The GRC occupies custom-designed BSL-2 research space within the University of Maryland’s Health Sciences Research Facility III, a state-of-the art research laboratory completed in 2018. The GRC has equipped the laboratory with custom-built laboratory automation platforms to increase efficiency, reduce error, and increase throughput. We utilize a customized Laboratory Information Management System (LIMS) for reagent, sample, and project tracking. The GRC LIMS increases efficiency and reduces errors through the use of custom workflows for each platform. These workflows provide process control and validation to ensure each sample is processed according to established and reviewed protocols. Our custom-designed facility, automation platforms, and LIMS support our high-throughput sequencing pipelines.

Illumina

Illumina sequencing technology has become the most widely adopted next-generation sequencing platform. Illumina sequencers provide high-throughput, short-read sequence data with the lowest cost-per-base of any commercially available platform. The technology uses sequencing-by-synthesis on a glass surface (flowcell) to produce billions of reads per run with read lengths up to 300bp and growing. This platform enables many applications, including whole genome sequencing, exome and custom targeted sequencing, transcriptome sequencing, epigenomic sequencing, and others. The GRC operates four types of Illumina sequencers: MiSeq, HiSeq 2500, HiSeq 4000, and the flagship NovaSeq 6000. A single 2 day NovaSeq 6000 dual S4 flowcell run can generate sufficient data to sequence 48 human genomes, more than 500 human exomes, more than 400 human transcriptomes, or thousands of microbial genomes. Both the MiSeq and HiSeq 2500 rapid run mode offer longer reads and more rapid turnaround time with lower overall throughput. Typical applications for these platforms include microbial genome sequencing, microbiome profiling, custom amplicon sequencing, and targeted panels.

PacBio

Pacific Biosciences (PacBio) is a single-molecule, real-time (SMRT) sequencing technology capable of detecting signal from an individual DNA molecule coupled with an individual DNA polymerase. It achieves the highest consensus accuracy and longest read lengths of any currently available sequencing technology and is free of amplification artifacts that can confound other sequencing technologies. While lower overall throughput than short-read technologies is a limitation with this platform, the fundamentally stochastic and unbiased error profile leads to rapid increases in accuracy as sequencing coverage increases.

The GRC is a PacBio Certified Service Provider and beta site for new applications. We operate all three PacBio sequencers, including the original RS II, the Sequel System, and the newest platform – the Sequel II System. With a mean read length greater than 20,000 bases, an N50 read length exceeding 35,000 bases on many runs, and a maximum read length >100,000 bases, the Sequel II platform enables many applications not possible with other technologies. The reaction kinetics data that are detected during the run can be used to natively detect modified nucleotides for epigenomic applications. Some applications ideally suited for this platform include de novo genome sequencing, variant detection and validation, methylation analysis, targeted/amplicon sequencing, and full-length transcript/cDNA sequencing (Iso-Seq).

10x Genomics Chromium

The 10x Genomics Chromium instrument is the most widely used commercial single-cell microfluidics system in the world. Capable of capturing hundreds to tens of thousands of single cells per sample, the technology enables both transcriptome and genome scale sequencing at the single cell level. Applications include gene expression profiling, chromatin accessibility assays (ATAC-Seq), immune profiling, genome-wide linked-read sequencing for de novo assembly, and more!

NanoString

The NanoString nCounter Analysis System has the ability to digitally quantify changes in up to 800 DNA, RNA, and protein targets using a single detector from samples of limited volume and concentration without the need for amplification or library preparation. The nCounter’s digital counting capability provides highly reproducible data over 5 logs of dynamic range and does not require any amplification steps that might introduce bias to the results. An additional advantage of nCounter chemistry is that it is highly tolerant of difficult sample types such as FFPE and crude-cell lysates. With a technology that expands our capabilities and complements our high-throughput sequencing platforms, we are excited to have the NanoString nCounter join our growing family of services.