NextGen Sequencing has been offered through the Molecular Genomics Core in the USC Norris Comprehensive Cancer Center for several years, but the service really took off in early 2011 with the hiring of dedicated sequencing staff, a director to oversee NextGen projects and the acquisition of a high-output sequencer from Illumina, the HiSeq 2000. Since then, the core has worked with dozens of USC Norris and Keck School of Medicine researchers to assist in the design, implementation and interpretation of NextGen sequencing experiments.

In summer 2014, the core acquired two of the latest sequencing instruments from Illumina, the NextSeq 500 and MiSeq, providing further access of cutting-edge instrumentation to the HSC research community.
The core is centrally located on the HSC campus in NRT G514. It is open during normal working hours and freely accessible through entrances off Eastlake (the main entrance with the traffic turnaround) and Pappas Quad.

Core Staff
The NextGen Sequencing Core has a friendly, interactive staff dedicated to the production and delivery of high-quality sequence data. Helen Truong and Selene Tyndale operate the instruments, construct and characterize sequencing libraries, and assist customers with quality control analysis of their libraries and starting materials such as RNA or genomic DNA. This is their 5th year performing these functions and they are a well-integrated, highly competent team. The core director, Charles Nicolet PhD has nearly 18 years of core management experience in the sequencing field. He oversees all core operations and has frequent meetings with core users or those interested in beginning or expanding their NextGen projects. His group has been together since January 2011, providing stability and continuity within the NextGen operation.

Major Services, Technologies, Equipment and Expertise Provided

Consultation
The core offers expert consultation on sequencing strategies, project design and technological core capabilities. The core director attends sequencing meetings (such as AGBT, ABRF and Illumina user’s group meetings), publishes in the sequencing field (1,2), and is part of several national sequencing consortia running large sequence technology comparison studies. Thus, customers can be assured they are hearing the most up-to-date information and recommendations for their experiments. Consultation does not stop once the samples are sequenced. The core director can assist in interpretation of sequencing quality and instruct users in the use of analysis routines that assess data quality in an alignment-independent fashion. Following more extensive bioinformatic analysis of their samples, users can also count on the core director to provide information about quality metrics associated with successful experiments. The goal is for users to know as soon as possible whether the data generated is suitable for further analyses or whether additional work needs to be done.

Direct Services
Next generation sequencing. The core operates three Illumina next generation sequencing instruments: the HiSeq 2000, NextSeq500 and MiSeq. All operate on the Illumina “sequencing by synthesis” paradigm. Illumina is the clear leader in the NextGen space, with greater than 80 percent of such data produced on Illumina instruments. Each instrument produces high-quality data but varies in ouput, timing and capabilities.

HiSeq 2000: This instrument has a more limited throughput compared to newer members in the Illumina family. However, it does produce data of exceptional quality and can be economically exploited for large projects by purchasing a whole sequencing run (that is, all eight lanes of a flow cell). At this level, the instrument will produce up to 1.6 billion reads comprising 100 times the coverage of the human genome in a single run.

NextSeq 500: This instrument has become our bread and butter machine; every night it is running. The output is substantial, up to 500 million reads with a maximum output of 45 times the coverage of a human genome in a single run. The nice thing about this instrument is speed — it can provide 45 times the coverage overnight, whereas on the HiSeq that would take nearly two weeks.

MiSeq: The output is substantially less than the other platforms (10 percent or lower), but it is designed and optimized for particular kinds of projects where huge amounts of data are not required. Examples of these would be analysis of microbial genomes, BAC sequencing, metagenomic studies and targeted resequencing.

Library Production. Experimental material to be analyzed on the NextGen sequencing platforms first needs to be engineered into a so-called sequencing library. A sequencing library can be generated from nearly any nucleic acid sample: DNA, RNA, material purified from cells, tissue, artificially generated or any combination. Due to the range of research taking place at USC, our core has acquired expertise in the preparation of a broad range of library types. The most common libraries produced in our core are RNA-seq, ChIP-seq, whole genome and whole genome bisulfite converted libraries. Within each of these broad types are several variations on each theme, many of which have been optimized in our core. For example, RNAseq library options with which we have experience includes polyA selected, ribosomal RNA depleted, very low quantity input or strand specific libraries. However, we are not limited to working with library types with which we have experience; we are also willing to develop new methods. In these cases, arrangements are made to split the research and development costs of new library types between the client and the core.

We also support and encourage the analysis of libraries generated in customers’ laboratories. The advantage here is typically a cost and time savings for the lab. If this is of interest, we can provide assistance and recommendations to help your lab successfully get library construction going.

Bioinformatics. Our core does not offer sufficient bioinformatics support to serve as an analytic endpoint for publication. However, we do have an analysis pipeline that will provide a sufficient amount of data to determine sample quality. This is important for time and economic considerations — customers don’t waste money at a bioinformatics core for substandard samples and the customer can find out rapidly if something does not work. Data metrics and file downloads are available through two avenues: our own ECDP interface and the Illumina BaseSpace interface. We can also assist in directing you to local bioinformatics resources, as we work closely with these groups.

Ancillary Services/Equipment. The core has equipment used to perform quality control on libraries, the primary platforms being a qPCR machine (LifeTech HT 9700) and an Agilent Bioanalyzer. These can be used by anyone during normal working hours once they have been appropriately trained. We assist in interpretation and provide guidance in optimal use of these machines. We can also run your samples on these platforms as a fee-for-service option.

Education. NextGen sequencing has become a standard indispensable tool in modern genomics and molecular biology. Although the NextGen tools are standard, they can be expensive, time consuming and fraught with pitfalls. We are available to help customers with all of these issues to enable a generation of high quality, scientifically interesting, as well as publishable and fundable, results. We pride ourselves on having an open and communicative core and encourage customers to contact us any time with questions before, during and after your NextGen sequencing projects.

1. “Multi-platform assessment of transcriptome profiling using RNA-seq in the ABRF next-generation sequencing study,” Li, S. et al. (2014). Nature Biotechnology 32: 915.
2. “Evaluation of Commercially Available RNA Amplification Kits for RNA Sequencing Using Very Low Input Amounts of Total RNA,” Shanker, S. et al (2015). J. Biomolecular Techniques Published online 2015 Jan 29. doi: 10.7171/jbt.15-2601-001