Illumina technology is based on massively parallel sequencing of millions of fragments of DNA using reversible terminator-based sequencing chemistry. This technology relies on the attachment of randomly fragmented DNA to a planar, optically transparent surface. Attached fragments are extended and bridge amplified to create an ultra-high density sequencing flow cell with millions of clusters, each containing ~1,000 copies of the same template. These templates are sequenced using a robust four-color DNA sequencing by synthesis technology that employs reversible terminators with removable fluorescent dyes. High-sensitivity fluorescence detection is achieved using laser excitation and high speed scanning optics. Alternative sample preparation methods allow the same system to be used for a range of applications including gene expression, small RNA discovery and protein-nucleic acid interactions. Our facility offers library preparation services from the point of purified and properly prepared DNA or RNA. We are able to make libraries for genomic, mRNA-seq, totalRNAseq and ChIP-seq samples. We offer target capture, both exon and custom, using the Agilent SureSelect system. The customer must supply the kit from Agilent, and we will provide the remainder of the reagents, consumables and labor. You may purchase Illumina compatible primers and adapters through our facility stock room.

Library Preparation
Library Preperation information is available on our Library Prep Overview
Quality Control
We run QC assays on all samples submitted for Next Generation Sequencing. You will receive a Hi-Sensitivity DNA BioAnalyzer file (in both .pdf and .xad formats) or a Hi-Sensitivity D1K TapeStation file (in both .pdf and .d1k formats). In addition, you will receive results from our SYBR qPCR assay stating what the functional concentration of your sample is as a result of this assay. Our staff will give you recommendations for deciding if you should run or not run your sample. This decision is based on expected cluster density and potential contaminating sequences or lack of insert sequence. If your sample passes our QC and you decide to move forward with sequencing, we can transition your samples directly to our sequencing queue. Should your sample fail QC and you wish to proceed with sequencing, we would require a waiver form to be signed stating that the experiment will be paid for regardless of the outcome. If your sample is low concentration and you submit additional material, we may require an additional round of QC to ensure that the material will yield sufficient clusters and quality sequence. Please note that multiple freeze-thaw cycles of prepared libraries have been shown to degrade library performance so it is important to keep these to a minimum.
Sequencing by Synthesis
Sequencing on the Illumina HiSeq 2500, NextSeq 500 and MiSeq instruments will be carried out only by facility personnel. Our facility is in constant contact with Illumina to be sure that we are utilizing the latest protocols and reagents on all platforms as well as upgrading instrument parts as necessary. Quality metrics generated during a run provide an indication of the sequence quality and the run can be stopped after the first base cycle to save reagent costs should the flow cell contents be determined to be of poor quality. Run times vary based on the number of cycles collected but range from one to twelve days for the HiSeq2000; from <1 day to 4 days for the HiSeq2500; and 6 to 26 hours for the MiSeq. For example a 50 cycle, indexed run would take 3 days on the HiSeq2000, one day on the HiSeq2500 and six hours on the MiSeq.

The Biopolymers Facility provides QC services multiple times per week. The table below lists the deadline for sample drop off and the day the service will be performed.

Service Drop Off Deadline Services Performed
TapeStation & QPCR QC Tuesday before Noon Wednesday (TS) & Thursday (QPCR)
TapeStation & QPCR QC Friday before Noon Tuesday (TS) & Wednesday (QPCR)
QPCR Only QC Wednesday Thursday
* When Harvard is closed the service deadline & execution will be the next business day.
** If we have too few samples to do a qPCR run by Thursday, qPCR will be run on Friday.

Sequencing services on the Illumina HiSeq2000, 2500 and MiSeq are performed as soon as possible in the order they are received. Certain Harvard & HHMI labs that assisted in equipment purchases will automatically receive preferential queuing.

  • One Illumina HiSeq 2500 sequencers
  • Two Illumina cBot stations
  • Three Illumina MiSeq sequencers
  • Four Illumina NextSeq 500 sequencer

All samples should meet the following criteria:

  • Submit at least 10µl of 0.5-1 ng/µl library.
  • Deliver your samples in lo-bind eppendorf tubes (ex. USA Scientific "Low Adhesion Microcentrifuge Tubes" #1415-2600).
  • Deliver your samples to a staff member, along with your order confirmation sheet.

If you have another concentration or volume, feel free to submit the libraries and we will let you know if there is enough material to run.

If the sample is deemed to be so concentrated that it is outside of these parameters and requires a dilution and an additional Tape Station run, there will be an additional charge.

Also note, because the functional library can vary from the concentration you have measured, we cannot tell you definitively whether or not your sample is sufficient to cluster until we have performed our QC.

The Illumina HiSeq 2500, NextSeq 500 and MiSeq do not allow us to save images. As a result, the output from the Real Time Analysis (RTA) base caller will be the final data output you receive. In order for the software to properly identify clusters and provide a high yield of high quality sequencing reads, it is important that your input library be "base balanced" in the first ten cycles of your insert DNA. This is especially important for individuals using indexes introduced into the beginning of the sequence read, as these initial cycles are used to identify cluster location and size. Please ensure, when pooling your own libraries, your indexes are balanced at each base position (i.e. 25% A, C, G, T).

If you have never placed an order for Illumina sequencing, please feel free to contact us with any questions and we are happy to assist you through the process.

Please contact us at nextgen@genome.med.harvard.edu for pricing information for your specific project.

Library Preperation Protocols are available in our Library Prep Protocols section.
Bioinformatics
The Harvard T.H. Chan School of Public Health Bioinformatics Core (HBC) http://hsphbio.ghost.io is the recommended data analysis support group for BPF customers.
A variety of support services are available through the HBC. Please contact them for further information and a possible consult at bioinformatics@hsph.harvard.edu.
The HBC will evaluate your needs and will answer questions regarding any potential fees that may be associated with your project analysis.
Data distribution
We process the data on the Harvard Medical School Orchestra cluster and we utilize Harvard Medical School's mass disk storage array as a data repository.
Harvard Users:
  • HMS Storage - We encourage you to create an account on the mass storage array and directly access your data there.
  • FTP - sequence and alignment files only (instructions).
  • External Hard Drive - If you are interested, you will need to make arrangements for file transfer to an external drive with our facility computer staff. There are additional fees associated with this.
External Users:
  • FTP - sequence and alignment files only (instructions).
  • External Hard Drive - If you are interested, you will need to make arrangements for file transfer to an external drive with our facility computer staff. There are additional fees associated with this.
Data Retention Policy
The volume of data that our facility will generate on an annual basis is too great for us to maintain long-term storage of next-gen data files.
All data will be erased after 90 days.
Contact us at nextgen@genome.med.harvard.edu for more information.