Difference between revisions of "BioMicroCenter:Illumina Sequencing"
m (→Platform Comparison)
(→Genome Analyzer IIx)
|Line 39:||Line 39:|
!width=75| cycles per day
!width=75| cycles per day
!width=75| cycles per kit
!width=75| cycles per kit
Revision as of 18:53, 26 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 firstname.lastname@example.org.
Illumina Massively Parallel Sequencing“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.
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
- Bisulfite sequencing
- Exome sequencing
The HiSeq2000 was donated to the BioMicro Center by Drs. Penny Chisholm and Chris Burge.
MiSeqphased sequencing. Highly unbalanced libraries, such as RRBS, should not be run on the MiSeq.
The strength of the MiSeq is its speed and read length. The MiSeq is able to sequence 12nt/hour which allows it to complete a 150+150nt paired end read, from cluster to fastq files, in a little over a day. This compares to 2-3 weeks of sequencing on the HiSeq. Because the chemistry is on the flowcell for less time, error rates are much lower for the MiSeq then the HiSeq. New kits should push read length even longer, with 250+250PE kits coming soon and the Broad Institute having reported 400+400PE runs.
The MiSeq is ideal for:
- Small genome resequencing
- Targeted resequencing
- barcode sequencing
The HiSeq2000 was donated to the BioMicro Center by Dr. Chris Love.
Genome Analyzer IIx
With the addition of the MiSeq, we have reworked how we are processing GAII flowcells. We have been able to create partial flowcells on the GAII by altering recipes. This has allowed us to move from a model like the HiSeq where we need a full flowcell before we run to a model where we can run as soon as the samples pass quality control, more like the MiSeq. However, unlike the MiSeq, we can run multiple lanes at once. Some critical caveats: First, these methods are not supported by Illumina so we cannot offer to replace failed runs. Second, unlike the HiSeq, the PhiX lane is *not* included. You must choose to sequence a lane of PhiX if you want to do control normalization. Finally, this service is completely "a la carte" so the pricing schema is quite different.
|# of Lanes||cycles per day||cycles per kit|
Using fewer lanes on each flowcell has allowed us to decrease the cycle time by not imaging all the lanes. In a typical 8 lane run, 20 minutes is spent doing chemistry followed by 40 minutes of imaging (each lane takes ~5 minutes to image). Therefore, a 2 lane flowcell runs twice as fast as an 8 lane flowcell. Also, since the chemistry is not running in to all of the lanes, each sequencing kit can go to a longer read length. The relationships are summarized in the chart on the left. Pricing is set on the number of lanes you are using, the number of days you are running the GAII, and the number of sequencing kits you are using. For example, if you wanted to run a 96+96 PE flowcell using 2 lanes, the cost would be the initial cost for the 2 lane PE flowcell plus an additional 3 days (one day is included in the original price) plus a second sequencing kit. That kit would not be completely used up (you would have an extra 48nt left that would be thrown away).
The GAII/GAIIx is ideal for:
- Unusual read lengths
- Protocol Prototyping
- Non-standard assays such as HITS-FLIP
This GAII pricing model is an experimental model and is subject to change
The Genome Analyzer IIs were donated to the BioMicro Center by Drs. Penny Chisholm, Chris Burge, Ernest Fraenkel and the Dept of Biology with contributions from many others
|Machine Names||FonZie|| Ryland
|# reads / lane||160-220m||20-40m||4-6m|
|# lanes coprocessed||7+PhiX||1 to 8||1|
|nt / day||24||24-72||288|
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.
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.
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.
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 for all supported technologies can be found here.
Pricing and Priority
Full pricing information is available on our price list.
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.
Initial page written by Summeet Gupta at the WI-CGT