High Throughput DNA Sequencing

Applications

We have two Illumina HiSeq 2000 sequencers that can be used for a broad range of applications, including but not limited to:

As there are many types of applications it is essential that you speak with us regarding the specific goals and technical aspects of your research projects. We can perform both single-end and paired-end sequencing at either 50bp or 100bp read lengths. Please contact us to discuss how longer reads or paired-end reads may be beneficial for your projects.

Service and Sample Submission

All users are required to fill the Service Request Form and email it to us at genomics@rockefeller.edu. Samples will not be accepted without the form. You will be entered into the sequencing queue upon receipt of this form and your samples having been dropped off at the center.

We can sequence up to 7 samples in a run. You can submit any number of samples and use any number of lanes for a given sample, as we can combine different users' samples in one run.

Full service - Library Preparation, Cluster Generation, and Sequencing

Currently, we only offer full service on genomic DNA-seq and RNA-seq. Users only need to provide us genomic DNA or total RNA, we will prepare libraries, generate clusters, and perform sequencing runs.

For other sequencing applications (such as ChIP-seq, targeted sequencing, and small RNA sequencing), users need to prepare the libraries and bring them to us for sequencing.

Genomic DNA sample submission
For each sample, a minimum of 2 micrograms of genomic DNA is required. The gDNA should be column purified and intact. An agarose gel picture is required and the A260:280 ratio needs to be above 1.8.

RNA-seq sample submission
For each sample, a minimum of 200 ng of total RNA is required. The RNA should be column purified and intact (RIN > 8.0; A260:280 ratio > 1.8)

Cluster Generation and Sequencing-only service

We can perform sequencing on customer generated libraries, provided they are constructed in accordance with standard Illumina protocols. Sample preparation kits can be purchased from Illumina, and protocols are available on the Illumina website (www.illumina.com). We strongly recommend the use of Illumina TruSeq library prep kits, which give the flexibility of performing either single read or paired-end runs as well as multiplexing several samples into one sequencing lane to save costs.

Pre-made library submission
For each sample, at least 10ul of 10nM template is required. This is to make sure that we can check the DNA concentration with the Agilent Bioanalyzer 2100 High-Sensitivity DNA kit. Higher concentrated samples (~100nM) are preferred to ensure accurate quantitation.

The proper size of the templates will depend on the application and the type of sequencing performed. It is best to consult with us prior to starting a project, but as a general guideline templates submitted to us should be no longer than 600bp.

A 260:280 ratio >1.8 and a 260:230 ratio >1.2 is required.

Data Output and Delivery

The standard outputs we generate are sequence files in FASTQ format with base-calling quality scores. We will deliver the files to users through Rockefeller CFS (Central File Storage), or directly to a laboratory's server if available. FASTQ files will be compressed with gzip prior to delivery, and sizes vary from 5Gb to 20Gb per lane depending on read length and the number of reads generated.

Data Analysis

We will perform initial data analysis to generate FASTQ files for all samples free of charge. We will also perform alignment to a reference genome free of charge upon user's requests. Further data analysis (such as peak finding, SNP finding, transcriptome analysis) as well as special requests (such as building lab database and pipeline) will incur a bioinformatic support charge of $90 per hour.

Data Retention

Due to the large size and high volume of sequencing data, the GRC has very limited capacity for data retention.

Image files: Updated Illumina software no longer retains image files. Images are analyzed in realtime and immediately deleted by the software. Users therefore do not have the option of requesting image files.

Intermediate analysis files: The standard procedure is to retain the intermediate files at the GRC only during the troubleshooting phase, which is generally less than a month. If users would like a copy of the intermediate analysis files, they must request this when submitting samples AND provide a portable hard drive for the file transfer.

Analyzed data (fastQ and aligned data): This data are delivered to users' CFS folders or designated servers, and will be stored at the GRC for six months. For operational purposes, the GRC may retain data beyond six months, but cannot guarantee retrievability after six months. All sequencing data will be permanently deleted from GRC computers/servers after one year.

Sample/Library Retention

If you would like to keep the samples or libraries that have already been sequenced, you need to get them back from us and store in your own freezers. We will keep them in our freezers for one month from the time that the sequencing data is delivered. After one month, leftover samples and libraries will be discarded.