that must be run together. In the BioMicro Center, lanes are grouped by read length to optimize throughput and keep costs low. This requirement for 7 samples has a major impact on queue time. If you do a common read length, such as 40nt SE, the queue length will be short. On the other hand, if you ask for an unusual read length (eg. 100nt SE), your samples may never come off the queue. By frequency, the most common read lengths are, in order: 40SE, 40+40PE, 80+80PE. We do fudge a little in some of the long read lengths to fit samples in (mixing 80+80 and 100+100PE for example), but we will always give priority to the samples of the read length we are going to do. We do not run incomplete flowcells and other options, such as GAII and MiSeq exist for unusual read lengths. NOTE: Barcoding is assumed to be done on all flowcells and we do NOT restrict pooling based on which samples are multiplexed and which are not.
Illumina Queue: Priority
The BioMicro Center queue operates on a tiered priority basis (see table). We hold to this priority schema as closely as we can while still needing to fill flowcells. Some caveats: Once a flowcell is quality controlled and "on deck" for clustering, that flowcell is considered locked and no new submissions will be added to it. Clustering can happen a week or more before loading. Once flowcells are clustered, their order is set barring the need for reruns.
The Illumina HiSeq is an unfortunately temperamental beast and failures do happen. The large majority of the failures on our sequencer are due to equipment failure and not due to operator failure, although those do happen as well. Once a failed run is identified, we must work with Illumina to identify the cause and prove to them that the cause is equipment based. This allows us to get replacement reagents which helps keep your costs down. Once a run has failed, the failed samples on that flowcell are given highest priority.
Failures create a large challenge for us in providing estimates of when your data will be ready. Because the failure rate is so high and failures tend to cluster, short sequencing estimates can quickly turn in to long waits, particularly if you are not at the top of the priority queue. The BioMicro Center works closely with the KI Biopolymer Core and the Whitehead Institute GTC to move samples to other machines whenever possible if we develop a backlog.
The final consideration in timing is best summarized as "Triage". This encompasses all of the many other factors we take in to account in creating flowcells. This can range from scheduling: Pushing a 40nt SE run on before an 80+80PE run so the turn around of the 80+80 times to be during the week instead of over a weekend - to flowcell logistics: Grabbing low priority 40+40PE samples to fill out a rerun flowcell while an 80+80PE run that does not have rerun samples waits in the queue. Our goal is to get everyone their data as fast as we possibly can. We look at every project every day and we are working as hard as we can to get you your data as fast as we can.
HOW DO I GET MY DATA
You will be notified by email that your data is ready. The data will be placed on one of our server for you the download.
- There is a Windows applications client called SSH Secure Shell available at :
- Instructions to use the application can be found Instructions to use SSH .
- There is a Macintosh client called Fetch 5.3.for Macintosh.
- It can found http://ist.mit.edu/
- Select software : File transfer – Macintosh
- Direction for installing and using Fetch are located here:
- Samples can be copied from our servers using scp
- Use the SSH client or Fetch to find the precise filenames / directory you want.
Illumina data is also made available by direct mount for many users on the MIT campus or who are running VPN. While we are transitioning to the Isilon system, not all labs may have access. Contact Stephen Goldman if you are interested.
Accessing the server
Your user name and password will be included in the email. To obtain your data, enter the following in your client:
Host: bmc-150.mit.edu user: provided in email Password: provided in email
Please contact Stephen Goldman if you have difficulty obtaining your data.
NON MIT USERS
Do you take samples from outside MIT?
The BioMicro Center is built to serve the MIT community. As such, members of the MIT community have priority on all of our services. However, if we have extra capacity, we are happy to make it available scientists not affiliated with MIT, with the caveats that MIT samples *always* have priority and that access is finite. The BioMicro Center retains the right to refuse any sample.
How can we ship samples to you?
Please email firstname.lastname@example.org to arrange a drop off date and time. DNA samples should be shipped at 4C and RNA samples should be shipped on dry ice.Samples should be submitted with a completed order form and shipped by overnight delivery to:
MIT BioMicro Center 31 Ames Street, Building 68-316 Cambridge, MA 02139
The pricing form says "NA". What does that mean?
Some of our services are specifically restricted to the MIT community and we cannot offer them to outside users. Others are restricted to academic labs. Please email us at email@example.com if you have any questions.
What is your billing address?
Payments should be made to our finance office at:
Massachusetts Institute of Technology Biology Finance Office, Attn: Alison Salie 77 Massachusetts Avenue, 68-157 Cambridge, MA 02139