Next Gen Sequencing
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 robust 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, amplicon, mRNA-seq, totalRNAseq, ChIP-seq, and more. Even if you do not see a compatible protocol listed, please contact us with details about your project, as sometimes we have a new method in the works or would be willing to implement a new option for your project.
Once samples are made into libraries, we can sequence them to a depth of 1M — 25B reads, offering incredible flexibility.
By ordering services from a non-profit academic core facility, you know that you are paying the actual cost of the experiment. It is important to us that you get the most from your grant dollars, and we will always suggest ways to save money. Most importantly, choosing a local core helps to keep expertise and low-cost sequencing available to you within walking distance!
Our turnaround time is very fast: Most samples sequence within 1 week of drop-off, including QC. The best way to improve turnaround time is to get libraries to us by Tuesday at noon, so they can be included in our weekly qPCR batch. Please communicate any relevant deadlines to our team and we will do our best to accommodate.
Library Preparation
Library Preparation information is available on our
Library Prep Overview
Quality Control
We run QC assays on all samples submitted for Next Generation Sequencing. Our QC has up to three parts: First, we run an Agilent
Tapestation High Sensitivity D5000 assay to evaluate the distribution of fragment sizes, which greatly impacts clustering behavior.
The Tapestation run also gives us an initial concentration reading. If the sample is above the qPCR's functional range (>5nM), we will
dilute and re-run the Tapestation to confirm the sample is now in range. Finally, we will run qPCR using primers complementary to the
p5/p7 sequencing adapters. This gives us a "functional concentration", amplifying only the DNA fragments which were successfully ligated
with adapters. Our team will evaluate the Tapestation trace, the qPCR value, and the difference in concentration between them, to advise
on the sample's success. If your sample has any warning flags for QC, we will not proceed until you confirm. If you choose to skip QC in
order to expedite your run, we cannot guarantee run success.
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 confirmation from you first, with the understanding 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.
Tapestation is run every day, but qPCR is run once a week in order to keep costs down for all users. In order to improve turnaround time for your sample, please be sure to have your sample to us by Tuesday at noon.
Sequencing by Synthesis
Sequencing on the Illumina 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. We run our
sequencers six days a week, to provide quick turnaround time without compromising run quality.
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 ~26 cycles of your insert DNA. This is especially important for the first ~5-10 bases of the sequence read, as these initial cycles are used to identify cluster location and size. Please ensure your indexes are balanced at each base position (i.e. 25% A, C, G, T). We also strongly recommend use of Unique Dual Indices, rather than single indexing.
Tapestation is run every day, but qPCR is run once a week in order to keep costs down for all users. In order to improve turnaround time for
your sample, please be sure to have your sample to us by Tuesday.
The table below lists the deadline for sample drop off and the day the service will be performed. QPCR data is usually distributed on Thursday, but can shift if a re-run is needed, if the batch had more samples than usual, or if there was a holiday.
| Service | Drop Off Deadline | Services Performed |
|---|---|---|
| TapeStation & QPCR QC | Tuesday before 5pm | All week (TS) & Wednesday (QPCR) |
* 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 Wednesday, qPCR will be run on Friday.
Sequencing services on the Illumina sequencers 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. We ask that you communicate any relevant deadlines to us as soon as possible! We cannot always move orders up in the queue, but we will always do what we can to expedite, and it can help with prioritizing if multiple orders come in at the same time.
- Illumina MiSeq sequencers (1M — 20M reads)
- Illumina NextSeq 2000 sequencers (100M — 1.8B reads)
- Illumina NovaSeq X Plus sequencer, with two stages for simultaneous runs. (675M reads — 25B reads)
All samples should meet the following criteria:
- Submit at least 20ul of 2ng/ul or 2nM library. More is better, as long as you can keep a backup in your lab in case we need additional volume. We hold onto samples for 3 months, so you can pick up extra volume.
- Deliver your samples in lo-bind 1.5mL tubes (ex. USA Scientific "Low Adhesion Microcentrifuge Tubes") labeled with the library ID number generated during the order placement process.
- Deliver your samples to our drop-off freezer (directions here), along with your order confirmation sheet.
If the sample is deemed to be so concentrated that it is outside of these parameters and requires a dilution and an additional TapeStation run, there will be an additional charge. This is already included on the order as the "QC Dilution Fee" which will be canceled if we do not need to run a dilution.
Also please 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.
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!
There are dozens of sequencing kits, and cost-per-sample decreases with bigger sample batch sizes. We will always help you find the fastest, most cost-effective option. As a non-profit facility, we only charge what the experiment costs, so you can know your grant dollars are being spent wisely.
Please contact us at nextgen@genome.med.harvard.edu for pricing information for your specific project.
Bioinformatics
The Harvard Chan School of Public Health Bioinformatics Core (HBC) is the recommended data analysis support resource for BPF customers.
HBC offers a wide range of services, including assistance with genomic, transcriptomic, epigenomic, and multimodal data analysis. They
also provide support for optimizing experimental designs and strengthening grant applications. For more information or to request a
consultation, visit their website at https://bioinformatics.sph.harvard.edu/ or contact them at bioinformatics@hsph.harvard.edu.
The Harvard T.H. Chan School of Public Health Bioinformatics Core (HBC) https://bioinformatics.sph.harvard.edu/ is the recommended data analysis support group for BPF customers.
The HBC will evaluate your needs and will answer questions regarding any potential fees that may be associated with your project
analysis.
Single Cell and Spatial RNA-Seq Analysis Tool
Cellenics is an open-source platform aimed at empowering research biologists to analyze their own single-cell RNA sequencing (scRNA-seq)
and spatial RNA sequencing datasets. This user-facing application for project management, data processing, quality control, and
visualization was developed as part of a collaborative effort between numerous Harvard Medical School departments, cores and centers.
Cellenics modules enable in-depth data exploration through differential expression and pathway analyses as well as the generation of
fully customizable plots for publication.
Sign up: https://cellenics.hms.harvard.edu
Contact for support: alex_pickering@hms.harvard.edu
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 should have access to O2, and should select O2 as their data distribution method in their account if so. Please contact Research Computing here, for assistance with an O2 account.
External Users can get data through Globus. Please contact us if you need assistance making a Globus account.
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 30 days. Please move the data to somewhere like a
hard drive, once you are notified the data is ready. We recommend keeping a few 1TB hard drives in your lab, in case one fails. A computing
cluster like O2 can also be a good storage solution while you are working on analysis, please contact Research Computing for assistance, at
rchelp@hms.harvard.edu.
Contact us at nextgen@genome.med.harvard.edu for more information.