Next-Generation Sequencing

Next-generation DNA sequencing and RNA-seq have made it possible not only to look at individual genomes, but also to rapidly compare genetic sequences among multiple genomes. These approaches can be used, for example, to determine differences in genomes and gene transcripts from person to person, between populations, and between normal and pathologic cells in cancer. As next-generation sequencing technologies have made it possible to generate high-resolution genomic data much more efficiently, researchers in many fields have turned to next-generation sequencing to identify particular features of the genome that contribute to specific phenotypes.

The Columbia Genome Center, located at Columbia University Medical Center in New York City, provides high-quality and cost-effective next-generation gene sequencing services. Our clients and collaborators include researchers in the Columbia University community, as well as investigators at other universities and in the biotechnology and pharmaceutical industries. We are an Illumina CSPro Certified Service Provider.

Please contact us to discuss your project and how next-generation sequencing can help you in your research.

Whole genome and whole exome sequencing

Next-generation DNA sequencing makes it possible to rapidly compare the genetic content among samples and identify germline and somatic variants of interest, such as single nucleotide polymorphisms (SNPs), insertions and deletions (indels), copy number variants (CNVs), and other structural variations.

In next-generation DNA sequencing, the DNA is first broken into a library of small fragments. These fragments are then attached to oligonucleotide adapters that facilitate the biochemistry necessary for the sequencing reaction. After being placed on a slide or in a flow cell, the strings of nucleotide bases that make up the fragments are then sequenced in hundreds of millions of parallel reactions. In re-sequencing studies, where a high-quality reference genome exists, the reads from the machine are mapped to the reference genome based on sequence alignment, which in turn is used for calling variants. In de novo genome sequencing studies, the reads are assembled to form a draft genome.

Next-generation technologies can quickly generate a sequence of a whole genome, or can be more targeted using an approach called exome sequencing. Exome sequencing focuses specifically on generating reads from known coding regions. In contrast to whole genome sequencing, which sequences the entire genome, exome sequencing is a cost-effective approach that can detect single nucleotide or short indel variants in coding regions, and provides sufficient information for many research needs.

RNA-seq

RNA-seq is a next-generation sequencing technique that measures the abundance of RNA transcripts in a sample. It is a powerful tool for understanding dynamics in the transcriptome, including gene expression level difference between different physiological conditions, or changes that occur during development or over the course of disease progression. Specifically, this application can be used to study phenomena such as:

gene expression changes
alternative splicing events
allele-specific gene expression
chimeric transcripts, including gene fusion events
novel transcripts
RNA editing

In a standard RNA-seq procedure, total RNA first goes through a poly-A pull-down for mRNA purification, and then goes through reverse transcription to generate cDNA. The cDNA is broken into a library of small fragments, attached to oligonucleotide adapters that facilitate the sequencing reaction, and then sequenced either single-ended or pair-ended. Finally, the reads are aligned to a reference genome to estimate the expression level of known or novel transcripts. The results can indicate differences in transcriptional structure and/or in expression levels of specific genes. To study noncoding RNAs that lack poly-A tails, the poly-A pull-down step is replaced with a ribosomal RNA reduction experiment.

Our sequencing platforms

We use a variety of successful and widely adopted tools for conducting next-generation sequencing. Our technical infrastructure includes:

Illumina HiSeq 2000
An industry-standard platform for next-generation sequencing, the HiSeq is designed for large-scale high-throughput experiments. Outfitted with two 8-lane flow cells, it can quickly generate large amounts of data (600 Gb per run as of summer 2012).

Illumina MiSeq
This benchtop technology makes it possible to perform smaller sequencing experiments, including test runs to evaluate samples before performing a more extensive sequencing project. It also offers flexibility that is important in clinical applications. A planned upgrade of the technology will generate longer reads (up to 500 bp), making it useful in a new set of interesting experiments, such as HLA allele typing, de novo genome sequence and assembly, and T cell receptor and immunoglobulin repertoire clonetyping.

Life Technologies Ion Torrent
This benchtop tool uses a newer semiconductor technology that senses the release of hydrogen ions to monitor the sequencing process.

Pricing that encourages experimentation

We welcome next-generation sequencing projects of all sizes, including both large and small runs. Unlike at larger industrial sequencing facilities, the Columbia Genome Center has no minimum sample size. This makes it cost-effective to perform advanced, higher-risk applications that are often more expensive at other centers.

We also encourage pilot projects: You can test an experiment and our facilities by sequencing just a small number of samples for the same price per sample as a more extensive experiment. This flexibility is often particularly useful for researchers who are new to the technology.

Advanced bioinformatics capabilities