Platforms

NextGen Sequencing Technologies

NISC currently employs the following NextGen sequencing technologies:

Illumina NovaSeq X Plus
Illumina MiSeq
PacBio Sequel II
Oxford Nanopore PromethION

Illumina NovaSeq X Plus

The NovaSeq X Plus offers scalable sequencing through multiple flow cell modules. The most cost-effective module is the 25B which produces ~8 Tb per flow cell or ~1 Tb per lane. Due to the extremely high quantity of sequence output, this technology is primarily useful for projects requiring a very large number of reads, such as whole genome, and multiplexed transcriptome experiments.

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 (2x300 b) with a yield of ~10 Gb. This capability is primarily useful for sequencing microbial genomes and amplicons.

PacBio Sequel II

The PacBio Sequel II 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 ~25 kb that can achieve accuracy > 99.9% (termed High Fidelity/HiFi reads). 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 15-35 Gb of data and completes in 20-30 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. Raw read accuracy is currently about 98% with new advances expected to push that over 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. Using this approach, a typical PromethION flowcell yields 80 Gb of quality reads with N50 approximately 72kb. Preliminary experiments have also been undertaken to explore direct RNA and full-length cDNA sequencing.