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 library preparation service for genomic DNA and RNA-seq.

Sample Submission

Chromatin

ChIP-Seq: Sonicated chromatin will undergo IP on the IPstar. RNase A, Proteinase K, Phenol Chloroform extraction and purification will be performed off of the instrument and submitted for QC on the Bioanalyzer. These ChIP samples may be picked up for qPCR or sent directly to SPRI for library construction and sequencing (options are listed on submission forms). Users will be contacted if ChIP samples fail QC. These samples will not be submitted to SPRI-works but will be charged for the IP.

DNA

There are three options when submitting samples for library prep:

BMC Fragmented DNA sample prep (SPRI) - 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.

BMC Intact DNA sample prep (NEXTERA) - Samples will be nanodropped, processed using the Epicentre Nextera kit, enriched using Nextera PCR primers (including molecular barcodes if requested) quality controlled and submitted for sequencing on the Illumina platform. At least 50ng of DNA is required for Nextera. Samples for Nextera cannot be pre-QC'ed on the BioAnalyzer because the large fragments will clog the microchannels.

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.

RNA

mRNA-seq - Samples will be QC'd, poly-A purification will be performed using the Illumina Tru-Seq protocol, cDNA will be run on the SPRI-works system using BMC stored adapters, enriched using BMC stored PCR primers, and submitted for sequencing on the Illumina platform.

Sample Submission Forms

Sample Submission Requirements

Chromatin – Submission of sonicated chromatin must be submitted as 5 million cells per 200uL volume. Antibodies must be submitted at the same time, ensuring that there is at least 3ug of antibody per IP. We have done months of testing and highly recommend using the following sonication conditions:

1. Resuspend fixed cells in 10mL of LB1:

  LB1

2. Rock cells at 4 degrees for 10 minutes
3. Pellet cells at 4 degrees for 5 minutes at 1350g
4. Remove supernatant and resuspend cells in 10mL of LB2:

  LB2

5. Rock cells at 4 degrees for 10 minutes
6. Pellet cells at 4 degrees for 5 minutes at 1350g
7. Remove supernatant and resuspend cells in a volume of LB3 where there is 5 million cells/200uL
**Example: Resuspend 25 million cells in 1mL of LB3**

  LB3

8. Sonicate cells
9. Pellet debris at 4 degrees for 10 minutes at 20,000g
10. Submit lysate for ChIP

Genomic DNA– Samples for Illumina must be submitted 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. The Nextera kit requires 50ng of material for preparation.

RNA-Seq – Submission of total RNA samples for RNA-Seq must be submitted in water or TE. Total RNA needs to be between 0.1-4ug and should have a RIN above 9.0.

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-dimer 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 sample preparation can be directed to Manlin Luo and Ryan Sinapius.