BioMicroCenter:Illumina Sequencing: Difference between revisions
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The MIT BioMicro Center has five high-throughput Illumina sequencers, including a HiSeq 2000, three Genome Analyzers and one MiSeq. We support a wide variety of applications, such as ChIP-Seq, miRNA sequencing and RNA-seq. Each lane can potentially accomodate dozens of barcoded samples (depending on sequence complexity and desired coverage). [[BioMicroCenter:CoverageCalculations|Read lengths]] vary, depending on users, between 36nt and 150nt per end. <br> | |||
All questions about Illumina Sequencing can be directed to Kevin Thai at kthai@mit.edu. | All questions about Illumina Sequencing can be directed to Kevin Thai at kthai@mit.edu. | ||
==Illumina Massively Parallel Sequencing== | ==Illumina Massively Parallel Sequencing== | ||
[[Image:cbot_left.jpg|left]]Illumina sequencing works by binding randomly fragmented DNA to an optical flowcell. Fragments are sequenced by sequentially incorporating and imaging fluorescently labeled nucleotides in a [http://illumina.com/pages.ilmn?ID=203 “Sequencing-By-Synthesis”] reaction. Illumina recently rolled out its [http://www.illumina.com/truseq/about_truseq/truseq_sequencing_by_synthesis.ilmn TruSeq v3] reagent kits, improving read quality and reducing GC bias at high cluster densities. For an in-depth overview of the Illumina sequencing chemistry, please refer to [http://www.ncbi.nlm.nih.gov/pubmed/19682367 Kirchner et al 2009.] | |||
== Platforms == | |||
=== HiSeq 2000 === | |||
[[Image:hiseq_2000.jpg|right]] The Illumina HiSeq 2000 is the workhorse of BMC's Illumina fleet and is optimized for maximum yield and the lowest price per basepair. Each lane on the HiSeq is typically produces between 160 and 220 million reads passing our quality filter (for high quality libraries). HiSeq flowcells have 8 lanes, one of which is committed to a control sample that is used for base normalization (lane 1). Read lengths on the HiSeq very between 40 and 100nt per side and nearly all flowcells use barcoding to run multiple samples in each lane. <BR><BR> | |||
In order to optimize work flow and keep costs under control, only full flowcells are run. Since all 8 lanes of the flowcell must be run at equal lengths, submissions of single lanes must be grouped with other similar read lengths. This means that some read lengths move through our queue faster then others because more samples of that length are submitted to the BioMicro Center for sequencing. 40nt single end (SE) samples are by far the most common and move through the queue rapidly followed by short paired end (40+40) runs. Many lengths are very unusual (eg. 100nt single end) and can wait months for sequencing. We strongly recommend moving samples with unusual read requirements to one of the other platforms. If you have questions about this (or any other aspect of sequencing) please do not hesitate to contact us.<BR> | |||
<BR> | |||
The HiSeq2000 is ideal for: | |||
* High numbers of multiplexed samples | |||
* De novo sequencing | |||
* SNP detection | |||
* ChIPseq | |||
* RNAseq | |||
* Exome sequencing | |||
''The HiSeq2000 was donated to the BioMicro Center by Drs. Penny Chisholm and Chris Burge. '' | |||
=== MiSeq === | |||
=== Genome Analyzer IIx === | |||
[[Image:GAIIxcollage.jpg|right]] | |||
=== Platform Comparison === | |||
== Applications == | == Applications == | ||
Revision as of 19:43, 24 May 2012
| HOME -- | SEQUENCING -- | LIBRARY PREP -- | HIGH-THROUGHPUT -- | COMPUTING -- | OTHER TECHNOLOGY |
The MIT BioMicro Center has five high-throughput Illumina sequencers, including a HiSeq 2000, three Genome Analyzers and one MiSeq. We support a wide variety of applications, such as ChIP-Seq, miRNA sequencing and RNA-seq. Each lane can potentially accomodate dozens of barcoded samples (depending on sequence complexity and desired coverage). Read lengths vary, depending on users, between 36nt and 150nt per end.
All questions about Illumina Sequencing can be directed to Kevin Thai at kthai@mit.edu.
Illumina Massively Parallel Sequencing
Illumina sequencing works by binding randomly fragmented DNA to an optical flowcell. Fragments are sequenced by sequentially incorporating and imaging fluorescently labeled nucleotides in a “Sequencing-By-Synthesis” reaction. Illumina recently rolled out its TruSeq v3 reagent kits, improving read quality and reducing GC bias at high cluster densities. For an in-depth overview of the Illumina sequencing chemistry, please refer to Kirchner et al 2009.
Platforms
HiSeq 2000
The Illumina HiSeq 2000 is the workhorse of BMC's Illumina fleet and is optimized for maximum yield and the lowest price per basepair. Each lane on the HiSeq is typically produces between 160 and 220 million reads passing our quality filter (for high quality libraries). HiSeq flowcells have 8 lanes, one of which is committed to a control sample that is used for base normalization (lane 1). Read lengths on the HiSeq very between 40 and 100nt per side and nearly all flowcells use barcoding to run multiple samples in each lane.
In order to optimize work flow and keep costs under control, only full flowcells are run. Since all 8 lanes of the flowcell must be run at equal lengths, submissions of single lanes must be grouped with other similar read lengths. This means that some read lengths move through our queue faster then others because more samples of that length are submitted to the BioMicro Center for sequencing. 40nt single end (SE) samples are by far the most common and move through the queue rapidly followed by short paired end (40+40) runs. Many lengths are very unusual (eg. 100nt single end) and can wait months for sequencing. We strongly recommend moving samples with unusual read requirements to one of the other platforms. If you have questions about this (or any other aspect of sequencing) please do not hesitate to contact us.
The HiSeq2000 is ideal for:
- High numbers of multiplexed samples
- De novo sequencing
- SNP detection
- ChIPseq
- RNAseq
- Exome sequencing
The HiSeq2000 was donated to the BioMicro Center by Drs. Penny Chisholm and Chris Burge.
MiSeq
Genome Analyzer IIx
Platform Comparison
Applications
Illumina currently provides reagents and support for four major sequencing applications:
The following application has been published but does not yet have a kit from Illumina:
- Genotyping: Protocols are being developed for detection of SNPs, chromosomal rearrangements and other genotyping applications.
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. The BioMicro Center accepts fully prepared libraries from users and also offers a variety of sample preparation services for different applications.
Information is also available about multiplexing.
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.
Data Analysis
Each flowcell lane flowcell should produce between 10 and 120 million DNA fragments as of September 2011. 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:
Protocols
Protocols for all supported technologies can be found here.
Pricing and Priority
Full pricing information is available on our price list.
Priority for Illumina sequencing is currently given to labs associated with the BioMicro Center Core departments. We are able offer our services to other MIT and non-MIT users as space allows.
Turnaround Time
Each Genome Analyzer processes 8 samples per run, or 7 samples plus a control. Multiplexed pools of samples count as one for this purpose. The control is typically used to improve sequence quality. Full flowcells can usually be run within two weeks of submission. Partial submissions of less than eight samples (or 7 with control) are put into a project queue, where they join existing samples or await others before processing. Wait times for partial submissions vary depending on demand from other users. Once processing begins, approximately six days are required for clustering, sequencing, and data analysis for a 36-base-pair read. Longer reads add approximately one hour per additional base-pair including a second read and the barcode read, if either are present.
MIT Core Collaboration
Because of the layout of Illumina flowcells, samples must be run in batches of 7 lanes (a pool of multiplexed samples counts as one lane). In order to ensure quick 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.
View current samples queuing for Illumina
Initial page written by Summeet Gupta at the WI-CGT
