BioMicroCenter:Illumina Library Preparation
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Generating high quality data on the Illumina sequencing platform begins with generating high quality libraries that have the desired insert size and required Illumina adapters. Prior to sequencing, these samples must pass the BioMicro Center’s Sequencing Quality Control process. The BMC currently offers genomic DNA library preparation as a service; RNA-seq is being tested in beta.
Sample Submission
There are two options when submitting samples for library prep:
BMC Illumina sample prep - Samples will be QC'd, run on the SPRI-works system using BMC stored adapters, enriched using BMC stored PCR primers, and submitted for sequencing on the Illumina platform. All samples submitted for this option will be QC’d using the BioAnalyzer to confirm sufficient fragmentation and concentration.
SPRI-Works Direct Submission- Samples will be run on the SPRI-works system using user supplied adapter mix and then returned for enrichment. If using this option please note that samples will need to be submitted separately for Illumina sequencing.
Sample Submission Requirements
Genomic DNA– Samples for Illumina must be sonicated and in either water or TE. The SPRIworks robot is not highly sensitive to the amount of DNA, however care must be taken in submitting an appropriate amount of adapter for ligation.
RNA-Seq – Submission of total RNA samples for RNA-Seq is currently in early stages of beta testing. RNA-Seq samples that have been mRNA selected, fragmented, and synthesized into dsDNA can be submitted for further library preparation as genomic DNA.
Barcoding
Samples submitted for Illumina preparation can be barcoded by request to allow sequencing of multiple samples per lane. Full information about barcoding can be found HERE.
Illumina Library Preparation Workflow
- Fragmentation - All samples must be fragmented to the point where the majority of the fragment distribution is between 100 to 300 base pairs. RNA-seq samples are usually fragmented after poly-A selection and before first strand synthesis.
- End Repair and 3’ dA Addition – Any damage end damage that may have occurred during fragmentation is repaired and then an extra A is added to all 3’ ends to increase efficiency during ligation.
- Adapter ligation – The ligation of partial Illumina adapter sequence to the sample fragments, a total addition of 66bp. Each adapter sequence has a T overhang to increase efficiency. It is very important to maintain a good ratio of sample material to adapter, Illumina recommends a 1:10 ratio, to avoid primer dimmer occurrences post enrichment.
- Size Selection – This step, usually consisting of an agarose gel, is designed so that only the fragments within the desired size range (now ~200 – 400bp) are included in the final library. Size selection is very important, fragments that are too large will create large clusters and can result in a loss of read count in the final data.
- Enrichment – The PCR reaction run during this step amplifies using the complete primer construct required for binding and clustering on the flowcell, an addition of 53bp for a final adapter length of 119bp. It is very important to optimize the number of PCR cycles to generate a sufficient amount of material for clustering but limit PCR biases.
Traditional Illumina protocols can be found on our protocols page
Pricing
Priority for Illumina sample prep is currently available for labs associated with the BioMicro Center Core departments. We are able to do Illumina sample prep for other MIT and non-MIT users as space allows on the sequencers. Full pricing information is available at our price list.
Protocols
Protocols for all of the supported technologies can be found by visiting the Protocols page
QC
Quality control is very important to help optimize the number of reads and quality of data produced. For information on QC methods and protocols please visit the Sequencing Quality Control page
All questions about Illumina Sequencing can be directed to Kevin Thai.
