The GTC offers Next-Generation sequencing on the Illumina platform. We have two HiSeq 2500 systems with Rapid mode capabilities.
There are two options for Illumina sequencing. HiSeq "Standard" and HiSeq "Rapid".
In Standard mode (a.k.a. "High Output" mode) the HiSeq typically yields 120-150 million reads per lane. Standard mode require 7 full lanes of samples before sequencing can proceed and takes about 1 day for every 25 bases of read length. Note: You do not need to fill out a full flow cell yourself, we will group you with other samples to fill out a run.
In "Rapid" mode, individual samples can be sequenced much more quickly without the need to fill out full flow cells. Read lengths in Rapid mode are also longer, up to 250x250 and can be completed in a day or two compared to up to two weeks in standard mode. Rapid runs cost approximately 50% more than standard mode runs for similar amounts of data.
LIBRARY PREP SERVICE
We offer many options for library prep for a wide range of applications and input levels down to RNA from single cells. The following chart can be used as a starting point but you should feel free to consult GTC staff about which prep is right for you.
|Sample Type||Application||Input amount (ng)**||Prep|
|Genomic DNA||Any||>100||TruSeq DNA|
|RNA (total)||Basic (PolyA) Expression||>100||TruSeq Stranded RNA|
|RNA (total)||With Ribozomal Deletion||100-4,000||TruSeq Stranded RNA with RiboZero|
|RNA (total)||Ultra Low Input||<10||Clonetech UltraLow Input|
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SEQUENCING SUBMISSION GUIDELINES
You can download detailed guidelines in word format from here.
STEP 1: REGISTRATION (FOR NEW CUSTOMERS ONLY)
- INTERNAL CUSTOMERS: Please click here to register in our system.
- EXTERNAL CUSTOMERS: Fill out a new customer form (Available Here) with billing information. Once the form is emailed to us, we will register you in our database and notify you when you can submit samples to us.
All external customers please obtain a purchase order number prior to your submission. If you need a quote, please email us at email@example.com or call us at 617-258-8803. Blanket purchase orders are preferred, if you plan to submit multiple projects over time.
STEP 2: PREPARATION OF SAMPLES
SUBMISSION OF SAMPLES FOR LIBRARY PREPARATION
Samples should be in a buffer that does not contain trace amounts of phenol or EDTA. We will save and return any unused input material after libraries are successfully prepared.
Below are our input requirements for Library Prep Service
|Sample Type||Sonication/Fragmentation*||Fragment Size (bp)||Input requirements (ng)**||Volume (ul)|
|Genomic||Required||200 to 400||500 to 1000||10-50|
|ChIP||Required||Variable (specify)||10 to 50||10-40|
|RNA (total)||NA||NA||100 to 1000||10-50|
*Unless special arrangements are made with us in advance, we will assume that your genomic DNA sample has been fragmented, by sonication or otherwise, prior to submission.
**We can work with less if necessary, but it is best if you elute your DNA in 50 ul or less if you believe we may need to use all of it. Please give us at least 10uL of sample to ensure we have enough for QC and prep.
SUBMISSION OF PRE-PREPARED LIBRARIES
We typically need 10-20 ul of each library at 5 ng/ul or greater. If your library is less concentrated than 5 ng/ul, we’ll quantify it to make sure we have enough to sequence.
STEP 3: ONLINE SUBMISSION OF SEQUENCING LIBRARIES
- Please complete the online submission form (Link To Form Available Here). Questions or problems related to the submission should be directed to Sumeet Gupta at firstname.lastname@example.org
- Every physical sample tube should have its own Genome Tech Core ID (GTC ID) number (automatically generated and emailed to you immediately after submission of the online form), even if you plan to multiplex them.
- Please write your name GTC ID numbers on the tubes. We prefer 1.5 ml tubes.
STEP 4: DROP-OFF OR SHIPPING
When you are ready, you may hand deliver or ship samples them on dry ice to:
Whitehead Institute Genome Technology Core
9 Cambridge Center, Room 325
Cambridge, MA 02142
Please do not ship without first contacting someone in the lab, to make sure that we are here to receive your package. Do not rely on voicemail. Please wait until you hear back from someone.
Images acquired from the sequencer are processed through the bundled Illumina image extraction pipeline to get the sequence and quality score for each base. An in-depth QC report is included in the package.
Each sequencer processes 8 lanes per run, or 7 lanes plus a control. Full flowcell submissions (7 lanes) can usually be started within one week of submission. Partial submissions of less than seven lanes are put into a project queue where they join existing samples or await others before processing. Wait times for partial submissions vary but are generally two weeks or less until the START of the run depending on the requested run configuration and demand from other users. The most common run configuration is short (40 bases) and single read. Longer read and paired-end samples may take longer to turn around as there are generally fewer similar samples in the queue to fill out runs. Once a run has started, estimate approximately 1 day per 20 bases for the run and an additional 24-48 hours for data processing. Samples requiring library prep will also take an additional one to two weeks for the prep.
Next-Generation Sequencing can be used for a wide variety of applications including ChIP-Seq, RNA-Seq, smallRNA-seq, GWAS and much more. Visit Illumina's web site or contact us to discuss your project goals.