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High Throughput DNA Sequencing

Applications

We have Illumina NovaSeq 6000, NextSeq 2000, NextSeq 500, and MiSeq sequencers that can be used for a broad range of applications, including but not limited to:

  • Whole genome sequencing
  • Exome sequencing
  • Targeted sequencing
  • RNA sequencing
  • Protein-DNA/RNA interactions (ChIP-Seq, CLIP, etc.)
  • Targeted sequencing
  • Small RNA sequencing
  • ATAC sequencing
  • Ribosomal profiling

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. Samples will be entered into the sequencing queue upon receiving the form and samples.

Full service – Library Preparation and Sequencing

Currently, we only offer library preparation service for genomic DNA sequencing, RNA sequencing, and PCR amplicon sequencing. Users only need to provide us genomic DNA, total RNA, or PCR amplicons, we will perform sample QC, prepare libraries, pool libraries, and perform sequencing runs.

For other sequencing applications, users need to prepare and pool the libraries and bring them to us for sequencing.

Genomic DNA sample submission
For each sample, a minimum of 100 ng genomic DNA is required. The gDNA should be column purified and intact. An agarose gel picture is required and the A260:280 as well as 260:230 ratios need to be above 1.8.

RNA-Seq sample submission
For standard RNA-Seq protocols, a minimum of 100 ng total RNA is required.  RNA should be column purified and intact (RIN > 8.0; A260:280 and 260:230 ratios > 1.8).  We also perform RNA-Seq library preparation with very small input material, as little as 1 ng of total RNA.

PCR amplicon sample submission
For each sample, a minimum of 100 ng PCR amplicons is required.  Amplicons should be column purified and the size range should be between 150 bp and 600 bp.

 

Sequencing-only service

We perform sequencing on user-prepared libraries.  Users are responsible to make sure that libraries are compatible with Illumina sequencers.  We can also use custom sequencing primers, and again users are responsible for the compatibility with Illumina sequencers.  Please contact Illumina technical support with any questions techsupport@illumina.com

Pre-made library submission
Users should pool libraries before submitting to us.  For each pool, at least 10ul of 10nM material is required for NextSeq, and 100 ul of 10nM for NovaSeq. We will check library quality and quantity on TapeStation and Qubit before sequencing.

With user-prepared libraries, we can’t guarantee sequencing quality.  If libraries need to be resequenced to obtain enough reads, 50% fees will apply for repeating.

Data Analysis and Delivery

We perform initial data analysis and demultiplexing to generate individual FASTQ file for each library in a pool. We deliver FASTQ files to users through Rockefeller CFS (Central File Storage), HPC cluster or directly to a laboratory’s server if available.

Sequencing Data Retention

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

BCL file retention: Illumina sequencers generate raw data in binary base call (BCL) format, which needs to be converted to FASTQ files for further data analysis.  Our center uses bcl2fastq Conversion Software, which is a standalone software offered by Illumina, to do demultiplexing and convert BCL files to standard FASTQ files.

BCL files would be retained for a very short period of time only for troubleshooting, usually less than a month. If users would like a copy of the BCL files, they must request this when submitting samples.

FASTQ file retention: FASTQ is a text-based sequencing data file format that stores both raw sequence data and quality scores. FASTQ files have become the standard format for storing NGS data from most sequencing systems, and can be used as input for a wide variety of secondary data analysis solutions.

FASTQ is usually the data format that delivered to users, and will be stored at our center for three months. Once delivered, it’s users’ responsibility to keep the data files.

Other Sequence File Formats: FASTQ files are the starting format for data analysis.  During secondary or tertiary analysis of NGS data, FASTQ files could be converted into other formats, for example, .bam, .vcf etc. Our center cooperates with Bioinformatics core to help you achieve the designated data format.

For Single Cell data, we use cellranger mkfastq to demultiplex the Illumina sequencer’s base call files(BCLs) into FASTQ files. This is a pipeline that wraps Illumina’s bcl2fastq and recommended by 10x for their planform.

Further data analysis pipeline, cellranger counts, aggr, vdj, could also be performed with prerequisite.

Sample/Library Retention

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