NISC’s role within NHGRI, and more broadly across NIH, aims to advance genome sequencing and its many applications, with a goal not simply to produce sequence data, but to produce the infrastructure required to bring genomic sequence to biology and medicine. We accomplish this by meeting with each NIH investigator to discuss the details of their project to determine which method(s) would work best. The most common types of sequencing projects include whole exome sequencing, RNA sequencing, custom capture sequencing, CHiP-seq and whole genome sequencing. However, we are always interested in exploring new methods and expanding our repertoire in this rapidly changing field. We also work closely with other investigators across the NHGRI Intramural Research Program to develop novel methods to analyze genomics data with applicability to clinical and basic science questions that were thought to be intractable only a few years ago.
• Whole Exome Sequencing and Analysis – FAQ
• Bulk RNA-Seq – FAQ
• Single-Cell RNA-Seq – FAQ
• ChIP-Seq – FAQ
• Amplicon sequencing
• Custom capture projects – FAQ
• Library Construction – NISC has a highly skilled library construction group able to make a wide range of libraries compatible with all of our sequencing platforms. You deliver high-quality DNA or RNA and we construct the library. We prefer to start a sequencing project at this point, which allows our experienced hands to control each step for optimal performance and ensure the highest rate of success at the end.
• Data Return – The raw data from the sequencers is extensively processed, and data is evaluated for quality. For Illumina data, the general deliverable is fastq files which contain the base calls as well as sequence quality scores.
• Illumina MiSeq - MiSeq is a small-scale benchtop sequencer. This instrument is compatible with any of the Illumina libraries described above. MiSeq produces reads up to 300 bases in length from a paired-end read run (2×300 b) with a yield of ~10 Gb. This capability is primarily useful for sequencing microbial genomes and amplicons.
• PacBio Revio - The PacBio Revio platform generates long read lengths while maintaining high consensus accuracy and unbiased coverage. Multi-pass sequencing of both strands of the template are used to generate circular consensus (CCS) reads up to ~15-20 kb that can achieve accuracy > 99.9% (termed High Fidelity/HiFi reads) and detect base modifications. This platform can generate sequence reads that support (1) high-quality assemblies from small bacterial genomes to diploid human genomes; (2) detection of variants and phasing haplotypes; (3) characterization of RNA including full-length isoforms, whole transcriptome analysis and annotation, full-length 16S, and single-cell transcripts; (4) amplicons; (5) epigenomics; (6) metagenomics. For most applications, one SMRTcell generates 60-90 Gb of data and completes in ~30 hours.
• Oxford Nanopore PromethION - The Oxford Nanopore platform is the newest and most rapidly evolving platform in our portfolio of sequencing technologies. Monitoring current fluctuations as native DNA or RNA molecules pass through nanopores, this approach has key advantages of being able to read ultra-long fragments (some > 1Mb) and detecting modified bases without special library construction. Base calling is done in real time making sequence data available right from the start of the run. DNA read accuracy, including modified base detection is currently >99%. The primary uses of this technology to date at NISC have taken advantage of the ultra-long reads to detect large structural variants and create de novo genome assemblies. The amount of data generated per flowcell is dependent on the sample type with ultra-long genome libraries typically yielding ~20 Gb of reads >100kb (~40 Gb total). Other capabilities beyond whole-genome sequencing and detection of base modifications include direct RNA and full-length cDNA sequencing.
The NIH Intramural Sequencing Center provides cost-recovery sequencing services to the NIH Intramural Community. Initiating A Project – If you are thinking about initiating a new sequencing project, please visit https://www.nisc.nih.gov/contact.cgi. After this form is submitted, we will set up a meeting with you to plan the best approach for your needs. Please note that our sequencing service is restricted to intramural investigators at the NIH. Human DNA samples will need to be properly consented and de-identified.