BioMicroCenter:Sequencing

ILLUMINA MASSIVELY PARALLEL SEQUENCING
The MIT BioMicro Center has four high-throughput Illumina sequencers, including the HiSeq 2000, which are currently being used for a variety of applications, including ChIP-Seq, miRNA sequencing and RNA-seq. Each next-generation sequencer can process up to 7 lanes of samples with multiple samples per lane (if multiplexed), with a data yield of over 25 million reads per lane for the GAIIx and over 120 million reads for the HiSeq (Single read). Read lengths vary, depending on users, between 36nt and 150nt per end on the GAIIx and between 40nt and 100nt per end on the HiSeq. Click here for recommendations on read length.

Illumina sequencing works by binding randomly fragmented DNA to an optical flowcell. Templates are sequenced by incorporating fluorescently labeled nucleotides in a “Sequencing-By-Synthesis” reaction. A detailed description of this process can be found at Illumina's website. Illumina recently rolled out its TruSeq v3 reagent kits, improving read quality and reducing GC bias at high cluster densities. As a result, the capacity of the HiSeq has been expanded dramatically, so that a single flowcell lane on the HiSeq produces as many reads as a whole GAIIx flowcell. The Genome Analyzer IIx system consists of a cluster generation station, a Paired-End module, and a Genome Analyzer sequencer, all of which work in concert to generate and analyze flowcells. The HiSeq system is similar in setup, but features expanded capacity and an integrated paired-end module. An overview of both systems can be found at the Illumina website: GAIIx HiSeq

For an in-depth overview of the Illumina sequencing chemistry, please refer to the following paper:

Kirchner et al 2009.



Sample Preparation
Illumina sequencing requires the input of libraries which have been properly fragmented, ligated to specific adapters, and, in the case of RNA inputs, converted into complementary DNA. BioMicro Center offers a variety of sample-preparation services for different applications, and we also accept fully prepared libraries from users. For information on Illumina library preparation techniques and services offered by the BMC please visit the Illumina Library Preparation page (please note that this page is currently under development).

Information is also available about multiplexing.



Applications
Illumina currently provides reagents and support for four major sequencing applications:


 * ChIP Seq
 * RNA Seq
 * Small RNA Sequencing
 * Genome sequencing and resequencing

Other DNA Sequencing applications: The following applications have been published but do not yet have kits from Illumina.


 * Genotyping: Protocols are being developed for detection of SNPs, chromosomal rearrangements and other genotyping applications.



Data Analysis
Each lane of the flowcell should produce between 10 and 120 million DNA fragments as of March 2010. Understanding this data often requires a significant investment in informatics and many applications require entirely different interpretations of the data. As part of our sequencing service we provide many of the early steps of bioinformatics for different applications. Further data processing can be arranged on a collaborative basis as resources are available. For more information, check out the links below:


 * Illumina pipeline - How it works!
 * Illumina pipeline output formats
 * Bioinformatic Consulting

Pricing
First priority for Illumina sequencing goes to BMC-associated core departments. We can do Illumina sequencing for other MIT and non-MIT users as space allows. Full pricing information is available at our price list.



Protocols
Protocols for all of the supported technologies can be found on the protocols page.



QC
Quality control is very important for optimizing the number of reads and the quality of data produced. We run Bioanalyzer and RT-PCR for all submitted cDNA libraries for Illumina sequencing. For more information on QC methods and protocols please visit the Sequencing Quality Control page.



MIT Core Collaboration
Illumina samples are run in batches of 7 flowcell lanes (if many samples are multiplexed into one lane, they count as one for the purpose of completing a batch). In order to optimize our throughput, we have established a collaboration that allows us to move partial flowcells between the various centers at MIT. For users with less then 4 samples, their samples may be moved between the BioMicro Center, the Whitehead Institute Center for Genome Technologies and the Koch Institute Biopolymer Center. Samples will be moved only to fill out runs or to expedite processing. The Centers are committed to working together to maintain consistent quality between the different cores, so you should see no difference whether your samples are run in BioMicro or at one of our sister centers. Transfers are only available for members of the MIT community.

[[Media:QueueReport.pdf| View current samples queuing for Illumina]] 

All questions about Illumina Sequencing can be directed to Kevin Thai at kthai@mit.edu.

Initial page written by Summeet Gupta at the WI-CGT